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Pharmacokinetics and Pulmonary Disposition of Clarithromycin and Tilmicosin in Foals

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PHARMACOKINETICS AND PU LMONARY DISPOSITION OF CLARITHROMYCIN AND TILMICOSIN IN FOALS. By ARIEL Y. WOMBLE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Ariel Y. Womble

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iii ACKNOWLEDGMENTS I would like to thank my family for th eir continuous support while I pursue my goals in veterinary medicine. Their unwa vering confidence in my abilities makes the accomplishment of this thesis even more va luable to me. I also want to thank my boyfriend Michael who pushed me to go furthe r, never doubting that I would make it. I especially want to thank Dr. Steeve Gigure for giving me this incredible opportunity. He opened the door to an experien ce that has forever shaped me. I have learned more than I imagined I would, not only about science and veterinary medicine but about myself. I greatly appreciate the support, guidance, and mentorship that he provided to me. I will be forever grateful.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Foal Pneumonia............................................................................................................3 Rhodococcus Equi ........................................................................................................5 Macrolides....................................................................................................................9 Clarithromycin.....................................................................................................10 Tilmicosin............................................................................................................13 3 PHARMACOKINETICS OF CLARITHROMYCIN AND CONCENTRATION IN BODY FLUIDS AND BRONCHOALVEOLAR CELLS IN FOALS.................17 Abstract.......................................................................................................................17 Introduction.................................................................................................................18 Materials and Methods...............................................................................................19 Horses and Experimental Design........................................................................19 Bronchoalveolar Lavage......................................................................................21 Drug Analysis by High Performance Liquid Chromatography (HPLC).............21 Measurement of Clarithromycin Activ ity Using a Microbiologic Assay...........23 Estimation of PELF and BAL Cell Volumes and Determination of Clarithromycin Concentrations in PELF and BAL Cells................................24 Pharmacokinetic Analysis...................................................................................25 Statistical Analysis..............................................................................................26 Results........................................................................................................................ .26 Discussion...................................................................................................................27

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v 4 PULMONARY DISPOSITION OF TILMICOSIN IN FOALS AND IN VITRO ACTIVITY AGAINST RHODOCOCCUS EQUI AND OTHER COMMON EQUINE BACTERIAL PATHOGENS.....................................................................36 Abstract.......................................................................................................................36 Introduction.................................................................................................................37 Material and Methods.................................................................................................38 Horses and experimental design..........................................................................38 Experimental design and sample collection........................................................39 Bronchoalveolar lavage.......................................................................................39 Drug analysis.......................................................................................................40 Estimation of PELF and BAL Cell Volume s and Determination of Tilmicosin Concentrations in PELF and BAL Cells..........................................................40 Pharmacokinetic Analysis...................................................................................41 Statistical Analysis..............................................................................................42 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of tilmicosin against R. equi....................42 Checkerboard assay.............................................................................................43 Time kill curve assay...........................................................................................43 In vitro activity of tilmicosin ag ainst equine bact erial pathogens.......................44 Results........................................................................................................................ .45 Serum and pulmonary disposition of tilmicosin in foals.....................................45 In vitro susceptibility testing a nd antimicrobial drug combinations...................45 Discussion...................................................................................................................46 5 SUMMARY AND CONCLUSIONS.........................................................................52 LIST OF REFERENCES...................................................................................................55 BIOGRAPHICAL SKETCH.............................................................................................64

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vi LIST OF TABLES Table page 1.1 MIC90 ( g/mL) of azithromycin, clarithromycin, and erythromycin against common equine bacterial pathogens........................................................................16 3.1 Pharmacokinetic variables (mean SD unless otherwise specified) for clarithromycin after IV or intragastric administration to 6 foals at dose of 7.5 mg/kg of body weight..............................................................................................33 3.2 Mean SD clarithromycin activity in body fluids and BAL cells of six foals after 6 intragastric administrati ons (7.5 mg /kg every 12 hours).............................33 4.1 Serum and pulmonary pharmacokinetic variables (mean SD unless otherwise specified) for tilmicosin after IM admini stration to seven foals at a dose of 10 mg/kg of body weight..............................................................................................50 4.2 Tilmicosin in vitro susceptibility of 183 bacterial is olates obtained from horses....50

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vii LIST OF FIGURES Figure page 1.1 Classification of macrolides according to the number of atoms comprising the lactone ring...............................................................................................................16 3.1 Mean (+ SD) serum clarithromycin concentration as measured by HPLC method or microbiologic assay in 6 foals admini stered a single IV dose of 7.5 mg/kg.......34 3.2 Mean (+ SD) serum clarithromycin activity ( g/ml) in 6 foals following intragastric clarithromycin (7.5 mg/kg) administration at 0, 24, 36, 48, 60, and 72 hours. Results are based on measurements with the microbiologic assay.........35 4.1 Mean SD tilmicosin concentrations in serum, BAL cells, PELF ( g/mL), and lung tissue ( g/g) of 7 foals following a single IM dose of tilmicosin (10 mg/kg of body weight)........................................................................................................51 4.2 Effect of time and tilmicosin concentration on in vitro survival of a clinical isolate of R. equi Identical results were obtaine d with 2 additional isolates...........51

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viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHARMACOKINETICS AND PU LMONARY DISPOSITION OF CLARITHROMYCIN AND TILMICOSIN IN FOALS. By Ariel Y. Womble August 2006 Chair: Steeve Gigure Major Department: Veterinary Medicine Bronchopneumonia is the leadi ng cause of morbidity and mortality in foals aged between 1 and 6 months. Gram-positive bacteria such as Streptococcus equi subspecies zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in foals. Erythromycin, a macrolide antimicr obial agent, is commonly used in equine medicine for treatment of foal pneumonia, es pecially in foals infected with Rhodococcus equi. Two other macrolides, clarithromycin and tilmicosin, may be useful alternatives to currently used antimicrobial agents owing to their accumulation in lung tissue and phagocytic cells, as well as their broad spectru m in vitro activity. The objectives of this study were to determine the pharmacokine tics and pulmonary distribution of clarithromycin and tilmicosin in foals, and to investigate the in vitro activity of tilmicosin against common bacterial pathogens of hor ses. Clarithromycin (7.5 mg/kg) was administered to six foals via intravenous (IV) and intragastric (IG) routes, in a cross-over design. Concentrations of clarithromycin and its 14-hydroxy-metabolite in serum were

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ix measured by HPLC. A microbio logic assay was used to measure clarithromycin activity in serum, urine, peritoneal fluid, syn ovial fluid, cerebrospi nal (CSF), pulmonary epithelial lining fluid (PELF), and bronc hoalveolar (BAL) ce lls. Following IV administration, clarithromycin had a t of 5.4 hours, a body clearance of 1.27 L/h/kg, and an apparent volume of distribution at steady state of 10.4 2.1 L/kg. Oral bioavailability of clarithromycin was 57.3 12.0 %. In a separate study, a single dose of a fatty acid salt formulation of tilmicosin (10 mg/kg) was administered by the intramuscular route to 7 healthy 5to 8-week-o ld foals. Concentrations of tilmicosin in serum were measured by HPLC and concentrat ions in lung tissue, PELF, and BAL cells were measured by mass spectrometry. Mean peak tilmicosin concentrations were significantly higher in BAL cells (20.1 5.1 g/mL) than in lung tissue (1.90 0.65 g/mL), PELF (2.91 1.15 g/mL), and serum (0.19 0.09 g/mL). Harmonic mean elimination half life in lung tissue (193.3 h) was significantly longer than that of serum (18.4 h). Elimination half lives in BAL cells and PELF were 62.2 h and 73.3 h, respectively. Tilmicosin was active in vitr o against most strept ococci, Staphylococcus spp., Actinobacillus spp., and Pasteurell a spp. The drug was not active against Rhodococcus equi, Pseudomonas spp., and En terobacteraceae. In conclusion, oral administration of clarithromycin at a dosag e of 7.5 mg/kg every 12 hours would maintain serum, PELF, and BAL cell concentrations a bove the minimum inhibitory concentration for R. equi and S. zooepidemicus isolates fo r the entire dosing inte rval. The formulation of tilmicosin investigated in the pres ent study resulted in high and sustained concentrations in the lung, P ELF, and BAL cells of foals and may be appropriate for the treatment of susceptible bacterial infections.

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1 CHAPTER 1 INTRODUCTION Bacterial pneumonia is the leading cause of morbidity and mortality in foals aged between 1 and 6 months. Gram-positive bacteria such as Streptococcus equi subspecies zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in foals. Gram-negative bacteria such as Pasteurella spp., Actinobacillus spp., Escherichia coli and Klebsiella pneumoniae may also occasionally be cu ltured from tracheobronchial aspirates of affected foals. Administration of antimicrobial agents is the most important part of the therapeutic plan. When R. equi is suspected or confirmed, therapy has historically consisted of ad ministration of the macrolide erythromycin in combination with rifampin. This combination has drama tically reduced foal mortality since its introduction. However, this treatment regi men is not without problems. Erythromycin has poor and variable oral bioavailability in foals, r equi res multiple daily dosing, and most importantly, has a high incidence of poten tially fatal adverse effects. Therefore, there is a tremendous need for other effective and potentially safer antimicrobial agents to combat infection caused by this devast ating pathogen. Two other macrolides, clarithromycin and tilmicosin, may be useful al ternatives to currently used antimicrobial agents. The documented pharmacokinetic advantages of clarithromycin over erythromycin in humans include higher oral bioavailabilit y, longer elimination half -life, larger volume of distribution, and improved ti ssue and phagocytic ce ll uptake. Tilmicosin may also be a useful alternative to the current antimicr obial agents used in horses owing to its

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2 accumulation in lung tissue and phagocytic cells, as well as in vitro activity against many Gram-positive and Gram-negative bacterial spec ies. In addition, ava ilability of a long acting antimicrobial agent such as tilmicosin would result in less fr equent administration, which in turn may improve client compliance. The overall goal of the work presented in this thesis is to determine the pharm acokinetics and pulmonary distribution of clarithromycin and tilmicosin in foals. This thesis includes two studies. The objectives and hypotheses of th e first study (Chapter 3) are: 1 To determine the pharmacokinetics of clarithromycin and its metabolite in foals. Our hypothesis is that oral clarithromycin is well absorbed in foals and is metabolized to 14-hydr oxy clarithromycin. 2To determine concentrations of clarithromycin in body fluids and bronchoalveolar cells. Our hypothesis is that oral clarithromycin provides serum and pulmonary drug concentrations above the minimu m inhibitory concentration of R. equi The objectives and hypotheses of the second study (Chapter 4) are: 1To determine the pulmonary disposition of tilmicosin in foals. Our hypothesis is that a new fatty acid salt formulation of tilmicosin provides high and sustained concentrati ons in the lungs of foals. 2To investigate the in vitro activity of tilmicosin against R. equi and other common bacterial pathogens of horses. Our hypothesis is that tilmicosin is active in vitro against common equine bacterial pathogens of the respiratory tract.

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3 CHAPTER 2 LITERATURE REVIEW Foal Pneumonia Lower respiratory tract infection is th e leading cause of both morbidity and mortality in foals aged between 1 and 6 months (Cohen, 1994). The morbidity rate is approximately 6% across the United States. It is likely, however, that the true incidence of infection is much higher and that many cases of infec tion go unrecognized and resolve spontaneously. Indeed, careful weekly physic al examination and cytologic examination of the lower respiratory tract in more than 200 Thoroughbred foals on 10 farms in Ontario, Canada demonstrated an average morbid ity from bacterial inf ection of the distal respiratory tract of 82% (Hoffman et al. 1993a). Increased susceptibility to disease in this age group may result from delay in the establishment of a competent immune system and environmental factors such as overcr owding, shipping, and sales (Wilson, 1992). The disease may be subclinical initially; however, as the infection progresses, clinical signs may include depression, inappetence, coughing, na sal discharge, and tachypnea. Fever is a common finding as well. Severely affected foals may develop respiratory distress. Common laboratory abnormalities in foal s with bacterial pneumonia include leukocytosis, hyperfibrinogenemia, and hyperg lobulinemia (Barr, 2003). Mild anemia may develop in chronic cases. Radiogra phy and ultrasonographic examination of the thorax are useful diagnostic imaging tools to detect a nd assess the severity of lung lesions. Culture of a tracheobronchial aspira te is necessary to determine the causative microorganism. The vast majority of cases of foal pneumonia are bacterial in origin.

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4 Viral agents such as influenza, equi ne herpesvirus-1 (EHV-1), EHV-2, EHV-4, rhinovirus, and adenovirus may cause primar y lung disease or predispose to secondary bacterial pneumonia (Wilson, 1992). However, in most cases of fo al pneumonia, viral agents cannot be isolated at the time of presentation (Hoffman et al. 1993b). Most bacteria associated with pneumonia are ubiqu itous in the foal's environment. The pathophysiology of bacterial pneumonia begins with either inhalation of environmental microbes or aspiration of oropharygeal bacter ia. The bacteria become pathogenic only when the pulmonary defense mechanisms are compromised or are overwhelmed by a large number of bacteria (Wilson, 1992). The inflammatory response induced by bacterial invasion will result in infiltration wi th neutrophils and other inflammatory cells into the airways and pulmonary parenchyma. Inflammatory cells and their mediators cause damage to the airway epithelium and capillary endothelium, leading to flooding of the terminal airways with inflammatory cells serum cellular debris and fibrin. This process is generally more severe in the cran ioventral portions of th e lung. These lesions interfere with gas exchange and, if severe enough, the resulting ventilation-perfusion mismatch leads to hypoxemia and clini cal signs of respiratory disease. Gram-positive bacteria such as Strept ococcus equi subsp. zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in foals (Hoffman et al., 1993b; Gigure et al., 2002; Barr, 2003). Gram -negative bacteria such as Pasteurella spp., Actinobacillus spp., Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Bordetella bronchiseptica may also be cultured from tracheobronchial aspirates of affected foals. Mixed bacter ial infections are common as well (Wilson, 1992; Hoffman et al., 1993b). Administration of antimicrobial agen ts is the most important part of the

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5 therapeutic plan. The choice of the antimicrobi al agent depends on the results of culture and susceptibility testing of tracheobronchial aspi rates, severity of th e clinical signs, cost, ease of administration, and history of response to therapy within the herd. Since a high percentage of pneumonia in foals older than 1 month is due to penicillin-sensitive bacteria such as S. equi subsp. zooepidemicus, penicillin is often us ed for initial therapy, pending culture results. Ceftiofur, a third ge neration cephalosporin, has a broad spectrum of activity that includes most of the etiologic agents of foal pneumonia, except R. equi. If resistant Gram-negative organisms are pres ent, an aminoglycoside (gentamicin or amikacin) is often combined with penicillin (Wilson, 1992). When R. equi is suspected or confirmed, therapy consists of administra tion of a macrolide in combination with rifampin. It is common practice to use a comb ination of a macrolide and rifampin as the first line of therapy on farms where R. equi is endemic as this combination is also active against streptococci. Rhodococcus Equi R. equi is a facultative intracellu lar pathogen that has the ability to survive and even replicate within macrophages (Zink et al., 1987). R. equi is closely related to Mycobacterium tuberculosis. Both R. equ i and M. tuberculosis are members of a phyogenetically distinct group called Mycolata which are characterized by a unique cell envelope that consists of mycolic acids (S utcliffe, 1997). This unique envelope forms a permeability barrier to hydrophilic compounds a nd promotes granuloma formation so the organism is able to multiply in and destr oy macrophages (Sutcliffe, 1997). The similarity between R. equi and M. tuberculosis is further emphasized by th e degree of homology of their genome sequence (Rahman et al. 2003).

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6 The most common manifestation of R. equi infections in foals is a chronic suppurative bronchopneumonia with extensive abscessation and associated suppurative lymphadenitis. Other, less-comm on clinical manifestations of R. equi infections in foals include ulcerative entero colitis, colonic or mesenteric lymphadenopathy, immunemediated synovitis and uveitis, osteomyelitis, a nd septic arthritis (Gigure and Prescott, 1997). R. equi has also been increasingly recognized as an important cause of pneumonia in immunosuppressed people, especial ly those infected with HIV. R. equi may also cause disease in other animal species such as cattle, sheep, goats, dogs, and cats; however infection is rare and usually associated with immunosuppresion (Prescott, 1991). The reasons for the peculiar susceptibility of young foals are not entirely clear. R. equi is a saprophytic inhabitant of soil. Although all ho rse farms are infected to various degrees with R. equi the clinical disease is enz ootic and devastating on some farms, sporadic on others, and unrecognized on most farms. On farms where the disease is enzootic, costs associated with veterinary care, early di agnosis, long-term therapy, and mortality of foals may be very high. In addition to significant immediate costs, R. equi pneumonia has a long-term detrimental effect on the equine industry because foals that recover from the disease are less lik ely to race as adults (Ainsworth et al. 1998). The ability of R. equi to induce disease in foals likely depends on both host and microbial factors. The key to the pathogenesis of R. equi is its ability to replicate within pulmonary macrophages apparently by inhi biting phagosome-lysosome fusion (Zink et al. 1987). Unlike most environmental R. equi isolates from pneum onic foals typically contain 80-90 kb plasmids. The plasmid enc odes seven related virulence-associated proteins designated VapA a nd VapC through VapH (Takai et al. 2000). Plasmid-cured

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7 derivatives of virulent R. equi strains lose their ability to replicate and survive in macrophages (Gigure et al. 1999). Plasmid-cured deriva tives also fail to induce pneumonia and are completely cleared from the lungs of foals two weeks following heavy intrabronchial challenge, confirming the absolute necessity of the large plasmid for the virulence of R. equi (Gigure et al. 1999). Vap A is highl y immunogenic, lipidmodified protein expressed on the surface of R. equi (Tan et al. 1995). An R. equi mutant lacking a 7.9 kb DNA region spanning 5 vap genes ( vapA, -C, -D, -E, -F ) was attenuated for virulence in mice and failed to replicate in macrophages (Jain et al. 2003). Complementation with vapA alone could restore full virule nce, whereas complementation with vapC vapD or vapE could not (Jain et al. 2003). More recently, attenuation of 2 other plasmid-encoded genes was also found to decrease virulence in mice despite enhanced transcription of vapA (Ren and Prescott, 2004). Th ese findings suggest that other plasmid-encoded genes besides vapA contribute to the virulence of R. equi Inhalation of virulent R. equi is the major route of pne umonic infection. Ingestion of the organism is a significant route of e xposure, and likely also of immunization, but rarely leads to hematogenously acquired pneumonia unless the foal has multiple exposures to large numbers of bacteria (Johnson et al. 1983). A major ity of foals may be exposed by ingesting the ba cterium; however, they proba bly develop a strong immune response and are protected against subse quent intrabronchial challenge (HooperMcGrevy et al. 2005). Control of R. equi infections on farms where the dis ease is enzootic is difficult. Attempts at actively immunizing foals against R. equi infections have consistently failed. Intravenous administration of hyperimmune plasma obtained from horses vaccinated

PAGE 17

8 against R. equi has given contradictory results. Ther efore, screening strategies promoting early recognition of R. equi cases with treatment of infected foals will reduce losses, decrease the spread of virulent organisms and limit the cost of therapy on farms where the disease is endemic. A wide variety of antimicrobial agents are effective against R. equi in vitro (Jacks et al. 2003). However, many of these drugs are ineffective in vivo The discrepancy in results is likely due to the intracellular nature of this bacterium and the fact that it causes abscesses where diffusion and activity of many antimicrobial agents is not optimal. In one study, all 17 foals with R. equi pneumonia treated with the combination of penicillin and gentamicin died despite all isolat es being susceptible to gentamicin in vitro (Sweeney et al. 1987). In the mid 1980s, the combinati on of erythromycin and rifampin became the treatment of choice (Hillidge, 1987). The combination of erythromycin and rifampin has become the prevalent treatment for R. equi infections in foals and has dramatically reduced foal mortality since its introduction (Hilli dge, 1987; Sweeney et al. 1987). Although both erythromycin and rifamp in are bacteriostatic against R. equi (Nordmann and Ronco, 1992), they are highly effective in vitro The combination of these two antimicrobials is synergistic as well, both in vitro and in vivo and when used in combination reduces the likelihood of resistan ce to either drug (Prescott and Nicholson 1984; Nordmann et al. 1992; Nordmann and Ronco, 1992). Rifampin and, to a lesser extent, erythromycin are lipid soluble, a llowing them to penetrate caseous material. Although combined therapy with erythromycin and rifampin has dramatically improved the survival rate of foals infected with R. equi this treatment regimen is not without problems. Erythromycin has poor and variable oral bioavailability in foals, requires

PAGE 18

9 multiple daily dosing, and most importantly, has a high incidence of potentially fatal adverse effects (Lakritz et al. 2000a; Lakritz et al. 2000b; Stratton-Phelps et al. 2000). The use of erythromycin to treat foals with pneumonia results in an increased risk of diarrhea, hyperthermia, and respiratory dist ress compared with pneumonic foals treated with either penicillin or trim ethoprim sulfa (Stratton-Phelps et al. 2000). Clostridium difficile enterocolitis has also been observed oc casionally in the dams of nursing foals while the foals are being treated with oral erythromycin presumably because of sufficient active erythromycin to perturb the intestinal flora (Baverud et al. 1998). Macrolides Macrolide antimicrobial agents are chemica lly comprised of a lactone ring with 14 to 16 carbons attached to 2 sugar moieties. Macrolide antimicrobials are typically classified according to the size of their macroc yclic lactone ring (Figure 1.1). Macrolides inhibit protein synthesis by re versibly binding to 50S subunits of the ribosome. Their binding sites on the 23S rRNA of the 50S ri bosomal subunit overlap with that of clindamycin but are different from those of chloramphenicol (Prescott, 2000). Macrolides are generally bacteriostatic ag ents. They may be bactericidal at high concentrations and against a low inoculum of highly susceptible bacteria. Their spectrum of activity includes mostly Gram-positive microorganisms, most Mycoplasma spp., some Chalmydiae as well as some Gram-negative pathogens such as Haemophilus influenzae Campylobacter jejuni Bordetella spp., and Mannheimia haemolytica (Alvarez-Elcoro and Enzler, 1999). This class of antimicrobials has been important in the treatment of respiratory tract, skin, and soft tissue infecti ons as well as venereal disease in humans. Some macrolides (tilmicosin, tulathromycin) are also approved for the treatment of bronchopneumonia in cattle.

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10 Macrolide antimicrobial agents other th an erythromycin may provide a suitable alternative for the treatment of Rhodococcus equi infections in foals. Azithromycin and clarithromycin are used with increasing fre quency in human medicine. Compared with erythromycin, azithromycin and clarithromycin have a higher oral bi oavailability, longer elimination half lives, larger volumes of distribution, as well as improved tissue and phagocytic cell uptakes (Whitm an and Tunkel, 1992; Conte et al. 1995; Rodvold, 1999). In humans, the incidence and the severity of adverse reactions for azithromycin and clarithromycin are also considerably decrea sed compared with erythromycin (Whitman and Tunkel, 1992). Another macrolide, tilmic osin, is not currently approved for use in horses but may provide a suitable a lternative for the treatment of R. equi The pharmacokinetics and pulmonary distributi on of azithromycin have been studied extensively in foals (Jacks et al. 2001; Davis et al. 2002). However, there is no information on the pulmonary distribution of clarithromycin and tilmicosin in foals. Clarithromycin Clarithromycin is a semi-synthetic antim icrobial agent that is derived from erythromycin. Clarithromycin has an O-met hyl ether substitution instead of the C-6 hydroxyl group of erythromycin at position 6 of the macrolide ring (Rodvold, 1999). This modification provides greater stability th an erythromycin in gastric content, thus improving oral bioavailability. The doc umented pharmacokinetic advantages of clarithromycin over erythromycin in humans include higher oral bioavailability, longer elimination half-life, larger volume of di stribution, and improved tissue and phagocytic cell uptake (Conte et al. 1995; Rodvold, 1999). Clarithromycin undergoes hepatic metabolis m as well as elimination by secretion into the intestinal lumen. In humans, clarithromycin is metabolized in the liver by

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11 cytochrome P-450 enzymes to the active me tabolite 14-hydroxy-clar ithromycin (Ferrero et al. 1990). This active metabolite contributes approximately 50% of the biological activity of clarithromycin and has synergis tic effects with clarithromycin (Fernandes et al. 1988). Other species including rats, mice or desert tortoise do not produce the 14hydroxy metabolite (Ferrero et al. 1990; Bedos et al. 1992; Wimsatt et al. 1999). Microorganisms with MIC 2 g/ml are generally regarded as susceptible and 8 g/ml as resistant to clarithromycin and erythromycin. The efficacy of clarithromycin in vitro is greatest against the aer obic and facultative anaerob ic non-spore-forming, Grampositive bacteria. It also has in vitro activity against several microorganisms such as, Mycoplasma spp., Chlamydia spp., and some Mycobacteria. The lack of activity against most Gram-negative bacteria is likely due to its inability to penetrate the bacterial cell wall. Clarithromycin is active against many Gram-positive bacterial pathogens of horses (Jacks et al. 2003). Of the macrolides tested so far, clarithromycin has the greatest in vitro activity against R. equi isolates cultured from pneumonic foals (Table 1.1). Approved indications for the use of clarit hromycin in people include the treatment of: pharyngitis/tons illitis due to Streptococcus pyogenes ; acute maxillary sinusitis due to Haemophilus influenzae, Moraxella catarrhalis, or Streptococcus pneumoniae ; community acquired pneumonia or bronchitis due to Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcu s pneumoniae, Moraxella catarrhalis, or Chlamydia pneumoniae ; uncomplicated skin infections due to Staphylococcus aureus, or Streptococcus pyogenes ; and disseminated mycobacterial infections due to Mycobacterium avium, or Mycobacterium intracellulare. Finally, clarithromycin

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12 combined with amoxicillin and a proton pump i nhibitor is also appr oved for the treatment of gastric and duodenal ulceration caused by Helicobacter pylori Clarithromycin is a macrolide antimicrobial agent which achieves low plasma concentrations relative to the minimum inhib itory concentration (MIC) of the pathogens it is used to treat. The physiochemical pr operties of clarithrom ycin, including its lipophilicity indicate that the drug concentrations at a pe ripheral site would be greater than concurrent serum concentrations (Drusa no, 2005). The disposition of clarithromycin in pulmonary epithelial lining fluid (PELF) and alveolar macrophages (AM) has been investigated extensively in healthy human volunteers. In humans, clarithromycin achieves considerably greater concentrations in pulmonary epithelial lining fluid and alveolar macrophages than either er ythromycin or azithromycin (Conte et al. 1995;Conte et al. 1996; Patel et al. 1996; Rodvold et al. 1997). However, the half-life of clarithromycin at these sites is much shorter than that of azithromycin. A recent preliminary study confirmed that therapeutic concentrations are achieved in serum following oral administration of clarithromycin to foals (Jacks et al ., 2002). In a retrospective study of foals presented to a referral institution, the combination of clarithromycin-rifampin was found to be superior to azithromycin-rifampin or erythromycin-rifampin for the treatment of pneumonia caused by R. equi (Gigure et al. 2004). However, concentrations of the drug in body fluids pulmonary epithelial lining fluid (PELF) and bronchoalveolar (BAL) cells have not been measured. Recent studies demonstrate that the concentration of macrolides at the site of inf ection may be a better indicator of clinical efficacy than se rum concentrations alone (Drusano, 2005).

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13 Tilmicosin Tilmicosin is a semi-synthetic derivative of tylosin. Typical of macrolides, it inhibits Gram-positive bacteria including Clostridium spp., Staphylococcus spp., and Streptococcus spp., some Gram-negative bacteria including Actinobacillus spp., Campylobacter spp., Histophilus spp., and Mannheimia/ Pasteurella spp. (Prescott, 2000). All Enterobacteriaceae are re sistant to tilmicosin. Mycoplasma susceptibility can be quite variable because of resistance (Vicca et al. 2004). Mannheimia / Pasteurella spp. isolated from cattle are regarded as susceptible if their MIC is < 8 g/ml, intermediate if MIC is 16 g/ml, and resistant if their MIC is > 32 g/ml (Shryock et al. 1996). The pharmacokinetic properties of tilmicosin are similar to that of macrolides in general, and are characterized by low seru m concentrations but large volumes of distribution (> 2 L/kg), with accumulation a nd persistence in tissues including the lung, which may concentrate drug 20-60 fold compared to serum (Ziv et al. 1995; Clark et al. 2004). Intracellular con centrations have been shown to be 40 times greater than that of serum (Ziv et al. 1995; Scorneaux and Shryock, 1999). Tilmicosin has been developed as a longacting formulation for use in bovine and ovine respiratory disease. A single SC dose of 10 mg/kg results in lung concentrations exceeding the MIC of M. haemolytica for 72 hours (Ziv et al. 1995; Scorneaux and Shryock, 1999). Experimental and field data support the value of a single-dose SC prophylaxis on arrival of cattle in feedlots a nd in the treatment in pneumonia of cattle (Ose and Tonkinson, 1988; Musser et al. 1996; Morck et al. 1997; Hoar et al. 1998). Repeat injections after three days are necessary in some animals (Laven and Andrews, 1991; Scott, 1994). Tilmicosin is not approved fo r use in lactating ca ttle because of the prolonged period (two to three weeks) duri ng which milk residues can be detected.

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14 Intramammary tilmicosin at drying-off have been shown to be efficacious in curing some existing S. aureus infection (Dingwell et al. 2003). Tilmicosin is also approved as an oral medication for the control of Actinobacillus spp or P. multocida pneumonia in swine (Paradis et al. 2004). It may also be useful in the control of atrophic rhinit is. In-feed, treatment with 400 ppm of tilmicosin phosphate significantly reduced the presence of A. pleuropneumoniae on the surface of tonsils but was unable to completely eliminate the organi sm from deeper tonsillar tissues and to prevent bacterial shedding by carrier animals (Fittipaldi et al. 2005). Macrolides have immunomodulatory eff ects that are beneficial for humans suffering from many inflammatory pulmonary di seases. These effects are independent of the antibacterial activity of these drugs. Ne utrophils play an important role in the destruction and elimination of bacterial invaders. However, they also release lipid mediators such as leukotriene B4 (LTB4) which induce a local inflammatory response. Lesions of the lung contain viable as well as necrotic neutrophils which add to the tissue damage caused by invading microorganisms. Apoptotic cell death is less damaging because the cells maintain cellular membrane s, preventing further release of damaging LTB4 (Nerland et al. 2005). Tilmicosin has been shown to induce apoptosis and reduce LTB4 as well as prostaglandin E2 concentrations in pulmonary fluid of cattle and swine with pneumonia (Lakritz et al. 2002; Nerland et al. 2005). These anti-inflammatory effects may contribute to the therapeutic efficacy of tilmicosin. Tilmicosin is potentially toxic to the car diovascular system, which varies to some extent with species. According to the produc t insert, the drug is fatal to swine when administered by IM injection at doses ra nging between 10-20 mg/ kg. The toxic dose for

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15 goats is only about 30 mg/kg SC. The toxic e ffects of tilmicosin are mediated through its effects on the heart, possibly by causi ng rapid depletion of calcium (Main et al. 1996). There are no published reports on the safety of tilmicosin in horses. The product insert suggests that the currently available tilmicos in formulation may be fatal in the equine species. This formulation is also toxic when us ed in cats. A tilmicosin-fatty acid salt has been developed as a safe and conve nient formulation for cats (Kordick et al. 2003). Tilmicosin may be a useful alternative to th e current antimicrobial agents used in horses owing to its accumulation in lung tiss ue and phagocytic cells, as well as in vitro activity against many Gram-positive and Gram-negative bacterial species. In addition, availability of a long acting antimicrobi al agent providing sustained therapeutic concentrations at the site of infection would result in less frequent administration, which in turn may improve client compliance.

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16 Table 1.1 MIC90 ( g/mL) of azithromycin, clarithromycin, and erythromycin against common equine bacterial pathogens. Organism (n) AzithromycinClarithromycinErythromycin Rhodococcus equi (60) 1.0 0.12 0.25 Streptococci (45) <0.12 <0.06 <0.25 Staphylococcus spp. (18) 0.5 0.25 0.25 Pasteurella spp. (10) 0.25 1.0 1.0 Klebsiella spp. (9) >8.0 >4.0 >4.0 Escherichia coli (16) >8.0 >4.0 >4.0 Salmonella enterica (11) 4.0 >4.0 >4.0 Adapted from Jacks and Gigure 2003 Macrolideantibiotics13-membered ring 15-membered ring 14-membered ring16-membered ring SemisyntheticSemisynthetic Semisynthetic Natural Semisynthetic Natural Tulathromycin(10%)Erythromycin Oleandomycin Clarithromycin Roxithromycin Dirithromycin Fluorithromycin Azithromycin Tulathromycin(90%)Spiramycin Tylosin Josamycin Midecamycin Tilmicosin Miokamycin Rokitamycin Macrolideantibiotics13-membered ring 15-membered ring 14-membered ring16-membered ring SemisyntheticSemisynthetic Semisynthetic Natural Semisynthetic Natural Tulathromycin(10%)Erythromycin Oleandomycin Clarithromycin Roxithromycin Dirithromycin Fluorithromycin Azithromycin Tulathromycin(90%)Spiramycin Tylosin Josamycin Midecamycin Tilmicosin Miokamycin Rokitamycin Figure 1.1 Classification of macrolides accordin g to the number of atoms comprising the lactone ring.

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17 CHAPTER 3 PHARMACOKINETICS OF CLARITHROMYCIN AND CONCENTRATION IN BODY FLUIDS AND BRONCHOAL VEOLAR CELLS IN FOALS Abstract The objective of this research was to de termine pharmacokinetics of clarithromycin and the concentrations achiev ed in body fluids and bronchoalveolar cells in foals. Six healthy 2to 3-week-old foals were used in this project. Clarit hromycin (7.5 mg/kg of body weight) was administered to each foal via intravenous (IV) and intragastric (IG) routes, in a cross-over design. After the first IG dose, 5 additional doses were administered at 12-hour intervals. Concentr ations of clarithromycin and its 14-hydroxy metabolite in serum were measured by HP LC. A microbiologic assay was used to measure clarithromycin activity in serum, urine, peritoneal fluid, synovial fluid, cerebrospinal (CSF), pulmonary epithelial lining fluid (PELF), and bronchoalveolar (BAL) cells. Following IV administration, clarithromycin had a t of 5.4 hours, a body clearance of 1.27 L/h/kg, and an apparent volume of distri bution at steady state of 10.4 2.1 L/kg. Detection of 14-hydroxy-clarithromycin was achieved in all 6 foals by 1 h post-administration. Oral bioavailability of clarithromycin was 57.3 12.0 %. Peak serum clarithromycin concentration following multiple IG administration was 0.88 0.19 g/mL. After multiple IG doses, peritoneal fl uid, CSF, and synovial fluid clarithromycin concentrations were similar to or lower than serum concentrations whereas urine, PELF, and BAL cell concentrations were signif icantly higher than concurrent serum concentrations. Oral administration at a dosage of 7.5 mg/kg every 12 hours would

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18 maintain serum, PELF, and BAL cell concentrations above the minimum inhibitory concentrations of Rhodococcus equi isolates for the entire dosing interval. Introduction Clarithromycin is a semi-synthetic macr olide antimicrobial agent chemically derived from erythromycin A. It differs from erythromycin A by having an O-methyl ether substitution at position 6 of the macr olide ring. This change provides greater stability in gastric acid resulting in enhanced absorption by the oral rout e. This structural difference also results in a longer elimination half life, a larger volume of distribution, as well as improved tissue and phagocytic cell up take compared to erythromycin (Conte et al ., 1995; Rodvold, 1999). Clarithromycin under goes extensive hepatic metabolism in people and is primarily metabolized to 14-hydroxy-clarithromycin (Ferrero et al ., 1990). This metabolite is responsible for approximate ly 50% of the total bi ological activity of clarithromycin and has an additive or s ynergistic effect with the parent compound (Fernandes et al ., 1988; Martin et al ., 2001). Macrolide antimicrobial agents in combin ation with rifampin are commonly used in equine medicine for treatment of Rhodococcus equi infections in foals. R. equi, a Gram-positive facultative intr acellular pathogen surviving in macrophages, is a common cause of pneumonia in foals be tween 3 weeks and 5 months of age. Combined therapy with erythromycin and rifampin has dramatical ly improved the historic al survival rate of affected foals (Hillidge, 1987). However, recen t evidences indicate that clarithromycin may be superior to erythromycin for the treatment of R. equi pneumonia in foals. Clarithromycin is more active against R. equi in vitro than either erythromycin or azithromycin (Jacks et al ., 2003). In addition, in a retros pective study of foals presented to a referral institution, the combination of clarithromycin-rifampin was found to be

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19 superior to azithromycin-rifampin or er ythromycin-rifampin for the treatment of pneumonia caused by R. equi (Gigure et al ., 2004). A recent preliminary study confirmed that therapeutic concentrations are achieved in serum following oral administration of clarithromycin to foals (Jacks et al ., 2002). However, a single oral dose was given precl uding accurate determination of steady state drug concentrations and calcula tion of important pharmacokinetic parameters such as oral bioavailability, clearance, and apparent volume of dist ribution. In addition, concentrations of the drug in body flui ds, pulmonary epithelial lining fluid and bronchoalveolar (BAL) cells were not measured. Recent studies demonstrate that the concentration of macrolides at the site of in fection may be a better indicator of clinical efficacy than serum concentrations al one (Drusano, 2005). Finally, the methodology used to measure drug concentration in the preliminary study did not allow detection of metabolites such as 14-hydroxy-clarithromycin. The objectives of the present study were to determine the pharmacokinetics and oral bioavailability of clarithromycin in foals as well as to measure drug concentrations in body fluids and BAL cells after a multi-dose in tragastric (IG) regimen. An additional objective was to determine if clarithromycin is converted to the 14-hydroxy metabolite in foals. Materials and Methods Horses and Experimental Design Four male and two female foals (5 Thoroughbred and 1 Quarter Horse) between 2 and 3 weeks of age and weighing between 71 and 100 kg were selected for use in the study. The foals were considered healthy on the basis of history, physical examination, complete blood count and plasma biochemical profile. The foals were kept with their

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20 dams in individual stalls during the experiment with ad libitum access to grass hay and water. Clarithromycin was administ ered at a dose of 7.5 mg/kg of body weight via the IV and the IG routes, using a cross-over design. For the IV study, pur ified clarithromycin powder (Courtesy of Franks Pharmacy, Ocala, FL, USA) was dissolved in sterile water (100mg/ml) and administered as a single bolus through a catheter placed into the left jugular vein. Blood samples were obtained from a catheter placed in the right jugular vein at 0 (prior to administration), 3, 6, 10, 20, 30, 60, 90 minutes and at 2, 3, 4, 6, 8, 12, and 24 hours after the drug was administered. For the IG route, clarithromycin ta blets (250 mg tablets; Biaxin, Abbott Laboratories, Chicago, IL, USA) were dissolved in 50 ml of water and administered by nasogastric tube. For the first 24 hours, bl ood samples were collected as described for the IV study. Afterwards, 5 additional doses were administered at 12 hour intervals (24, 36, 48, 60, 72 hours after the initial dose). Blood samples were collected immediately before each additional dose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after dose 2, 4, and 6. Bronchoalveolar lavage was performed and samples of synovial fluid, peritoneal fluid, cerebrospinal fluid (CSF), and urine we re collected aseptically 2 and 12 hours after administration of the last IG dose. Foal s were sedated by administration of xylazine hydrochloride (1.0 mg/kg, IV), and butorphano l tartrate (0.07 mg/kg, IV). Immediately after collection of BAL fluid (see below) general anesthesia was induced by IV administration of diazepam (0.1 mg/kg, IV) and ketamine (2.5 mg/kg). Samples of synovial fluid were obtained from the inter carpal or radiocarpal joint by use of a 20-

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21 gauge needle. Samples of CSF were collected from the atlantooccipital space by use of a 3.5-inch, 20-gauge spinal needle. Abdominal fluid was collected by use of an 18-gauge needle. A flexible 8-F Foley catheter was used to collect urine directly from the bladder. Samples were centrifuged and the supernatants were stored at C until analysis. Bronchoalveolar Lavage A 10 mm diameter, 1.8 m bronchoscope (P entax, Welch Allen, Orangeburg, NY, USA) was passed via nasal appro ach into either the left or right lung until wedged in a fourth to sixth generation bronchus. The lavage solution consisted of 4 aliquots of 50 ml physiologic saline (0.9% NaCl) solution in fused and aspirated immediately. The bronchoscope was passed alternating into either the left or right lung to prevent the effect of repeated bronchoalveolar lavages on differe ntial cell counts. Tota l nucleated cell count in BAL fluid was determined by use of a hemacytometer. Bronchoalveolar fluid was centrifuged at 200 X g for 10 minutes. Bronchoalv eolar cells were washed, re-suspended in 1 mL of phosphate-buffered solution, vor texed and frozen at -80C until assayed. Supernatant BAL fluid was also frozen at -8 0C until assayed. Before assaying, the cell pellet samples were thawed, vortexed vigorousl y, and sonicated for 2 minutes to ensure complete cell lysis. The resulting suspension was centrifuged at 500 g for 10 minutes and the supernatant fluid was used for determination of intracellular clarithromycin concentrations. Drug Analysis by High Performanc e Liquid Chromatography (HPLC) Serum samples underwent a two-step extr action procedure prior to analysis by HPLC. Samples (500 l) of serum were th awed and mixed with an equal volume of internal standard roxithromycin (Sigma, St-Louis, MO, USA) (4 g/ml in 10 mM NaH2PO4 buffer, pH 3) and acidified with 2 N HC l to a final pH of 3. Each acidified

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22 sample was mixed briefly and transferred quantitatively onto a so lid phase extraction column (Varian Bond Elut C18, 500 mg. Varian, Inc. Palo Alto, CA, USA) that had been pre-conditioned with 5 ml of methanol and 10 mM phosph ate buffer (pH 3). Following sample loading, each column was rinsed w ith 5 ml of 10 mM phosphate buffer (pH 3) prior to elution of drugs with 5 ml of alka linized methanol (99:1 mix of methanol: 1 N NaOH). Methanolic eluates were collected and evaporated to dryness in a vacuum concentrator at ambient temperature. Dr ied samples were reconstituted in 4 N NaOH (500 l) by incubation at room temperature for 30 min with intermittent vortex mixing. Thereafter, 3 ml of hexane:ethyl aceta te (50:50) was added and samples mixed vigorously. Aqueous and organi c layers were separated by centrifugation (4 min at 4000 g) and a portion of the organic layer was re moved and evaporated to dryness. Dried samples were reconstituted in mobile phase and analyzed immediately by HPLC (Beckman System Gold; Beckman Coulte r, Inc. Fullerton, CA, USA) with electrochemical detection (HPLC-EC). Sa mples were injected and separated on a reversed phase column (Supelco Discovery C18, 150 x 5.6 mm, 5 m particle size) using a filtered (0.2 m) degassed mobile phase containing a 55:45 mixture (v/v) of 1 mM sodium phosphate (pH 7.0) and acetonitrile (final adjusted pH 7.5) at a flow rate of 1ml/min. Concentrations of the three macrolide antibiotics were measured by amperometric detection using an LC-4C elect rochemical detector (BAS, Lafayette, IN, USA) with a platinum electrode set at +1100m V potential (1nA full s cale). Peak areas for all three compounds exhibited a linear relationship versus drug concentration over the ranges of 0.25 5.00 g/ml for 14-hydroxy-clarithromycin (Courtesy of Abbott Laboratories, Abbott Park, IL, USA) and 0.50 -5.00 g/ml for clarithromycin (US

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23 Pharmacopeia, Rockville, MD, USA) and roxi thromycin with a correlation coefficient ( r ) value 0.99. Each sample was run in duplicate and drug concentrations were estimated by comparison of peak areas against linear sta ndard curves for each analyte. Therefore, 0.5 g/ml was used as the lowest limit of qua ntification for clarithromycin. Average retention times were 3.8 (14hydroxy-clarithromycin), 9.0 (c larithromycin) and 11.0 min (roxithromycin). In spiked serum samples, drug extraction yields of clarithromycin (76 3.2 %) and the internal standard roxithromycin (77 3.9 %) were highly correlated (r = 0.98). In contrast, extraction yield for 14-hydr oxy-clarithromycin was greater but more variable 92 11.4 % and was poorly correlated (r = 0.78) with internal standard and, for that reason, exact concentrations of the metabolite are not reported here. Measurement of Clarithromycin Acti vity Using a Microbiologic Assay Concentrations of clarithromycin were determined in serum, synovial fluid, peritoneal fluid, CSF, BAL fluid, and BAL cells, using an agar well diffusion microbiologic assay with Micrococcus luteus (ATCC 9341, American Type Culture Collection, Rockville, MA, USA) as the assa y organism. One millili ter of a bacterial suspension was grown overnight in trypticase so y broth and adjusted to an optical density of 0.5 at 550 nm. This suspension was added to tempered neomycin assay agar (Neomycin assay agar, Fische r Scientific Inc, Pittsburgh, PA, USA) and distributed evenly over the assay plates. The plates were allowed to solidify for 45 minutes, and 0.5 mm wells were punched and filled with 50 l of samples or clarithromycin standards (US Pharmacopeia, Rockville, MD, USA) ra nging in concentrations from 0.02 to 5.0 g/ml. Known amount of purified clar ithromycin were added to eq uine serum, synovial fluid, and urine to produce standard curves for each type of substrate. Bronchoalveolar cells,

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24 BAL fluid, CSF, and peritoneal fluid were assayed with standard s diluted in phosphatebuffered saline. The agar plates were incuba ted for 36 hours at 30 C. Zones of bacterial inhibition were measured to the nearest 0.1 cm Each sample or standard was assayed in triplicate and mean values for 3 measurements of the zone diameters were determined. The lower limit of quantification of the assay was 0.02 g/ml for serum, BAL cells, and body fluid samples. Negative control samples did not cause bacterial inhibition, which indicated no antibacterial activity of e quine serum, or body fluids, or BAL cell supernatants. Plots of zone diameters versus standard clarithromycin concentrations were linear between 0.02 and 5 g/ml with r values ranging between 0.993 and 0.998. The coefficients of variation for repeatedly assayed samples at concentrations > 0.1 g/ml and < 0.1 g/ml were < 5% and < 10%, respectively. Estimation of PELF and BAL Cell Volume s and Determination of Clarithromycin Concentrations in PELF and BAL Cells Pulmonary distribution of clarithromycin was determined as reported (Baldwin et al., 1992). Estimation of the volume of PELF was done by urea dilution method (Conte et al ., 1996; Jacks et al ., 2001). Serum urea nitroge n concentrations (UreaSERUM) were determined by use of enzymatic methodology (Labsco Laboratory Supply Company; Louisville, KY, USA) on a chemistry an alyzer (Hitachi 911 analyzer, Boehringer Mannheim Inc, Indianapolis, IN, USA). For measurement of urea concentration in BAL fluid (UreaBAL), the proportion of reagents to specimen was changed from 300 l/3 l in serum to 225 l/50 l. The volume of PELF (VPELF) in BAL fluid was derived from the following equation: VPELF = VBAL X (UreaBAL/UreaSERUM), where VBAL is the volume of recovered BAL fluid. The concentration of clarit hromycin in PELF (CLRPELF) was derived form the following

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25 relationship: CLRPELF =CLRBAL X (VBAL/ VPELF), where CLRBAL is the measured concentration of clarithromycin in BAL fluid. The concentration of clarithromycin in BAL cells (CLRBAL) was calculated using the following relationship: CLRBAL = (CLRPELLET/VBALC) where CLRPELLET is the concentration of antimicrobial in the BAL cell pellet supernatant and VBALC is the mean volume of foal BAL cells. A VBALC of 1.20 l/106 cells was used for calculations based on a previous study in foals (Jacks et al. 2001). Pharmacokinetic Analysis For each foal, the plasma concentration versus time data were analyzed based on noncompartmental pharmacokinetics using computer software. (PK Solutions 2.0, Summit Research Services, Montrose, CO, USA). The elimination rate constant (Kel) was determined by linear regression of the term inal phase of the logarithmic plasma concentration versus time curve using a minimu m of 3 data points. Elimination half-life (t) was calculated as the natura l logarithm of 2 divided by Kel. Pharmacokinetic values were calculated as reported by Gibaldi a nd Perrier (1982). Th e area under the concentration-time curve (AUC) and the area under the first moment of the concentration-time curve (AUMC) were calc ulated using the trapezoidal rule, with extrapolation to infinity using Cmin/ Kel, where Cmin was the final measurable plasma concentration. Mean residence time (MRT ) was calculated as: AUMC/AUC. Apparent volume of distribution based on the AUC (Vdarea) was calculated as: dose /AUC Kel, apparent volume of distribution at steady state (VDss) was calculated as (dose/AUC)/(AUMC/AUC), and systemic clearance (CL) was calculated from: dose/AUC. Bioavailability was calculated as (AUCIG/AUCIV) x (doseIV/doseIG).

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26 Statistical Analysis Pharmacokinetic-derived data are pres ented as mean SD unless otherwise specified. The paired t -test was used to compare differences in Kel between IV and IG administration as well as peak serum concentration after the first dose (Cmax 0-24h) and peak serum concentrations after the last dose (Cmax 72-84h). The Friedman repeated measures ANOVA on ranks was used to compar e clarithromycin concentrations between sampling sites (serum, synovial fluid, periton eal fluid, CSF, urine, PELF, BAL cells). When indicated, multiple pairwise compar isons were done using the Student-NewmanKeuls test. Differences were considered significant at P < 0.05. Results Following IV administration of clar ithromycin (7.5 mg/kg), serum drug concentrations were similar when measured by HPLC or the microbiologic assay; although, HPLC-based measurements tended to be higher immediately following drug administration (Figure 2.1). Pharmacokinetic parameters were calculated based on data obtained with the microbiologic assay because the high limit of qua ntification of the HPLC method did not allow accura te evaluation of the terminal elimination phase of the drug. Clarithromycin had a t of 5.4 hours (harmonic mean), a body clearance of 1.27 L/h/kg, and a Vdss of 10.4 2.1 L/kg (Table 1). De tection of 14-hydroxy-clarithromycin was first achieved 0.5 h after IV administrati on in 3 foals and by 1 h post-administration in all 6 foals. Time to maximum concentration (Tmax) of 14-hydroxy-clarithromycin following IV administration was 1.7 1.2 h. After IG administration, quantifiable cl arithromycin activity was found in 4 of 6 foals at 10 minutes and in all 6 foals at 15 minutes. The time to peak serum clarithromycin concentration (Tmax) was 1.6 0.4 h and F was 57.3 12.0 % (Table 1).

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27 Peak serum clarithromycin concentrati on following multiple IG administration (Cmax 7284h: 0.88 0.19 g/mL) was significantly higher ( P = 0.011) that that achieved after the first IG dose (Cmax 0-24h: 0.52 0.17 g/mL) (Figure 2). Differences between Kel after IV and IG administration were not significant ( P = 0.617). After multiple IG doses, peritoneal fluid, CSF, and synovi al fluid clarithromycin concen trations were similar to or lower than serum concentrations whereas urin e, PELF, and BAL cell concentrations were significantly higher than concur rent serum concentrations (T able 2). Detection of 14hydroxy-clarithromycin was first achieved 0.5 h af ter IG administration in 2 foals and by 2 h post-administration in all 6 foals with a Tmax of 1.7 1.2 h. One foal developed transient tachyp nea and profuse sweating 5 min after administration of the IV bolus. One foal de veloped diarrhea after the third IG dose and another foal developed diarrhea af ter the last intragastric dose. In both foals, the diarrhea resolved without therapy within 36 h. Discussion Clarithromycin undergoes extensive metabo lism in people. Of the 8 metabolites that have been identified, 14-hydroxy-clarithromycin is th e most abundant and the only one with substantial antimi crobial activity (Fernandes et al ., 1988; Ferrero et al ., 1990). The metabolism of clarithromycin is unique in people since it is the only 14-membered macrolide to demonstrate 14-hydroxyl ation. The 14-hydroxy metabolite of clarithromycin is also produced in monkeys but not in rats, mice, or desert tortoises (Ferrero et al ., 1990; Bedos et al ., 1992; Wimsatt et al ., 1999). The present study confirms the production of 14-hydr oxy-clarithromycin in foals with peak concentrations detected approximately 1.7 h following IV or IG administration. In people, peak 14-

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28 hydroxy-clarithromycin concentrat ions at approximately 1.3 g/ml were detected 3 h following oral administration of a do se of 7.5 mg/kg of body weight (Gan et al ., 1992). Unfortunately, exact concentrations of 14-hydroxy-clarithromyc in could not be determined in the present study due to the inab ility to find an internal standard exhibiting parallel recovery efficiency. The microbiological assay used to calcul ate pharmacokinetic parameters in the present study only allows an approximation of the drug disposition because it does not differentiate between clarithromycin and its 14-hydroxy metabolite. However, in a clinical situation, the total antimicrobial activity meas ured by the microbiological assay is adequate to determine a dosage regimen. The or al bioavailability of clarithromycin in the present study (57%) is similar to that reported in people (55%) but lower than the 70-75% reported in dogs (Chu et al ., 1992; Vilmanyi et al ., 1996). The oral bioavailability of clarithromycin in the present study is similar to that of azithromycin (38-56%) and much higher than that of erythromyc in (14%) in foals (Lakritz et al ., 2000:1011-1015; Jacks et al ., 2001; Davis et al ., 2002). Clarithromycin eliminati on half-life in the present study (5.4 h) was slightly longer than that reported after oral ad ministration to dogs (3.9 h) (Vilmanyi et al ., 1996). Elimination halflives reported in people range between 3 to 5 h for clarithromycin and 4 to 9 h for 14-hydroxy-clarithromycin (Rodvold, 1999). The elimination half-life of clarith romycin in the present study is longer than that reported for erythromycin (1 h) but considerably shorter than that of azithromycin (16-20 h) in foals (Prescott et al., 1983; Lakr itz et al., 1999; Lakritz et al ., 2000:914-919; Jacks et al ., 2001; Davis et al ., 2002).

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29 The optimal dosing of antimicrobial ag ents is dependent not only on the pharmacokinetics, but also on the ph armacodynamics of the drug. The pharmacodynamic properties of a drug a ddress the relationship between drug concentration and antimicrobial activit y. Much confusion exists over the pharmacodynamics of macrolides and azalides b ecause their concentrat ion-time profile is low relative to the minimum inhibitory concen trations of the pathogens for which they are used typically. An importa nt factor in determining the efficacy of many macrolides in animal models of infection with extracellu lar bacteria is the length of time that serum concentrations exceed the MIC of the pa thogen (T > MIC) (Rodvold, 1999). In a mouse thigh model of Streptococcus pneumoniae infection, T > MIC for at least 60% of the dosage interval with clarithromycin was the be st predictor of efficacy (Craig, 1997). In a murine model of pneumococcal pneumonia, T > MIC of 50-70%, Cmax/MIC of 3-7, and AUC0-24/MIC 40-100 were all comparable in predicting efficacy (Tessier et al ., 2002). Recent data suggest that traditional pharmacodynamic parameters based on plasma concentrations of macrolides may not best ap ply to the treatment of pulmonary infections and infections caused by facultative intracellular pathogens such as R. equi (Drusano, 2005). While drug concentration in plasma is clear ly a driving force for penetration to the site of infection, the actual drug-concentration time profile in a peripheral site may be quite different from that of plasma (Dru sano, 2005). Macrolides cross the cellular membranes primarily by passive diffusion (Fietta et al ., 1997). They are potent weak bases that become ion-trapped within acidic intracellular compartments such as lysosomes and phagosomes. A number of in vitro and in vivo studies support the notion

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30 that white blood cells act as carriers for the deliv ery of macrolides to the site of infection (Retsema et al ., 1993; Mandell & Coleman, 2001). However, the white blood cell delivery theory does not explain the very high concentrations of these drugs in PELF, as this was demonstrated in hea lthy subjects where trafficking of white blood cells to the PELF should have been minimal (Conte et al ., 1995; Rodvold et al ., 1997). A high concentration of macrolides in PELF has l ong been proposed as a key factor in their efficacy against respiratory pathogens in people. The pref erential activity of clarithromycin in the lung was recently demonstrated in mice infected with S. pneumoniae isolates with efflux-mediated macrolide resist ance. Consistent bacterial kill was observed in the lung model whereas no drug effect was seen in the thigh model (Maglio et al ., 2004). These differences in bact erial activity betw een sites were explained by the higher concentrations in PELF than in serum (Maglio et al ., 2004). In the present study, administration of cl arithromycin at 7.5 mg/kg every 12 h resulted in serum concentrations above the MIC inhibiting 90% of R. equi isolates (MIC90 =0.12 g/ml) throughout the entire dosing interval, a mean Cmax/MIC90 ratio of 7, and mean AUC0-24/MIC90 ratio of 57. Because serum concentrations alone should not be used to determine the likelihood of clin ical efficacy in the treatment of R. equi pneumonia of foals, clarithromy cin concentrations were also measured in PELF and BAL cells. Estimation of PELF volume by use of th e urea dilution method may result in falsely increased BAL fluid urea concentration by di ffusion of urea from the interstitium and blood if BAL fluid dwell-time is prolonged (Baldwin et al ., 1992). Prolonged BAL fluid dwell-time was minimized in our study by us e of rapid infusion of 100 ml of saline solution followed by immediate aspiration. Ov erestimation of urea concentrations in

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31 BAL fluid would falsely increase the volume of PELF, which would in turn result in an underestimation of clarithromycin c oncentrations in PELF (Baldwin et al ., 1992). Concentrations of clarithromycin in P ELF and BAL cells in the present study considerably exceeded the MIC90 of R. equi isolates obtained from foals with pneumonia. Clarithromycin concentrations in PELF and BAL cells in the present study were also considerably higher than concentrations reported following multiple daily administration of azithromycin to foals. In the present study, clarithromycin concentrations in BAL cells and PELF had decreased considerably 12 h following administration. This is in contrast to azithromycin concentrations in PELF and BAL cells which do not decrease for at least 48 h following administration to foals (Jacks et al ., 2001). Collectively, these findings in foals are consistent with st udies in people showing much higher peak clarithromycin concentrations in PELF a nd BAL cells compared to azithromycin, but much longer persistence of azithromycin th an clarithromycin at these sites (Conte et al ., 1996; Patel et al ., 1996; Rodvold et al ., 1997). Following administ ration of a single oral dose to people, clarithromycin is no longer detectable in PELF after 24 h and in BAL cells after 48 h (Conte et al ., 1996). The release of azithromycin from cells is much slower than that of erythromycin a nd clarithromycin, resu lting in sustained concentrations of azithromyci n in tissues for days followi ng discontinuation of therapy (Fietta et al ., 1997). Clarithromycin concentrations in peritoneal fluid, synovial fluid, and CSF were significantly lower than P ELF concentrations in the present study indicating preferential diffusion of cl arithromycin into pulmonary fluid. Adverse effects in humans r eceiving clarithromycin are ra re and usually related to the gastrointestinal tract w ith diarrhea, nausea, and abdominal pain being the most

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32 frequently reported (Alvarez-Elcoro & Enzler, 1999). Two of 6 foals in the present study developed mild self-limiting diarrhea. The incidence of diarrhea in the present study was similar to that of a retrospective study in which 5 of 18 foals (28%) with R. equi pneumonia treated with clarithromycin and rifa mpin also developed diarrhea (Gigure et al ., 2004). This is similar to the incidence of diarrhea reported in foals being treated with erythromycin-rifampin (17 to 36%) (Stratton-Phelps et al ., 2000; Gigure et al ., 2004). In contrast, the incidence of gastrointestinal adverse effects in pe ople is significantly lower during clarithromycin (4 %) than during erythromycin (19%) therapy (Anderson et al ., 1991).

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33 Table 3.1 Pharmacokinetic variables (mean SD unless otherwise specified) for clarithromycin after IV or intragastric administration to 6 foals at dose of 7.5 mg/kg of body weight. Variable IV Intragastric Kel (h-1) 0.129 0.022 0.141 0.05 AUC0 ( gh/mL) 6.2 1.5 3.4 1.1 AUMC0 ( gh2/mL) 51.1 16.2 24.4 9.7 MRT (h) 8.25.989 7.1.70 t (h) 5.4* NA Vdarea (L/kg) 9.9 1.8 NA Vdss (L/kg) 10.4 2.1 NA Clearance (L/h/kg) 1.27 0.25 NA Tmax (h) NA 1.6 0.4 Cmax 0-24h ( g/mL) NA 0.52 0.17 Cmax 72-84h ( g/mL) NA 0.88 0.19 C84h NA 0.20 0.06 F (%) NA 57.3 12.0 NA = Not applicable *Harmonic mean. Kel = Elimination rate constant. t = Elimination half-life. AUC = Area under the serum concentration versus time curve. AUMC = Area under the first moment of the concentration versus time curve. MRT = Mean residence time. Vdarea = Apparent volume of distribution (area) Vdss = Apparent volume of dist ribution (steady-state) tabs = Absorption half-life. Tmax = Time to peak serum concentration. Cmax 0-24h = Peak serum concentration after the first dose. Cmax 72-84h = Peak serum concentration after repeated doses. C84h = Minimum serum concentration 12 h after the last dose. F = Oral bioavailability. Table 3.2 Mean SD clarithromycin activity in body fluids and BAL cells of six foals after 6 intragastric administra tions (7.5 mg /kg every 12 hours). Time after administration (h) Sample 2 12 Serum ( g/mL) 0.83 0.18a 0.20 0.06a Synovial fluid ( g/mL) 0.27 0.06b 0.08 0.02a Peritoneal fluid ( g/mL)* 0.43 0.32b 0.11 0.06a Urine ( g/mL) 36.8 46.4c 2.53 0.82b CSF ( g/mL) 0.22 0.09b 0.13 0.09 a Pulmonary epithelial lining fluid ( g/mL) 76.2 59.4 c 21.4 20.5c Bronchoalveolar cells ( g/mL) 269 232d 117 107d *n=3 at 2 h and n=5 at 12 h Drug concentrations are in g/m l of bronchoalveolar cell volume a,b,c,dDifferent letters within a column indicate statistically significant difference in clarithromycin concentrations ( P < 0.05)

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34 0.01 0.1 1 10 04812162024 Time (h)Clarithromycin ( g/mL) HPLC Microbiologic Figure 3.1 Mean (+ SD) serum clarithromycin concentration as measured by HPLC method or microbiologic assay in 6 foal s administered a single IV dose of 7.5 mg/kg.

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35 Figure 3.2 Mean (+ SD) serum clarithromycin activity ( g/ml) in 6 foals following intragastric clarithromycin (7.5 mg/kg) administration at 0, 24, 36, 48, 60, and 72 hours. Results are based on measurements with the microbiologic assay. 0.001 0.01 0.1 1 10 012243648607284 Time (h)Clarithromycin activity ( g/mL)

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36 CHAPTER 4 PULMONARY DISPOSITION OF TILMICOSIN IN FOALS AND IN VITRO ACTIVITY AGAINST RHODOCOCCUS EQUI AND OTHER COMMON EQUINE BACTERIAL PATHOGENS Abstract Tilmicosin is a long-acting macrolide currently approved for treatment of respiratory disease in cattle, sheep and swin e. The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against R. equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to 7 healthy 5to 8-week-old foal s by the intramuscular route. Concentrations of tilmicosin in serum were measured by HPLC and concentrations in lung tissue, pulmona ry epithelial lining fluid (PELF), and bronchoalveolar (BAL) cells were measur ed by mass spectrometry. Mean peak tilmicosin concentrations were signif icantly higher in BAL cells (20.1 5.1 g/mL) than in lung tissue (1.90 0.65 g/mL), PELF (2.91 1.15 g/mL), and serum (0.19 0.09 g/mL). Harmonic mean elimination half lif e in lung tissue (193.3 h) was significantly longer than that of serum (18.4 h). Elimina tion half lives in BAL cells and PELF were 62.2 h and 73.3 h, respectively. The MIC90 of 56 R. equi isolates was 32 g/mL. Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus spp., Pseudomonas spp., and Enterobacteraceae In conclusion, the formulation of

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37 tilmicosin investigated in the present study re sulted in high and sustained concentrations of tilmicosin in the lung, PELF and BAL cells of foals. Introduction Tilmicosin is a semi-synthetic 16-membered lactone ring macrolide chemically derived from tylosin (Prescott, 2000). Tilm icosin is approved as a suspension for subcutaneous administration in the th erapy or control of pneumonia caused by Mannheimia haemolytica in cattle and sheep. It is also approved for use in feed for the control of swine respirator y disease associated with Actinobacillus pleuropneumoniae and Pasteurella multocida In addition, the drug is active in vitro against a variety of pathogens of cattle and swine including Histophilus somni Haemophilus parasuis Actinobacillus suis Arcanobacterium pyogenes Erysipelothrix rhusiopathiae Staphylococcus spp., some Streptococcus spp., and many Mycoplasma spp. (Watts et al. 1994; DeRosa et al. 2000; Prescott, 2000). The pharmac okinetic properties of tilmicosin are similar to that of macrolides in ge neral, and are characterized by low serum concentrations but large volumes of distri bution, with accumulation and persistence in many tissues including the lung, which may c oncentrate the drug 60-fold compared to serum (Ziv et al. 1995; Scorneaux and Shryock, 1999; Clark et al. 2004). Despite low extracellular concentrations, tilmicosin accumu lates substantially in phagocytic cells of cattle and swine (Scorneaux and Shryock, 1998; Scorneaux and Shryock, 1999). Pneumonia is a leading cause of morbid ity and mortality in foals (Cohen, 1994). Gram-positive bacteria such as Streptococcus equi subspecies zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in foals between 1 and 6 months of age (Hoffman et al. 1993; Gigure et al. 2002). Gram-negative bacteria such as Pasteurella spp., Actinobacillus spp., Bordetella bronchiseptica Escherichia coli

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38 Klebsiella pneumoniae and Salmonella enterica may also be cultured from tracheobronchial aspirates of affected foals (Wilson, 1992). Macrolide antimicrobial agents are commonly used in equine medi cine for treatment of foal pneumonia, particularly when infection with Rhodococcus equi is suspected or confirmed. Tilmicosin may be a useful alternative to currently used antimicrobial agents owing to its accumulation in lung tissue and phagocytic cells, as well as in vitro activity against many Gram-positive and Gram negative bacterial spec ies. In addition, av ailability of a long acting antimicrobial agent providi ng sustained therapeutic concentrations at the site of infection would result in less frequent administration, which in turn may improve client compliance. However, the lack of pharmacokinetic studies and in vitro susceptibility data with bacterial pathogens of horses prec ludes the rational use of this antimicrobial agent in foals. The objectives of the stu dy reported here were to determine the pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against R. equi and other common bacteria l pathogens of horses. Material and Methods Horses and experimental design Four male and three female Thoroughbred foals between 5 and 8 weeks of age and weighing between 80 and 135 kg were selected fo r this study. The foals were considered healthy on the basis of history, physical ex amination, complete blood count and plasma biochemical profile. The foals were kept with their dams in indivi dual stalls during the experiment with ad libitum access to grass hay and water. The study was approved by the Institutional Animal Care and Use Committee at the University of Florida.

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39 Experimental design and sample collection A proprietary fatty acid salt formulat ion of tilmicosin (250 mg/mL; Idexx Pharmaceuticals, Durham, NC) was administer ed as a single dose of 10 mg/kg of body weight via the intramuscular route in th e semimembranosus/semitendinosus muscles. Blood samples (8 mL) were obtained from a jugular catheter at 3, 6, 10, 20, 30, 60, 90 minutes and at 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 168, and 288 hours after the drug was administered. Bronchoa lveolar lavage (BAL) was performed 24, 48, 72, 168, and 288 hours and samples of cerebro spinal fluid (CSF) were collected aseptically 4, 24, and 72 hours after administration of tilmicosin. Lung tissue was obtained 24, 72, 168, and 288 hours after administ ration of the drug. Prior to collection of BAL, lung tissue, and CSF, foals were sedated by administration of xylazine hydrochloride (1 mg/kg, IV) and butorphanol ta rtrate (0.07 mg/kg, IV). Immediately after collection of BAL fluid, gene ral anesthesia was induced by IV administration of diazepam (0.1 mg/kg) and ketamine (2.5 mg/ kg) for collection of lung tissue and CSF fluid. Using sterile techniques, CSF was colle cted from the atlantoocciptal space by use of a 3.5 inch, 20-gauge spinal needle. Bl ood and CSF samples were centrifuged and serum and CSF supernatants were stored at -80C until analysis. Lung tissue was obtained aseptically from the 8th intercostal space at the level of the point of the shoulder using a 16 gauge spring activated biopsy in strument with a 20 mm specimen notch (J528a, Jorgenson laboratories, Loveland, CO). Bronchoalveolar lavage A 10 mm diameter, 2.4 m bronchoalveolar lava ge catheter (Jorgenson laboratories, Loveland, CO) was passed via nasal approach until wedged into a bronchus. The lavage solution consisted of 4 aliquots of 50 mL phys iologic saline (0.9% NaCl) solution infused

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40 and aspirated immediately. Total nucleated cell count in BAL fluid was determined by use of a hemacytometer. Bronchoalveola r fluid was centrifuged at 200 X g for 10 minutes. Bronchoalveolar cells were wash ed, re-suspended in 500 l of phosphatebuffered solution, vortexed and frozen at -80 C until assayed. Supernatant BAL fluid was also frozen at -80C until assayed. Before a ssaying, the cell pellet samples were thawed, vortexed vigorously and sonicated for 3 minut es to ensure complete cell lysis. The resulting suspension was centrifuged at 500 X g for 10 minutes and the supernatant fluid was used to determine the intracellular concentrations of tilmicosin. Drug analysis The serum and other tissue samples were analyzed by validated methods at Idexx Pharmaceuticals (Durham, NC). Serum concentrations of tilmicosin were determined by HPLC analysis. The extraction efficiency from serum was 98%. The limit of quantification (LOQ) was 0.08 g/mL. The tilm icosin concentrations in all other body fluids or tissues were determined by ma ss spectrometry. The LOQ were 0.5 ng/mL, 1.44 ng/mL, 1.9 ng/mL, and 0.6 ng/mL for lung ti ssue, BAL fluid, CSF, and BAL cells, respectively. Estimation of PELF and BAL Cell Volu mes and Determination of Tilmicosin Concentrations in PELF and BAL Cells Pulmonary distribution of tilmicosin was determined as reported (Baldwin et al. 1992). Estimation of the volume of PELF wa s done by urea dilution method (Rennard et al. 1986; Conte et al. 1996). Serum urea nitrogen concentrations (UreaSERUM) were determined by use of enzymatic methodology (Labsco Laboratory Supply Company; Louisville, KY, USA) on a chemistry an alyzer (Hitachi 911 analyzer, Boehringer Mannheim Inc, Indianapolis, IN, USA). For measurement of urea concentration in BAL

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41 fluid (UreaBAL), the proportion of reagents to specimen was changed from 300 l/3 l in serum to 225 l/50 l. The volume of PELF (VPELF) in BAL fluid was derived from the following equation: VPELF = VBAL X (UreaBAL/UreaSERUM), where VBAL is the volume of recovered BAL fluid. The concentr ation of tilmicosin PELF (TILPELF) was derived form the following relationship: TILPELF =TILBAL X (VBAL/ VPELF), where TILBAL is the measured concentration of tilmicosin in BAL fluid. The concentration of tilmicosin in BAL cells (TILBAL) was calculated using the following relationship: TILBAL = (TILPELLET/VBALC) where TILPELLET is the concentration of antimicrobial in the BAL cell pellet supernatant and VBALC is the mean volume of foal BAL cells. A VBALC of 1.20 l/106 cells was used for calcula tions based on a previous study in foals (Jacks et al. 2001). Pharmacokinetic Analysis For each foal, serum, lung tissue, PELF, or BAL cells tilmicosin concentration versus time data were analyzed base d on noncompartmental pharmacokinetics using computer software (PK Solutions 2.0, Summit Research Services, Montrose, CO, USA). The elimination rate constant (Kel) was determined by linear regression of the terminal phase of the logarithmic con centration versus time curve using a minimum of 3 data points. Elimination half-life (t) was calculated as the natura l logarithm of 2 divided by Kel. Pharmacokinetic values were calculated as reported by Gibaldi and Perrier (1982). The area under the concentra tion-time curve (AUC) and the area under the first moment of the concentration-time curve (AUMC) were calculated using the tr apezoidal rule, with extrapolation to infinity using Cmin/ Kel, where Cmin was the final measurable tilmicosin concentration. Mean residence time (MRT) was calculated as: AUMC/AUC.

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42 Statistical Analysis Normality of the data and equality of variances were assessed using the Kolmogorov-Smirnov and Levene's tests, resp ectively. A one way repeated measure ANOVA was used to compare each pharmacokinetic parameter between sampling sites (serum, lung tissue, PELF, BAL cells). In ra re instances when the assumptions of the ANOVA were not met, a Friedman repeated measure ANOVA on ranks was used. When indicated, multiple pairwise comparisons were done using the Student-Newman-Keuls test. Differences were considered significant at P < 0.05. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of tilmicosin against R. equi R. equi isolates (n =56) were obtained from tracheobronchial aspirates or postmortem specimens from pneumonic foals. For each isolate, MIC and MBC were determined by a macrodilution broth dilution t echnique in glass tubes in accordance to the guidelines established by the Clinical a nd Laboratory Standard Institute (formerly NCCLS) (NCCLS, 1999a; NCCL S, 1999b; NCCLS, 2000) A standard inoculum of 5 x 105 was used for each isolate. Concentrations of tilmicosin tested ranged between 256 and 0.03 g/mL. All MIC and MBC determinations were performed in triplicate for each isolate. MIC was determined as the first di lution with no bacteria l growth after 24 h of incubation at 37C (National Committee for Clinical Laboratory Standards, 2000). MBC was calculated as the lower concentration of drug resulting in a 99.9% reduction of the original inoculum(National Committee for C linical Laboratory Standards, 1999a). Control strains used to validate the assay were Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212 (Odland et al.

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43 2000). The MIC r equi red to inhibit growth of 50% of isolates (MIC50) and the MIC r equi red to inhibit growth of 90% of isolates (MIC90) were determined. Checkerboard assay Activity of tilmicosin in combination with rifampin, gentamicin, amikacin, doxycycline, enrofloxacin, trimethoprim-sulfa, vancomycin, imipenem, or ceftiofur against R. equi was assessed using the modified ch eckerboard technique as previously described (Pillai et al. 2005). Three isolates of R. equi were randomly selected for this assay. All experiments were performed in trip licate for each of the isolate. For each antimicrobial agent, concentrations of 64-, 16-, 4-, 1-, and 0.5-times the MIC were used to study antibiotic combinations. An inoculum of 5 x 105 was used for each R. equi isolate. For each combination, the fractional inhibitory concentration (FIC) index after 24 h of incubation was calculated using the following formula: FIC index = FIC A + FIC B = (MIC of A in combination/MIC of A al one) + MIC of B in combination/MIC of B alone). A FIC index of 0.5 indicates synergism, a FIC index > 0.5-4 indicates indifference and a FIC index > 4 indicates antagonism (Pillai et al. 2005). Time kill curve assay A time kill curve assay was used to eval uate the effect of time and tilmicosin concentration on in vitro survival of R. equi All experiments were performed in triplicate using the same 3 R. equi isolates as for the checkerboard assay. An inoculum of 5 X 105 CFU/mL was used for each isolate. All e xperiments were perfor med with 4 mL of Mueller-Hinton broth in glass tubes. Afte r 0, 2, 6 and 24 hours of incubation, aliquots were collected from each tube. The aliquots were centrifuged, the bacterial pellets were washed twice to prevent antimicrobial carry over, and the CFU was counted.

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44 In vitro activity of tilmicosin against equine bacterial pathogens A total of 183 bacterial isolates from various equi ne clinical samples were examined. Isolates were obtained from clin ical samples submitted to the microbiology laboratory at the University of Florida Ve terinary Medical Center from July 2005 to January 2006. Susceptibility te sting was performed using the disk diffusion method. Briefly, fresh isolates were grown on blood ag ar plates, and colonies were suspended in sterile water to achieve turbidity equal to that of a 0.5 McFarland standard (final bacterial concentration of approximately 1 X 105 CFU/mL). A sterile swab was dipped into the inoculum suspension and used to inoculat e the entire surface of 100 mm Mueller-Hinton plates 3 times by rotating the plate approximately 60o for each inoculation to ensure an even distribution. After allowing the excess mo isture to dry (approx 10 to 15 minutes), 15 g tilmicosin disks (BBL Sensi-Disc, Hardy Di agnostics, Santa Maria, CA) were applied to the agar. The plates were incubated for 18 to 24 hours at 37oC. A test was considered valid only when there was adequate grow th on the plate. The zone diameter was measured to the nearest millimeter. Control strains used weekly to validate the assay were Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212. Results were consid ered valid only when zone diameters obtained with the control stains were within the re ference range proposed (Odland et al. 2000). According to CLSI guideline s, isolates with a zone diameter 14 mm (corresponding to a MIC 8 g/mL) were considered susceptible (Shryock et al. 1996).

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45 Results Serum and pulmonary disposition of tilmicosin in foals Quantifiable tilmicosin concentrations were found in 2 of 7 foals at 3 minutes after IM injection and in 5 of 7 foals at 10 mi nutes post-injection. Serum concentrations remained below the limit of quantification throughout the sampling period in 2 foals. Concentrations below the limit of quantifica tion were reported as 0 for calculation of mean SD (Figure 3.1). Serum pharmacokine tic parameters were derived from the 5 foals with quantifiable serum concentra tions (Table 3.1). Maximum tilmicosin concentrations (Cmax) and AUC were significantly higher in BAL cells than in serum, lung tissue, and PELF (Table 3.1). Similarly, Cmax and AUC were significantly higher in PELF and lung tissue than in serum. Elim ination half life in lung tissue (193.3 h) was significantly longer than that of serum (18.4 h). One foal died as a result of hemothorax within minutes of collection of the 72 h lung biopsy. One foal developed tachypnea a nd profuse sweating approximately 2 h after injection. The clinical signs persisted for approximately 45 min. Two foals developed a 10-15 cm in diameter area of painful swelli ng at the injection site within 12-24 h of injection. In one foal, the lesion was associ ated with hind limb lameness that persisted for 48 h. Three foals developed a small 1-2 cm in diameter hard nodule at the injection site. Four foals developed watery diarrhea 36-48 h after administration of tilmicosin. Diarrhea resolved without therapy within 48 h of onset. In vitro susceptibility testing a nd antimicrobial drug combinations Both the MIC50 and MIC90 of 56 R. equi isolates were 32 g/mL (range 16-64 g/mL). Tilmicosin was not bactericidal against R. equi at concentrations up to 256 g/mL. Combination of tilmicosin with rifampin, gentamicin, amikacin, doxycycline,

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46 enrofloxacin, trimethoprim-sulfa, vancomyci n, imipenem, or ceftiofur did not result in synergistic or antagonistic activity with median FIC indices ranging between 0.53 and 1.5. The time-kill experiment revealed that t ilmicosin is a time dependent antimicrobial agent with no benefit from increasing drug c oncentrations above 4 times the MIC (Figure 3.2). Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. (Table 3.2). Discussion A safe antimicrobial agent providing high a nd sustained drug concentrations in the lungs would be a useful addi tion to currently available antimicrobial agents for the treatment or prevention of pneumonia in foals. Tilmicosin has been approved for the control and treatment of respiratory disease in cattle, sheep, and swine. Tilmicosin has also been shown to be effective for the treatment of mastitis in cattle and sheep, pasteurellosis in rabbits, and Mycoplasma gallisepticum infections in chicken (McKay et al. 1996; Kempf et al. 1997; Croft et al. 2000; Dingwell et al. 2003). The currently available injectable tilmicosin formulation has been advocated as potentially fatal when administered to horses, swine, and goat s (Micotil 300 package insert, 1995). The cardiovascular system is the target of toxic ity in laboratory and domestic animals with tachycardia and decreased cardiac contracti lity being reported following parenteral administration of tilmicosin (Main et al. 1996). To minimize the risk of toxicity, a fatty acid salt formulation of tilmicosin newly deve loped as a safer and convenient formulation for use in cats (Kordick et al. 2003) was used in the present study. Mean peak serum concentrations and AUC achieved in the present study (0.19 g/mL) were considerably lower that that ac hieved after subcutane ous administration of

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47 the same dose to cattle (0.87 g/mL), sheep (0.82 g/mL), and goats (1.56 g/mL) (Ramadan, 1997; Modric et al. 1998). Peak serum concentrations in foals were also lower that that observed after administrati on of the same fatty acid salt formulation administered at a dose of 10 mg/kg SC to cats (0.73 g/mL) (Kordick et al. 2003). Tilmicosin serum elimination half-life in th e present study (18.4 h) was slightly shorter than that reported after SC administrati on to cattle (29.4 h), sheep (34.6 h), and goats (29.3 h) (Ramadan, 1997; Modric et al. 1998). Recent data suggest that traditional pharmacodynamic parameters based on plasma concentrations of macrolides may not best ap ply to the treatment of pulmonary infections and infections caused by facultative intracellular pathogens such as R. equi (Drusano, 2005). Serum concentrations of tilmicosin in cattle and sw ine are much lower than its MICs for common respiratory tract pathogens Nevertheless, multiple studies have demonstrated the efficacy of tilmicosin in the treatment of respiratory disease in these species (Musser et al. 1996; Paradis et al. 2004). Lung concentrations of tilmicosin remain above the MIC of Mannheimia haemolytica (3.15 g/mL) for at least 72 hours following a single SC injection at a dose of 10 mg/kg (Micotil 300 package insert, 1995). In cats, maximum lung concentrations of tilmicosin of 5.62 g/mL are achieved on day two following administration of the fatty acid salt formulation and measurable concentrations are still present in the lungs on day 21 (Kordick et al. 2003). While drug concentration in plasma is clear ly a driving force for penetration to the site of infection, the actual drug-concentration time profile at a peripheral site may be quite different from that of plasma. Macrol ides cross the cellular membranes primarily by passive diffusion (Fietta et al. 1997). Tilmicosin, like othe r macrolides, is a potent

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48 weak base that becomes ion-trapped within acidic intracellular compartments such as lysosomes (Scorneaux and Shryock, 1999). Th e ratio of cellular to extracellular concentration of tilmicosin is 193, 43, and 13, respectively, in bovine alveolar macrophages, monocyte-derived macrophages, and mammary epithelia l cells (Scorneaux and Shryock, 1999). Consistent with these fi ndings, peak tilmicosin concentrations in BAL cells of foals were approximately 107 times higher than peak serum concentrations. A number of in vitro and in vivo studies support the notion that white blood cells act as carriers for the delivery of macrolides to the site of infection (Retsema et al. 1993; Mandell and Coleman, 2001). Studies with tilm icosin in rats support this concept as drug concentrations in the lung of rats inoculated with Mycoplasma pulmonis were significantly higher that those of noninfected controls (Modric et al. 1999). Macrolides inhibit protein synthesis by re versibly binding to 50S subunits of the ribosome. Macrolides are generally bacteriostat ic agents but they may be bactericidal at high concentrations (Presco tt, 2000). In the presen t study, tilmicosin was only bacteriostatic against R. equi at concentrations up to 256 g/mL. The MIC90 of tilmicosin against foal isolates of R. equi (32 g/mL) in the present study was similar to that of a previous study looking at a combin ation of human and equine isolates (> 32 g/mL) (Bowersock et al. 2000). Consistent with a bacter iostatic antimicrobial agent, tilmicosin exerted time dependent activity against R. equi in vitro Even if tilmicosin concentrated more than 100-fold in BAL cells of foals, drug concentrations achieved in lung tissue, PELF, and BAL cells we re consistently below the MIC90 of R. equi Tilmicosin was active in vitro against all -hemolytic streptococci and Pasteurella spp., and most -hemolytic streptococci, Staphylococcus spp., and Actinobacillus spp.

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49 Additional studies will be required to determine the clinical efficacy of this fatty acid salt formulation of tilmicosin against these pathogens in foals. Adverse effects observed in the present study consisted mainly of swelling at the injection site in 5 foals and self limiting diarrhea in 4 foals. One foal developed tachypnea and profuse sweating approximately 2 h after injection. In swine, IM administration of the commercially availabl e formulation at a dose of 10 mg/kg has resulted in tachypnea, and convulsions, and death occurs with dosages 20 mg/kg (Micotil 300 package insert 1995). Tilmicosin included in the diet of horses at concentrations of 400, 1200, and 2000 ppm has result ed in gastrointestin al disturbance in all groups and death of 1 horse consuming the 2000 ppm diet (Pulmotil 90 package insert, 1995). In another study, SC admi nistration of the commerciall y available formulation of tilmicosin to foals at a dose of 10 mg/kg resu lted in immediate loss of the normal fecal streptococcal population and a corresponding massive overgrowth of coliform bacteria (Clark and Dowling, 2004). The fecal flora slow ly recovered over the next 7 days. Mild self-limiting diarrhea was observed in one foal (Clark and Dowling, 2004). In conclusion, the fatty acid salt formul ation of tilmicosin investigated in the present study resulted in high and sustained concentrations of tilmicosin in the lung, PELF, and BAL cells of foals following a si ngle IM administration. The drug was active in vitro against a variety of bacter ial pathogens. These data wa rrant further investigations into the clinical efficacy of th is formulation of tilmicosin in foals with respiratory disease.

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50 Table 4.1 Serum and pulmonary pharmacokinetic variables (mean SD unless otherwise specified) for tilmicosin after IM administration to seven foals at a dose of 10 mg/kg of body weight. Variable Serum1 Lung2 PELF BAL cells Kel (h-1) 0.04 0.02a 0.004 0.001b 0.009 0.004b 0.01 0.003b AUC0 ( gh/mL) 5.76 1.87a 711 351b 461 115b 2342 1006c MRT (h) 34.5 18.0a 323 91.0b 180 48.9c 117 29.6c t (h)* 18.4a 193.3b 73.1a,b 62.2a,b Tmax (h) 5.50 3.43a 30.8 18.1a,b 52.0 18.1b 54.9 33.1b Cmax ( g/mL or g/g) 0.19 0.09a 1.90 0.65b 2.91 1.15b 20.1 5.1c 1n=5 because 2 foals had serum tilmicosin concentrations below the limit of quantification. 2n=5 because one foal died after the 72 h sa mple and lung samples were too small for drug analysis in one foal. *harmonic mean. a,b,c,dDifferent letters within a row indicate a statistically significant difference between sampling sites ( P < 0.05). Kel = Elimination rate constant. AUC = Area under the serum concentration versus time curve. MRT = Mean residence time. t = Elimination half-life. Tmax = Time to peak serum concentration. Cmax = peak serum concentration. Table 4.2 Tilmicosin in vitro susceptibility of 183 bacteria l isolates obtained from horses. Zone diameter (mm) Microorganism (n) Median 25th percentile Range Susceptibility (%) Gram positives -hemolytic streptococci (7) 19 11 0-20 71 -hemolytic streptococci (37) 19 18 15-26 100 Enterococcus spp. (5) 0 0 0 0 Rhodococcus equi (9) 0 0 0 0 Staphylococcus spp. (25) 18 16 0-28 96 Gram-negatives Actinobacillus spp. (9) 16 12 0-19 67 Enterobacter spp. (9) 0 0 0 Escherichia coli (11) 0 0 0-14 9 Klebsiella spp. (5) 0 0 0 0 Pasteurella spp. (6) 23 18 18-34 100 Pseudomonas spp. (12) 0 0 0-13 0 Salmonella enterica (48) 0 0 0-11 0

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51 Figure 4.1 Mean SD tilmicosin concentrations in serum, BAL cells, PELF ( g/mL), and lung tissue ( g/g) of 7 foals following a single IM dose of tilmicosin (10 mg/kg of body weight). 4 5 6 7 8 06121824 Time (h)Log (CFU/mL) Control 0.25 X MIC 1 X MIC 4 X MIC 16 X MIC 64 X MIC Figure 4.2 Effect of time a nd tilmicosin concentration on in vitro survival of a clinical isolate of R. equi Identical results were obtaine d with 2 additional isolates. 0.001 0.01 0.1 1 10 100 04896144192240288 Time (h)Tilmicosin concentrations Serum BAL cells PELF Lung tissue

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52 CHAPTER 5 SUMMARY AND CONCLUSIONS The present study investigated the pharm acokinetics and pulmonary disposition of clarithromycin and tilmicosin in foals. Th e optimal dosing of antimicrobial agents is dependent not only on their pharmacokinetics but also on the pharmacodynamics of the drug. The pharmacodynamic properties of a drug address the relationship between drug concentration and antimicrobial activit y. Much confusion exists over the pharmacodynamics of macrolides. An important factor in determining the efficacy of many macrolides in animal models of infection with extracellular bacteria is the length of time that serum concentrations exceed the MI C of a given pathogen (T > MIC) (Rodvold, 1999). However, recent data suggest that traditional pharmacodynamic parameters based on plasma concentrations of macrolides may not best apply to the treatment of pulmonary infections and infections caused by facultative intrace llular pathogens such as R. equi and that concentrations at the site of infecti on are more important in predicting efficacy (Drusano, 2005). Macrolides enter phagocytic cells by passive diffusion and they accumulate in acidic intracellular compartments such as lysosomes and phagosomes. A number of in vitro and in vivo studies support the notion th at white blood cells act as carriers for the delivery of macr olides to the site of infec tion (Mandell et al., 2001). To provide a better assessment of the pot ential usefulness of clarithromycin and tilmicosin for the treatment of bronchopneumonia in foals, we measured drug concentrations in PELF, BAL cells, and lung tissue. Oral clarithromycin was wellabsorbed in foals and resulted in mean PELF concentrations approximately 95 times

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53 higher and mean BAL cell concentrations approximately 335 times higher than concurrent serum concentrations. Clarith romycin undergoes extensive metabolism in people. Of the 8 metabolites that have b een identified, 14-hydroxyclarithromycin is the most abundant and the only one with subs tantial antimicrobial activity (Fernandes et al ., 1988; Ferrero et al ., 1990). Although clar ithromycin is not co nverted to14-hydroxy clarithromycin in rodents and reptiles, the present study confirmed production of 14hydroxy-clarithromycin in foals. A new fatty acid salt formulation of tilmicos in, developed as a safer and convenient formulation for use in cats (Kordick et al. 2003), was investigated in the present study. Serum concentrations of tilmicosin in cattle and swine are much lower than its MICs for common respiratory tract pathogens. Neverthe less, multiple studies have demonstrated the efficacy of tilmicosin in the treatment of respiratory disease in these species because the drug concentrates in lung ti ssue and phagocytic cells (Musser et al. 1996; Paradis et al. 2004). Tilmicosin accumulated in the lungs of foals. BAL cells achieved the highest concentrations with Cmax approximately 100 times higher than that achieved in plasma. Tilmicosin concentrations in PELF were almo st identical to that of lung concentrations, indicating that measurement of drugs in PELF may represent a less invasive alternative to lung biopsies. Lung tissue and PELF tilmicos in concentrations were approximately 1015 times higher than peak serum concentratio ns. However, tilmicosin concentrations at all of the times sampled remained considerably below the MIC90 of R. equi Therefore, tilmicosin, at the dose used in the present st udy, would not be adequate for the treatment of R. equi infections in foals.

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54 In conclusion, oral administration of clar ithromycin at a dosage of 7.5 mg/kg every 12 hours would maintain serum, PELF, and BA L cell concentrations above the minimum inhibitory concentration for R. equi and S. zooepidemicus isolates for the entire dosing interval. The formulation of tilmicosin invest igated in the present study resulted in high and sustained concentrations in the lung, PELF, and BAL cells of foals and may be appropriate for the treatment of susceptible ba cterial infections. Additional studies will be required to establish the safety and determin e the efficacy of these drugs in a clinical setting.

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55 LIST OF REFERENCES Ainsworth, D.M., Eicker, S. W., Yeagar, A.E ., Sweeney, C.R., Viel, L., Tesarowski, D., Lavoie, J.P., Hoffman, A., Paradis, M.R ., Reed, S.M., Erb, H.N., Davidow, E. & Nalevanko, M. (1998) Associations between physical examination, laboratory, and radiographic findings and outcome and s ubsequent racing performance of foals with Rhodococcus equi Infection: 115 Cases (1984-1992). Journal of the American Veterinary Medical Association 213, 510-515. Alvarez-Elcoro, S. & Enzler, M.J. (1999) Th e macrolides: erythromycin, clarithromycin, and azithromycin. Mayo Clinic Proceedings, 74, 613-634. Anderson, G., Esmonde, T.S., Cole, S., Mackli n, J. & Carnegie, C. (1991) A comparative safety and efficacy study of clarithrom ycin and erythromycin stearate in communiy-acquired pneumonia. Journal of Antimicrobial Chemotherapy, 27, (Suppl. A), 117-124. Baldwin, D.R., Honeybourne, D. & Wise, R. (1992) Pulmonary disposition of antimicrobial agents: met hodological considerations. Antimicrobial Agents and Chemotherapy, 36, 1171-1175. Barr, B.S. (2003) Pneumonia in weanlings. Veterinary Clinic of North American Equine Practicioner, 19, 35-49. Baverud, V., Franklin, A., Gunnarsson, A ., Gustafsson, A. & Hellander-Edman, A. (1998) Clostridium Difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for Rhodococcus equi pneumonia. Equine Veterinary Journal, 30, 482-488. Bedos, J.P., Azoulay-Dupuis, E., Vallee, E., Vebe r, B. & Pocidalo, J. J. (1992) Individual efficacy of clarithromycin (A-56268) a nd its major human metabolite 14-hydroxy clarithromycin (A-62671) in experimental pneumococcal pneumonia in the mouse. Journal of Antimicrobial Chemotherapy, 29, 677-685. Bowersock, T.L., Salmon, S.A., Portis, E.S., Prescott, J.F., Robison, D.A., Ford, C.W. & Watts, J. L. (2000) MICs of oxazolidinones for Rhodococcus equi strains isolated from humans and animals. Antimicrobial Agents and Chemotherapy, 44, 13671369. Chu, S.Y., Deaton, R. & Cavanaugh, J. (1992) Ab solute bioavailability of clarithromycin after oral administration in humans. Antimicrobial Agents and Chemotherapy, 36, 1147-1150.

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56 Clark, C., and Dowling, P.M. (2004) Antimic robial-associated diarrhea in horses. [Proceedings of the 23rd ACVIM Forum], 169-171. Clark, C., Woodbury, M., Dowling, P., Ross, S. & Boison, J.O. (2004) A preliminary investigation of the dispos ition of tilmicosin residues in elk tissues and serum. Journal of Veterinnary Pharmacology and Therapeutics, 27, 385-387. Cohen, N.D. (1994) Causes of and farm manage ment factors associated with disease and death in foals. Journal of the American Vete rinary Medica l Association, 204, 16441651. Conte, J.E., Golden, J., Duncan, S., McKenna E., Lin, E. & Zurlinden, E. (1996) Singledose intrapulmonary pharmacokinetics of azithromycin, clarithromycin, ciprofloxacin, and cefuroxime in volunteer subjects. Antimicrobial Agents and Chemotherapy, 40, 1617-1622. Conte, J.E., Golden, J.A., Duncan, S., McKenna, E. & Zurlinden, E. (1995) Intrapulmonary pharmacokinetics of clarithromycin and of erythromycin. Antimicrobial Agents and Chemotherapy, 39, 334-338. Craig, W.A. (1997) Postantibiotic effects and the dosing of macrolides, azalides, and streptogramins. In Expanding indications for th e new macrolides, azalides, and streptogramins 3rd edn. Eds Zinner, S.H., Young, L.S., Acar, J.F. &Neu, H.C. pp. 27-38. Marcel Dekker, New York. Croft, A., Duffield, T., Menzies, P., Leslie, K., Bagg, R. & Dick, P. (2000) The effect of tilmicosin administered to ewes prior to lambing on incidence of clinical Mastitis and Subsequent Lamb Performance. Canadian Veterinary Journal, 41, 306-311. Davis, J.L., Gardner, S.Y., Jones, S.L ., Schwabenton, B.A. & Papich, M.G. (2002) Pharmacokinetics of azithromycin in foals after I.V. and oral dose and disposition into phagocytes. Journal of Veterinary Pharmacology and Therapeutics, 25, 99104. DeRosa, D.C., Veenhuizen, M.F., Ba de, D.J. & Shryock, T.R. (2000) In vitro susceptibility of porcine respir atory pathogens to tilmicosin. Journal of Veterinary Diagnostic Investigation, 12, 541-546. Dingwell, R.T., Leslie, K.E., Duffield, T. F., Schukken, Y.H., DesCoteaux, L., Keefe, G.P., Kelton, D.F., Lissemore, K.D., Shew felt, W., Dick, P. & Bagg, R. (2003) Efficacy of intramammary tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period. Journal of Dairy Science, 86, 159-168. Drusano, G.L. (2005) Infection site concentra tions: Their therapeutic importance and the macrolide and macrolide-like class of antibiotics. Pharmacotherapy 25, 150S158S.

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57 Fernandes, P.B., Ramer, N., Rode, R.A. & Freiberg, L. (1988) Bioassay for A-56268 (TE-031) and identification of its ma jor metabolite, 14-hydroxy-6-O-methyl erythromycin. European Journal of Clinical Micr obiology and Infectious Disease, 7, 73-76. Ferrero, J.L., Bopp, B. A., Marsh, K.C., Qu igley, S.C., Johnson, M.J., Anderson, D.J., Lamm, J.E., Tolman, K.G., Sanders, S.W ., Cavanaugh, J.H. (1990) Metabolism and disposition of clarithromycin in man. Drug Metabolism and Disposition, 18, 441446. Fietta, A., Merlini, C. & Gialdroni, G.G. (1997) Requi rements for intracellular accumulation and release of clarithromycin and azithromycin by human phagocytes. Journal of Chemotherapy, 9, 23-31. Fittipaldi, N., Klopfenstein, C., Gottschalk, M ., Broes, A., Paradis, M.A. & Dick, C.P. (2005) Assessment of the efficacy of tilmicosin phosphate to eliminate Actinobacillus pleuropneumoniae from carrier pigs. Canadian Journal of Veterinary Research, 69, 146-150. Gan, V.N., Chu, S. Y., Kusmiesz, H. T. & Cr aft, J.C. (1999) Pharmacokinetics of a clarithromycin suspension in infants and children. Antimicrobial Agents and Chemotherapy, 36, 2478-2480. Gibaldi, M. & Perrier, D. (1982) Noncompart mental analysis based on statistical moment theory. Pharmacokinetics 2, 409. Gigure, S., Gaskin, J.M., Miller, C. & Bowman, J.L. (2002) Evaluation of a commercially available hyperimmune plas ma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals. Journal of the American Veterinary Medical Association, 220, 59-63. Gigure, S., Hondalus, M.K., Yager, J.A., Darrah, P., Mosser, D.M. & Prescott, J.F. (1999) Role of the 85-Kilobase plasmid a nd plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi Infectious Immunity, 67, 3548-3557. Gigure, S., Jacks, S., Roberts, G.D., Hern andez, J., Long, M.T. & Ellis, C. (2004) Retrospective comparison of azithromycin, clarithromycin, and erythromycin for the treatment of foals with Rhodococcus equi pneumonia. Journal of Veterinary Internal Medicine, 18, 568-573. Gigure, S. & Prescott, J.F. (1997) Clinical manifestati ons, diagnosis, treatment, and prevention of Rhodococcus equi i nfections in foals. Veterinary Microbiology, 56, 313-334. Hillidge, C.J. (1987) Use of erythromycin -rifampin combination in treatment of Rhodococcus equi pneumonia. Veterinary Microbiology, 14, 337-342.

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58 Hoar, B.R., Jelinski, M.D., Ribble, C.S ., Janzen, E.D. & Johnson, J.C. (1998) A comparison of the clinical field efficacy a nd safety of florfenicol and tilmicosin for the treatment of undifferentiated bovine re spiratory disease of cattle in western Canada. Canada Veterinary Journal, 39, 161-166. Hoffman, A.M., Viel, L., Juniper, E. & Pres cott, J.F. (1993a) Clinical and endoscopic study to estimate the incidence of distal respiratory tract inf ection in Thoroughbred foals on Ontario breeding farms. American Journal of Veterinary Research, 54, 1602-1607. Hoffman, A.M., Viel, L., Prescott, J.F., Rose ndal, S. & Thorsen, J. (1993b) Association of microbiologic flora with clinical, endoscopic, and pulmonary cytologic findings in foals with distal re spiratory tract infection. American Journal of Veterinary Research, 54, 1615-1622. Hooper-McGrevy, K.E., Wilkie, B.N. & Presco tt, J.F. (2005) Virulence-associated protein-specific serum immunoglobulin G-Isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immuni zation with virulent R. equi. v accine, 23, 5760-5767. Jacks, S., Gigure, S., Gronwall, R.R., Brow n, M.P. & Merritt, K.A. (2002) Disposition of oral clarithromycin in foals. Journal of Veterinary Pharmacology and Therapeutics, 25, 359-362. Jacks, S., Gigure, S., Gronwall, R.R., Brown, M.P. & Merritt, K. A. (2001) Pharmacokinetics of azithromycin a nd concentration in body fluids and bronchoalveolar cells in foals. American Journal of Veterinary Research, 62, 18701875. Jacks, S., Gigure, S. & Nguyen, A. (2003) In vitro susceptibilities of Rhodococcus equi and other common equine pathogens to azi thromycin, clarithromycin and 20 other antimicrobials. Antimicrobial Agents and Chemotherapy, 47, 1742-1745. Jain, S., Bloom, B.R. & Hondalus, M.K. (2003) Deletion of VapA encoding virulence associated protein A attenuates the intracellular actinomycete Rhodococcus equi Molecular Microbiology, 50, 115-128. Johnson, J.A., Prescott, J.F. & Markham, R. J. (1983) The pathology of experimental Corynebacterium equi infection in foals following intragastric challenge. Veterinary Pathology, 20, 450-459. Kordick, D.L., Murthy, Y., & Henley, K. (2003) Biodistribution of a novel formulation of tilmicosin in cats. American Society for Microbiology [103rd General Meeting], Z014. Kempf, I., Reeve-Johnson, L., Gesbert, F. & Guittet, M. (1997) Efficacy of tilmicosin in the control of experimental Mycoplasma gallisepticum infection in chickens. Avian Disease, 41, 802-807.

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59 Lakritz, J., Tyler, J.W., Marsh, A.E., Rome sburg-Cockrell, M., Smith, K. & Holle, J.M. (2002) Tilmicosin reduces lipopolys accharide-stimulated bovine alveolar macrophage prostaglandin E(2) pr oduction via a mechanism involving phospholipases. Veterinary Therapeutics, 3, 7-21. Lakritz, J., Wilson, W.D., Marsh, A.E. & Mihaly i, J.E. (2000a) Eff ects of prior feeding on pharmacokinetics and estimated bioavail ability after oral administration of a single dose of microencap sulated erythromycin base in healthy foals. American Journal of Veterinary Research, 61, 1011-1015. Lakritz, J., Wilson, W.D., Marsh, A.E. & Mi halyi, J.E. (2000b) Pharmacokinetics of erythromycin estolate and erythromycin phosphate after intragas tric administration to healthy foals [In process citation]. American Journal of Veterinary Research, 61, 914-919. Lakritz, J., Wilson, W.D. & Mihalyi, J.E. (1999) Comparison of microbiologic and highperformance liquid chromatography assays to determine plasma concentrations, pharmacokinetics, and bioavailability of eryt hromycin base in plasma of foals after intravenous or intragastric administration. American Journal of Veterinary Research 60, 414-419. Laven, R. & Andrews, A.H. (1991) Long-acting antibiotic formulations in the treatment of calf pneumonia: a comparative study of tilmicosin and oxytetracycline. The Veterinary Record 129, 109-111. Maglio, D., Capitano, B., Banevicius, M.A., Geng, Q., Nightingale, C.H. & Nicolau, D.P. (2004) Differential efficacy of clarithromycin in lung versus thigh infection models. Chemotherapy 50, 63-66. Main, B.W., Means, J.R., Rinkema, L.E., Smith, W.C. & Sarazan, R.D. (1996) Cardiovascular effects of the macrolide antibiotic tilmicosin, administered alone and in combination with propranolol or dobutamine, in conscious unrestrained dogs. Journal of Veterinary P harmacology and Therapeutics, 19, 225-232. Mandell, G.L., Coleman, E. (2001) Uptake, transport, and delivery of antimicrobial agents by human polymor phonuclear neutrophils. Antimicrobial Agents and Chemotherapy, 45, 1794-1798. Martin, S.J., Garvin, C.G., McBurney, C.R. & Sahloff, E.G. (2001) The activity of 14hydroxy clarithromycin, alone and in comb ination with clarithromycin, against penicillinand erythromycin-resistant Streptococcus pneumoniae Journal of Antimicrobial Chemotherapy, 47, 581-587. McKay, S.G., Morck, D.W., Merrill, J.K., Ol son, M.E., Chan, S.C. & Pap, K.M. (1996) Use of tilmicosin for treatment of pasteurellosis in rabbits. American Journal of Veterinary Research, 57, 1180-1184.

PAGE 69

60 Modric, S., Webb, A.I. & Davidson, M. (1999) Effect of respiratory tract disease on pharmacokinetics of tilmicosin in rats. Lab Animal Science 49, 248-253. Modric, S., Webb, A.I. & Derendorf, H. (1998) Pharmacokinetics and pharmacodynamics of tilmicosin in sheep and cattle. Journal of Veterinary Pharmacology and Therapeutics, 21, 444-452. Morck, D.W., Merrill, J.K., Gard, M.S., Ols on, M.E. & Nation, P.N. (1997) Treatment of experimentally induced pneumonic pasteure llosis of young calves with tilmicosin. Canadian Journal of Veterinary Research, 61, 187-192. Musser, J., Mechor, G.D., Grohn, Y.T., Dubovi, E.J. & Shin, S. (1996) Comparison of tilmicosin with long-acting oxytetracycline fo r treatment of respir atory tract disease in calves. Journal of the American Vete rinary Medical Association, 208, 102-106. National Committee for Clinical Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents; approved guidelines. [M26-A]. 1999a. Wayne, PA. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptib ility tests for bacteria isolated from animals;approved standards. [M31-A]. 1999b. Wayne, Pa. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for b acteria that grow aerobically; approved standard. 5th Edition. [M7-A5]. 2000. Wayne, Pa. Nerland, E.M., LeBlanc, J.M., Fedwick, J.P ., Morck, D.W., Merrill, J.K., Dick, P., Paradis, M.A. & Buret, A.G. (2005) Effect s of oral administration of tilmicosin on pulmonary inflammation in piglets experimentally infected with Actinobacillus Pleuropneumoniae. American Journal of Veterinary Research, 66, 100-107. Nordmann, P., Kerestedjian, J.J. & Ronco, E. (1992) Therapy of Rhodococcus equi disseminated infections in nude mice. Antimicrobial Agents Chemotherapy, 36, 1244-1248. Nordmann, P. & Ronco, E. (1992) In-vit ro antimicrobial susceptibility of Rhodococcus equi. Journal of Antimicrobial Chemotherapy, 29, 383-393. Odland, B.A., Erwin, M.E. & Jones, R.N. ( 2000) Quality control guidelines for disk diffusion and broth microdilution antimicrobial susceptibility tests with seven drugs for veterinary applications. Journal of Clinical Microbiology, 38, 453-455. Ose, E.E. & Tonkinson, L.V. (1988) Singledose treatment of ne onatal calf pneumonia with the new macrolide antibiotic tilmicosin. The Veterinary Record, 123, 367-369.

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61 Paradis, M.A., Vessie, G.H., Merrill, J.K ., Dick, C.P., Moore, C., Charbonneau, G., Gottschalk, M., MacInnes, J.I., Higgins, R., Mittal, K.R., Girard, C., Aramini, J.J. & Wilson, J.B. (2004) Efficacy of tilmicosin in the control of experimentally induced actinobacillus pleuropneumoniae infection in swine. Canadian Journal of Veterinary Research, 68, 7-11. Patel, K.B., Xuan, D., Tessier, P.R., Russo manno, J.H., Quintiliani, R. & Nightingale, C.H. (1996) Comparison of bronchopulmonary pharmacokinetics of clarithromycin and azithromycin. Antimicrobial Agents and Chemotherapy, 40, 2375-2379. Pillai, S.K., Moellering, R.C. & Eliopoulos, G. M. (2005) Antimicrobial combinations, in Antibiotics in Laboratory Medicine (Lorian V ed), 365-440, Lippincott Williams & Wilkins, Philadelphia. Prescott, J.F., Hoover, D.J. & Dohoo, I.R. (1983) Pharmacokinetics of erythromycin in foals and in adult horses. Journal of Veterinary P harmacology and Therapeutics, 6, 67-73. Prescott, J.F. (2000) Lincosamides, macrolides, and pleuromutilins. Antimicrobial Therapy in Veterinary Medicine (Prescott J.F., Baggot DJ and Walker RD eds), 229-262. Prescott, J.F. (1991) Rhodococcus equi : an animal and human pathogen. Clinical Microbiology, 4, 20-34. Prescott, J.F. & Nicholson, V.M. (1984) Th e effects of combinations of selected antibiotics on the growth of Corynebacterium equi Journal of Veterinary Pharmacology and Therapeutics, 7, 61-64. Rahman, M.T., Herron, L.L., Kapur, V., Meijer, W.G., Byrne, B.A., Ren, J., Nicholson, V.M. & Prescott, J.F. (2003) Partial genome sequencing of Rhodococcus equi ATCC 33701. Veterinary Microbiology, 94, 143-158. Ramadan, A. (1997) Pharmacokinetics of tilmicosin in serum and milk of goats. Research Veterinary Science, 62, 48-50. Ren, J. & Prescott, J.F. (2004) The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence. Veterinary Microbiology, 103, 219230. Rennard, S.I., Basset, G., Lecossier, D., O' Donnell, K.M., Pinkston, P., Martin, P.G. & Crystal, R.G. (1986) Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. Journal of Applied Physiology, 60, 532538. Retsema, J.A., Bergeron, J.M., Girard, D ., Milisen, W.B. & Girard, A.E. (1993) Preferential concentration of azithromyci n in an infected mouse thigh model. Journal of Antimicrobial Chemotherapy, 31, 5-16.

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62 Rodvold, K.A. (1999) Clinical pharm acokinetics of clarithromycin. Clinical Pharmacokinetics, 37, 385-398. Rodvold, K.A., Gotfried, M.H., Danziger, L.H. & Servi, R.J. (1997) Intrapulmonary steady-state concentrations of clarithrom ycin and azithromycin in healthy adult volunteers. Antimicrobial Agents Chemotherapy, 41, 1399-1402. Scorneaux, B. & Shryock, T.R. (1999) Intracellular accumu lation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells. Journal of Dairy Science, 82, 1202-1212. Scorneaux, B. & Shryock, T.R. (1998) Intrace llular accumulation, sub cellular distribution and efflux of tilmicosin in swine phagocytes. Journal of Veterinary Pharmacology Therapeutics 21, 257-268. Scott, P.R. (1994) Field study of undifferentia ted respiratory disease in housed beef calves. The Veterinary Record, 134, 325-327. Shryock, T.R., White, D.W., Staples, J.M. & Werner, C.S. (1996) Minimum inhibitory concentration breakpoints and disk diffusion i nhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella Spp. associated with bovine respiratory disease. Journal of Veterinary Diagnostic Investigation, 8, 337-344. Stratton-Phelps, M., Wilson, W.D. & Gardner, I.A. (2000) Risk of adverse effects in pneumonic foals treated with erythromyc in versus other antibiotics: 143 cases (1986-1996). Journal of the American Vete rinary Medica l Association, 217, 68-73. Sutcliffe, I.C. (1997) Macroamphiph ilic cell envelope components of Rhodococcus equi and closely related bacteria. Veterinary Microbiology, 56, 287-299. Sweeney, C.R., Sweeney, R.W. & Divers, T.J. (1987) Rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy. Veterinary Microbiology, 14, 329-336. Takai, S., Hines, S.A., Sekizaki, T., Nicholson, V.M., Alperin, D.A., Osaki, M., Takamatsu, D., Nakamura, M., Suzuki, K., Ogino, N., Kakuda, T., Dan, H. & Prescott, J.F. (2000) DNA sequence and co mparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infectious Immun ity, 68, 6840-6847. Tan, C., Prescott, J.F., Patterson, M.C. & Nicholson, V.M. (1995) Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity. Canadian Journal of Veterinary Research, 59, 51-59. Tessier, P.R., Kim, M.K., Zhou, W., Xuan, D., Li, C., Ye, M., Nightingale, C.H. & Nicolau, D.P. (2002) Pharmacodynamic asse ssment of clarithromy cin in a murine model of pneumococcal pneumonia. Antimicrobial Agents and Chemotherapy, 46, 1425-1434.

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63 Tilmicosin for swine feed package insert. Pu lmotil 90, Elanco Animal Health-USA. Rev 02-1995. Tilmicosin injection package insert. Micot il 300, Elanco animal Health-USA. Rev 101995. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F. (2004) In vitro susceptibilities of Mycoplasma hyopneumoniae field isolates. Antimicrobial Agents and Chemotherapy, 48, 4470-4472. Vilmanyi, E., Kung, K., Riond, J.L., Trumpi, B. & Wanner, M. (1996) Clarithromycin pharmacokinetics after oral administration with or without fasting in crossbred beagles. Journal of Small Animal Practice, 37, 535-539. Watts, J.L., Yancey, R.J., Jr., Salmon, S.A. & Case, C.A. (1994) A 4-Year survey of antimicrobial susceptibility trends for isolates from cattle with bovine respiratory disease in North America. Journal of Clinical Microbiology, 32, 725-731. Whitman, M.S. & Tunkel, A.R. (1992) Azithromycin and clarithromycin: overview and comparison with erythromycin. Infection Control and Ho spital Epidemiology, 13, 357-368. Wilson, W.D. (1992) Foal pneumonia: an overview. Proceedings from The American Association of Equine Practitioners, 38, 203-229. Wimsatt, J.H., Johnson, J., Mangone, B. A., Tothill, A., Childs, J.M. & Peloquin, C.A. (1999) Clarithromycin pharmacokinetics in the desert tortoise (Gopherus Agassizii). Journal of Zoo Wildlife Medicine, 30, 36-43. Zink, M.C., Yager, J.A., Prescott, J.F. & Fe rnando, M.A. (1987) El ectron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages. Veterinary Microbiology, 14, 295-305. Ziv, G., Shem-Tov, M., Glickman, A., Winkler M. & Saran, A. (1995) Tilmicosin antibacterial activity and pharmacokinetics in cows. Journal of Veterinary Pharmacology and Therapeutics, 18, 340-345.

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64 BIOGRAPHICAL SKETCH Ariel Womble was born in Charlotte, N.C., to David Womble and Connie Harris. She moved to Palm Harbor, Florida when sh e was eight years old where she lived and attended high school. While in high school she earned a scholarship to attend the University of Florida where she earned a Bach elor of Science in animal science. During her years as an undergraduate she worked part -time at the Veterinary Teaching Hospital where she met Dr. Steeve Gigure. Through this meeting she became interested in research and pursuing a Master of Science de gree in veterinary medical s cience. After graduation she began work in graduate studies.


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Title: Pharmacokinetics and Pulmonary Disposition of Clarithromycin and Tilmicosin in Foals
Physical Description: Mixed Material
Copyright Date: 2008

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PHARMACOKINETICS AND PULMONARY DISPOSITION OF
CLARITHROMYCIN AND TILMICOSIN IN FOALS.
















By

ARIEL Y. WOMBLE


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

UNIVERSITY OF FLORIDA


2006





























Copyright 2006

by

Ariel Y. Womble















ACKNOWLEDGMENTS

I would like to thank my family for their continuous support while I pursue my

goals in veterinary medicine. Their unwavering confidence in my abilities makes the

accomplishment of this thesis even more valuable to me. I also want to thank my

boyfriend Michael who pushed me to go further, never doubting that I would make it.

I especially want to thank Dr. Steeve Giguere for giving me this incredible

opportunity. He opened the door to an experience that has forever shaped me. I have

learned more than I imagined I would, not only about science and veterinary medicine

but about myself. I greatly appreciate the support, guidance, and mentorship that he

provided to me. I will be forever grateful.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ......... .................................................................................... iii

L IST O F TA B L E S ........ ................... ........ ................................. ............ vi

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. vii

A B S T R A C T .......................................... .................................................. v iii

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 L ITER A TU R E R E V IEW .................................................................. .....................3

F oal P neum onia ....................................................... 3
R hodococcus E qui ................. .................................... ...... ........ .......... .......
M acrolides ......................................................................... . 9
C larithrom ycin........... ............................. ........ .... .. ........ .... 10
Tilmicosin ...... ............... ........................... 13

3 PHARMACOKINETICS OF CLARITHROMYCIN AND CONCENTRATION
IN BODY FLUIDS AND BRONCHOALVEOLAR CELLS IN FOALS .................17

A b stra ct ............ ......... ... .. ......... ... .. ................................................... 17
In tro du ctio n .......................... .. ......... ... .. ................................................18
M materials and M ethods ....................................................................... .................. 19
H orses and Experim ental D esign .............................................. ............... 19
B ronchoalveolar L avage .............. .......... ....... ............ ..... ................................2 1
Drug Analysis by High Performance Liquid Chromatography (HPLC).............21
Measurement of Clarithromycin Activity Using a Microbiologic Assay ...........23
Estimation of PELF and BAL Cell Volumes and Determination of
Clarithromycin Concentrations in PELF and BAL Cells ................................24
Pharm acokinetic Analysis .........................................................................25
Statistical A naly sis ........................................ .. .. .... ........... 26
R e su lts .........................................................................................................................2 6
D isc u ssio n ............................................................................................................. 2 7









4 PULMONARY DISPOSITION OF TILMICOSIN IN FOALS AND IN VITRO
ACTIVITY AGAINST RHODOCOCCUS EQUI AND OTHER COMMON
EQUINE BACTERIAL PATHOGEN S ........................................ .....................36

A b stra c t ..........................................................................................3 6
In tro d u ctio n .......................................................................................3 7
M material and M methods ........................................................................ ....... ...........38
H orses and experim ental design................................... .................................... 38
Experimental design and sample collection ................................................39
B ronchoalveolar lavage .......................................... .... ................. ... ............ 39
Drug analysis...................................... .......................40
Estimation of PELF and BAL Cell Volumes and Determination of Tilmicosin
Concentrations in PELF and BAL Cells............................... ............... 40
Pharm acokinetic Analysis ............................................................................41
Statistical A analysis ............... ................ .. .. ...................... .... .... .......... .. 42
Determination of minimum inhibitory concentration (MIC) and minimum
bactericidal concentrations (MBC) of tilmicosin against R. equi ..................42
Checkerboard assay ............................................. .. ....... .. ........ .... 43
T im e kill curve assay ................. ................... ...... .................. ......... .......... 43
In vitro activity of tilmicosin against equine bacterial pathogens.....................44
Results ......................... ............. ...... ...... ... .. ......................... 45
Serum and pulmonary disposition of tilmicosin in foals................................45
In vitro susceptibility testing and antimicrobial drug combinations .................45
D isc u ssio n ................................................... ................... ................ 4 6

5 SUMMARY AND CONCLUSIONS............................... ................ ..............52

LIST OF REFEREN CES ........................................... ...... ..................... ............... 55

B IO G R A PH IC A L SK E TCH ..................................................................... ..................64






















v















LIST OF TABLES


Table p

1.1 MIC90 ([tg/mL) of azithromycin, clarithromycin, and erythromycin against
common equine bacterial pathogens. ............................................ ............... 16

3.1 Pharmacokinetic variables (mean SD unless otherwise specified) for
clarithromycin after IV or intragastric administration to 6 foals at dose of 7.5
m g/kg of body w eight. ..................... ................ .............................33

3.2 Mean SD clarithromycin activity in body fluids and BAL cells of six foals
after 6 intragastric administrations (7.5 mg /kg every 12 hours). ..........................33

4.1 Serum and pulmonary pharmacokinetic variables (mean SD unless otherwise
specified) for tilmicosin after IM administration to seven foals at a dose of 10
m g/kg of body w eight. .................................................................... ...................50

4.2 Tilmicosin in vitro susceptibility of 183 bacterial isolates obtained from horses....50















LIST OF FIGURES


Figure page

1.1 Classification of macrolides according to the number of atoms comprising the
lactone ring ............... ........ .............................................................. ...... 16

3.1 Mean (+ SD) serum clarithromycin concentration as measured by HPLC method
or microbiologic assay in 6 foals administered a single IV dose of 7.5 mg/kg. ......34

3.2 Mean (+ SD) serum clarithromycin activity ([tg/ml) in 6 foals following
intragastric clarithromycin (7.5 mg/kg) administration at 0, 24, 36, 48, 60, and
72 hours. Results are based on measurements with the microbiologic assay........35

4.1 Mean SD tilmicosin concentrations in serum, BAL cells, PELF ([tg/mL), and
lung tissue ([tg/g) of 7 foals following a single IM dose of tilmicosin (10 mg/kg
of body w eight). .................................................... ................. 5 1

4.2 Effect of time and tilmicosin concentration on in vitro survival of a clinical
isolate of R. equi. Identical results were obtained with 2 additional isolates...........51















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

PHARMACOKINETICS AND PULMONARY DISPOSITION OF
CLARITHROMYCIN AND TILMICOSIN IN FOALS.

By

Ariel Y. Womble

August 2006
Chair: Steeve Giguere
Major Department: Veterinary Medicine

Bronchopneumonia is the leading cause of morbidity and mortality in foals aged

between 1 and 6 months. Gram-positive bacteria such as Streptococcus equi subspecies

zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in

foals. Erythromycin, a macrolide antimicrobial agent, is commonly used in equine

medicine for treatment of foal pneumonia, especially in foals infected with Rhodococcus

equi. Two other macrolides, clarithromycin and tilmicosin, may be useful alternatives to

currently used antimicrobial agents owing to their accumulation in lung tissue and

phagocytic cells, as well as their broad spectrum in vitro activity. The objectives of this

study were to determine the pharmacokinetics and pulmonary distribution of

clarithromycin and tilmicosin in foals, and to investigate the in vitro activity of tilmicosin

against common bacterial pathogens of horses. Clarithromycin (7.5 mg/kg) was

administered to six foals via intravenous (IV) and intragastric (IG) routes, in a cross-over

design. Concentrations of clarithromycin and its 14-hydroxy-metabolite in serum were









measured by HPLC. A microbiologic assay was used to measure clarithromycin activity

in serum, urine, peritoneal fluid, synovial fluid, cerebrospinal (CSF), pulmonary

epithelial lining fluid pelfF), and bronchoalveolar (BAL) cells. Following IV

administration, clarithromycin had a t/2 of 5.4 hours, a body clearance of 1.27 L/h/kg,

and an apparent volume of distribution at steady state of 10.4 + 2.1 L/kg. Oral

bioavailability of clarithromycin was 57.3 12.0 %. In a separate study, a single dose of

a fatty acid salt formulation of tilmicosin (10 mg/kg) was administered by the

intramuscular route to 7 healthy 5- to 8-week-old foals. Concentrations of tilmicosin in

serum were measured by HPLC and concentrations in lung tissue, PELF, and BAL cells

were measured by mass spectrometry. Mean peak tilmicosin concentrations were

significantly higher in BAL cells (20.1 5.1 Dg/mL) than in lung tissue (1.90 + 0.65

Sg/mL), PELF (2.91 + 1.15 1g/mL), and serum (0.19 0.09 1g/mL). Harmonic mean

elimination half life in lung tissue (193.3 h) was significantly longer than that of serum

(18.4 h). Elimination half lives in BAL cells and PELF were 62.2 h and 73.3 h,

respectively. Tilmicosin was active in vitro against most streptococci, Staphylococcus

spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against

Rhodococcus equi, Pseudomonas spp., and Enterobacteraceae. In conclusion, oral

administration of clarithromycin at a dosage of 7.5 mg/kg every 12 hours would maintain

serum, PELF, and BAL cell concentrations above the minimum inhibitory concentration

for R. equi and S. zooepidemicus isolates for the entire dosing interval. The formulation

of tilmicosin investigated in the present study resulted in high and sustained

concentrations in the lung, PELF, and BAL cells of foals and may be appropriate for the

treatment of susceptible bacterial infections.














CHAPTER 1
INTRODUCTION

Bacterial pneumonia is the leading cause of morbidity and mortality in foals aged

between 1 and 6 months. Gram-positive bacteria such as Streptococcus equi subspecies

zooepidemicus and Rhodococcus equi are the most common causes of pneumonia in

foals. Gram-negative bacteria such as Pasteurella spp., Actinobacillus spp., Escherichia

coli, and Klebsiellapneumoniae may also occasionally be cultured from tracheobronchial

aspirates of affected foals. Administration of antimicrobial agents is the most important

part of the therapeutic plan. When R. equi is suspected or confirmed, therapy has

historically consisted of administration of the macrolide erythromycin in combination

with rifampin. This combination has dramatically reduced foal mortality since its

introduction. However, this treatment regimen is not without problems. Erythromycin

has poor and variable oral bioavailability in foals, requires multiple daily dosing, and

most importantly, has a high incidence of potentially fatal adverse effects. Therefore,

there is a tremendous need for other effective and potentially safer antimicrobial agents to

combat infection caused by this devastating pathogen. Two other macrolides,

clarithromycin and tilmicosin, may be useful alternatives to currently used antimicrobial

agents.

The documented pharmacokinetic advantages of clarithromycin over erythromycin

in humans include higher oral bioavailability, longer elimination half-life, larger volume

of distribution, and improved tissue and phagocytic cell uptake. Tilmicosin may also be a

useful alternative to the current antimicrobial agents used in horses owing to its









accumulation in lung tissue and phagocytic cells, as well as in vitro activity against many

Gram-positive and Gram-negative bacterial species. In addition, availability of a long

acting antimicrobial agent such as tilmicosin would result in less frequent administration,

which in turn may improve client compliance. The overall goal of the work presented in

this thesis is to determine the pharmacokinetics and pulmonary distribution of

clarithromycin and tilmicosin in foals. This thesis includes two studies.

The objectives and hypotheses of the first study (Chapter 3) are:

1- To determine the pharmacokinetics of clarithromycin and its metabolite in
foals.
Our liypolthe'i is that oral clarithromycin is well absorbed in foals and is
metabolized to 14-hydroxy clarithromycin.
2- To determine concentrations of clarithromycin in body fluids and
bronchoalveolar cells.
Our hypothesis is that oral clarithromycin provides serum and pulmonary drug
concentrations above the minimum inhibitory concentration of R. equi.
The objectives and hypotheses of the second study (Chapter 4) are:

1- To determine the pulmonary disposition of tilmicosin in foals.
Our hypothesis is that a new fatty acid salt formulation of tilmicosin provides
high and sustained concentrations in the lungs of foals.
2- To investigate the in vitro activity of tilmicosin against R. equi and other
common bacterial pathogens of horses.
Our hypothesis is that tilmicosin is active in vitro against common equine
bacterial pathogens of the respiratory tract.














CHAPTER 2
LITERATURE REVIEW

Foal Pneumonia

Lower respiratory tract infection is the leading cause of both morbidity and

mortality in foals aged between 1 and 6 months (Cohen, 1994). The morbidity rate is

approximately 6% across the United States. It is likely, however, that the true incidence

of infection is much higher and that many cases of infection go unrecognized and resolve

spontaneously. Indeed, careful weekly physical examination and cytologic examination

of the lower respiratory tract in more than 200 Thoroughbred foals on 10 farms in

Ontario, Canada demonstrated an average morbidity from bacterial infection of the distal

respiratory tract of 82% (Hoffman et al., 1993a). Increased susceptibility to disease in

this age group may result from delay in the establishment of a competent immune system

and environmental factors such as overcrowding, shipping, and sales (Wilson, 1992). The

disease may be subclinical initially; however, as the infection progresses, clinical signs

may include depression, inappetence, coughing, nasal discharge, and tachypnea. Fever is

a common finding as well. Severely affected foals may develop respiratory distress.

Common laboratory abnormalities in foals with bacterial pneumonia include

leukocytosis, hyperfibrinogenemia, and hyperglobulinemia (Barr, 2003). Mild anemia

may develop in chronic cases. Radiography and ultrasonographic examination of the

thorax are useful diagnostic imaging tools to detect and assess the severity of lung

lesions. Culture of a tracheobronchial aspirate is necessary to determine the causative

microorganism. The vast majority of cases of foal pneumonia are bacterial in origin.









Viral agents such as influenza, equine herpesvirus-1 (EHV-1), EHV-2, EHV-4,

rhinovirus, and adenovirus may cause primary lung disease or predispose to secondary

bacterial pneumonia (Wilson, 1992). However, in most cases of foal pneumonia, viral

agents cannot be isolated at the time of presentation (Hoffman et al., 1993b). Most

bacteria associated with pneumonia are ubiquitous in the foal's environment. The

pathophysiology of bacterial pneumonia begins with either inhalation of environmental

microbes or aspiration of oropharygeal bacteria. The bacteria become pathogenic only

when the pulmonary defense mechanisms are compromised or are overwhelmed by a

large number of bacteria (Wilson, 1992). The inflammatory response induced by

bacterial invasion will result in infiltration with neutrophils and other inflammatory cells

into the airways and pulmonary parenchyma. Inflammatory cells and their mediators

cause damage to the airway epithelium and capillary endothelium, leading to flooding of

the terminal airways with inflammatory cells, serum cellular debris and fibrin. This

process is generally more severe in the cranioventral portions of the lung. These lesions

interfere with gas exchange and, if severe enough, the resulting ventilation-perfusion

mismatch leads to hypoxemia and clinical signs of respiratory disease.

Gram-positive bacteria such as Streptococcus equi subsp. zooepidemicus and

Rhodococcus equi are the most common causes of pneumonia in foals (Hoffman et al.,

1993b; Giguere et al., 2002; Barr, 2003). Gram-negative bacteria such as Pasteurella

spp., Actinobacillus spp., Escherichia coli, Klebsiella pneumoniae, Salmonella enterica,

and Bordetella bronchiseptica may also be cultured from tracheobronchial aspirates of

affected foals. Mixed bacterial infections are common as well (Wilson, 1992; Hoffman et

al., 1993b). Administration of antimicrobial agents is the most important part of the









therapeutic plan. The choice of the antimicrobial agent depends on the results of culture

and susceptibility testing of tracheobronchial aspirates, severity of the clinical signs, cost,

ease of administration, and history of response to therapy within the herd. Since a high

percentage of pneumonia in foals older than 1 month is due to penicillin-sensitive

bacteria such as S. equi subsp. zooepidemicus, penicillin is often used for initial therapy,

pending culture results. Ceftiofur, a third generation cephalosporin, has a broad spectrum

of activity that includes most of the etiologic agents of foal pneumonia, except R. equi. If

resistant Gram-negative organisms are present, an aminoglycoside (gentamicin or

amikacin) is often combined with penicillin (Wilson, 1992). When R. equi is suspected or

confirmed, therapy consists of administration of a macrolide in combination with

rifampin. It is common practice to use a combination of a macrolide and rifampin as the

first line of therapy on farms where R. equi is endemic as this combination is also active

against streptococci.

Rhodococcus Equi

R. equi is a facultative intracellular pathogen that has the ability to survive and even

replicate within macrophages (Zink et al., 1987). R. equi is closely related to

Mycobacterium tuberculosis. Both R. equi and M. tuberculosis are members of a

phyogenetically distinct group called Mycolata, which are characterized by a unique cell

envelope that consists of mycolic acids (Sutcliffe, 1997). This unique envelope forms a

permeability barrier to hydrophilic compounds and promotes granuloma formation so the

organism is able to multiply in and destroy macrophages (Sutcliffe, 1997). The similarity

between R. equi and M. tuberculosis is further emphasized by the degree of homology of

their genome sequence (Rahman et al., 2003).









The most common manifestation ofR. equi infections in foals is a chronic

suppurative bronchopneumonia with extensive abscessation and associated suppurative

lymphadenitis. Other, less-common clinical manifestations of R. equi infections in foals

include ulcerative enterocolitis, colonic or mesenteric lymphadenopathy, immune-

mediated synovitis and uveitis, osteomyelitis, and septic arthritis (Giguere and Prescott,

1997). R. equi has also been increasingly recognized as an important cause of pneumonia

in immunosuppressed people, especially those infected with HIV. R. equi may also cause

disease in other animal species such as cattle, sheep, goats, dogs, and cats; however

infection is rare and usually associated with immunosuppresion (Prescott, 1991). The

reasons for the peculiar susceptibility of young foals are not entirely clear.

R. equi is a saprophytic inhabitant of soil. Although all horse farms are infected to

various degrees with R. equi, the clinical disease is enzootic and devastating on some

farms, sporadic on others, and unrecognized on most farms. On farms where the disease

is enzootic, costs associated with veterinary care, early diagnosis, long-term therapy, and

mortality of foals may be very high. In addition to significant immediate costs, R. equi

pneumonia has a long-term detrimental effect on the equine industry because foals that

recover from the disease are less likely to race as adults (Ainsworth et al., 1998).

The ability ofR. equi to induce disease in foals likely depends on both host and

microbial factors. The key to the pathogenesis ofR. equi is its ability to replicate within

pulmonary macrophages apparently by inhibiting phagosome-lysosome fusion (Zink et

al., 1987). Unlike most environmental R. equi, isolates from pneumonic foals typically

contain 80-90 kb plasmids. The plasmid encodes seven related virulence-associated

proteins designated VapA and VapC through VapH (Takai et al., 2000). Plasmid-cured









derivatives of virulent R. equi strains lose their ability to replicate and survive in

macrophages (Giguere et al., 1999). Plasmid-cured derivatives also fail to induce

pneumonia and are completely cleared from the lungs of foals two weeks following

heavy intrabronchial challenge, confirming the absolute necessity of the large plasmid for

the virulence of R. equi (Giguere et al., 1999). Vap A is highly immunogenic, lipid-

modified protein expressed on the surface of R. equi (Tan et al., 1995). An R. equi mutant

lacking a 7.9 kb DNA region spanning 5 vap genes (vapA, -C, -D, -E, -F) was attenuated

for virulence in mice and failed to replicate in macrophages (Jain et al., 2003).

Complementation with vapA alone could restore full virulence, whereas complementation

with vapC, vapD or vapE could not (Jain et al., 2003). More recently, attenuation of 2

other plasmid-encoded genes was also found to decrease virulence in mice despite

enhanced transcription of vapA (Ren and Prescott, 2004). These findings suggest that

other plasmid-encoded genes besides vapA contribute to the virulence of R. equi.

Inhalation of virulent R. equi is the major route of pneumonic infection. Ingestion

of the organism is a significant route of exposure, and likely also of immunization, but

rarely leads to hematogenously acquired pneumonia unless the foal has multiple

exposures to large numbers of bacteria (Johnson et al., 1983). A majority of foals may

be exposed by ingesting the bacterium; however, they probably develop a strong immune

response and are protected against subsequent intrabronchial challenge (Hooper-

McGrevy et al., 2005).

Control of R. equi infections on farms where the disease is enzootic is difficult.

Attempts at actively immunizing foals against R. equi infections have consistently failed.

Intravenous administration of hyperimmune plasma obtained from horses vaccinated









against R. equi has given contradictory results. Therefore, screening strategies promoting

early recognition of R. equi cases with treatment of infected foals will reduce losses,

decrease the spread of virulent organisms and limit the cost of therapy on farms where

the disease is endemic.

A wide variety of antimicrobial agents are effective against R. equi in vitro (Jacks

et al., 2003). However, many of these drugs are ineffective in vivo. The discrepancy in

results is likely due to the intracellular nature of this bacterium and the fact that it causes

abscesses where diffusion and activity of many antimicrobial agents is not optimal. In

one study, all 17 foals with R. equi pneumonia treated with the combination of penicillin

and gentamicin died despite all isolates being susceptible to gentamicin in vitro (Sweeney

et al., 1987). In the mid 1980s, the combination of erythromycin and rifampin became

the treatment of choice (Hillidge, 1987). The combination of erythromycin and rifampin

has become the prevalent treatment for R. equi infections in foals and has dramatically

reduced foal mortality since its introduction (Hillidge, 1987; Sweeney et al., 1987).

Although both erythromycin and rifampin are bacteriostatic against R. equi (Nordmann

and Ronco, 1992), they are highly effective in vitro. The combination of these two

antimicrobials is synergistic as well, both in vitro and in vivo and when used in

combination reduces the likelihood of resistance to either drug (Prescott and Nicholson

1984; Nordmann et al., 1992; Nordmann and Ronco, 1992). Rifampin and, to a lesser

extent, erythromycin are lipid soluble, allowing them to penetrate caseous material.

Although combined therapy with erythromycin and rifampin has dramatically improved

the survival rate of foals infected with R. equi, this treatment regimen is not without

problems. Erythromycin has poor and variable oral bioavailability in foals, requires









multiple daily dosing, and most importantly, has a high incidence of potentially fatal

adverse effects (Lakritz et al., 2000a; Lakritz et al., 2000b; Stratton-Phelps et al., 2000).

The use of erythromycin to treat foals with pneumonia results in an increased risk of

diarrhea, hyperthermia, and respiratory distress compared with pneumonic foals treated

with either penicillin or trimethoprim sulfa (Stratton-Phelps et al., 2000). Clostridium

difficile enterocolitis has also been observed occasionally in the dams of nursing foals

while the foals are being treated with oral erythromycin presumably because of sufficient

active erythromycin to perturb the intestinal flora (Baverud et al., 1998).

Macrolides

Macrolide antimicrobial agents are chemically comprised of a lactone ring with 14

to 16 carbons attached to 2 sugar moieties. Macrolide antimicrobials are typically

classified according to the size of their macrocyclic lactone ring (Figure 1.1). Macrolides

inhibit protein synthesis by reversibly binding to 50S subunits of the ribosome. Their

binding sites on the 23S rRNA of the 50S ribosomal subunit overlap with that of

clindamycin but are different from those of chloramphenicol (Prescott, 2000).

Macrolides are generally bacteriostatic agents. They may be bactericidal at high

concentrations and against a low inoculum of highly susceptible bacteria. Their spectrum

of activity includes mostly Gram-positive microorganisms, most Mycoplasma spp., some

Chalmydiae as well as some Gram-negative pathogens such as Haemophilus influenza,

Campylobacterjejuni, Bordetella spp., and Mannheimia haemolytica (Alvarez-Elcoro

and Enzler, 1999). This class of antimicrobials has been important in the treatment of

respiratory tract, skin, and soft tissue infections as well as venereal disease in humans.

Some macrolides (tilmicosin, tulathromycin) are also approved for the treatment of

bronchopneumonia in cattle.









Macrolide antimicrobial agents other than erythromycin may provide a suitable

alternative for the treatment ofRhodococcus equi infections in foals. Azithromycin and

clarithromycin are used with increasing frequency in human medicine. Compared with

erythromycin, azithromycin and clarithromycin have a higher oral bioavailability, longer

elimination half lives, larger volumes of distribution, as well as improved tissue and

phagocytic cell uptakes (Whitman and Tunkel, 1992; Conte et al., 1995; Rodvold, 1999).

In humans, the incidence and the severity of adverse reactions for azithromycin and

clarithromycin are also considerably decreased compared with erythromycin (Whitman

and Tunkel, 1992). Another macrolide, tilmicosin, is not currently approved for use in

horses but may provide a suitable alternative for the treatment ofR. equi. The

pharmacokinetics and pulmonary distribution of azithromycin have been studied

extensively in foals (Jacks et al., 2001; Davis et al., 2002). However, there is no

information on the pulmonary distribution of clarithromycin and tilmicosin in foals.

Clarithromycin

Clarithromycin is a semi-synthetic antimicrobial agent that is derived from

erythromycin. Clarithromycin has an O-methyl ether substitution instead of the C-6

hydroxyl group of erythromycin at position 6 of the macrolide ring (Rodvold, 1999).

This modification provides greater stability than erythromycin in gastric content, thus

improving oral bioavailability. The documented pharmacokinetic advantages of

clarithromycin over erythromycin in humans include higher oral bioavailability, longer

elimination half-life, larger volume of distribution, and improved tissue and phagocytic

cell uptake (Conte et al., 1995; Rodvold, 1999).

Clarithromycin undergoes hepatic metabolism as well as elimination by secretion

into the intestinal lumen. In humans, clarithromycin is metabolized in the liver by









cytochrome P-450 enzymes to the active metabolite 14-hydroxy-clarithromycin (Ferrero

et al., 1990). This active metabolite contributes approximately 50% of the biological

activity of clarithromycin and has synergistic effects with clarithromycin (Fernandes et

al., 1988). Other species including rats, mice or desert tortoise do not produce the 14-

hydroxy metabolite (Ferrero et al., 1990; Bedos et al., 1992; Wimsatt et al., 1999).

Microorganisms with MIC < 2 .g/ml are generally regarded as susceptible and > 8

.g/ml as resistant to clarithromycin and erythromycin. The efficacy of clarithromycin in

vitro is greatest against the aerobic and facultative anaerobic non-spore-forming, Gram-

positive bacteria. It also has in vitro activity against several microorganisms such as,

Mycoplasma spp., Chlamydia spp., and some Mycobacteria. The lack of activity against

most Gram-negative bacteria is likely due to its inability to penetrate the bacterial cell

wall. Clarithromycin is active against many Gram-positive bacterial pathogens of horses

(Jacks et al., 2003). Of the macrolides tested so far, clarithromycin has the greatest in

vitro activity against R. equi isolates cultured from pneumonic foals (Table 1.1).

Approved indications for the use of clarithromycin in people include the treatment

of: pharyngitis/tonsillitis due to Streptococcus pyogenes; acute maxillary sinusitis due to

Haemophilus influenzae, Moraxella catarrhalis, or Streptococcus pneumoniae;

community acquired pneumonia or bronchitis due to Haemophilus influenzae,

Mycoplasma pneumoniae, Streptococcus pneumoniae, Moraxella catarrhalis, or

Chlamydiapneumoniae; uncomplicated skin infections due to Staphylococcus aureus, or

Streptococcus pyogenes; and disseminated mycobacterial infections due to

Mycobacterium avium, or Mycobacterium intracellulare. Finally, clarithromycin









combined with amoxicillin and a proton pump inhibitor is also approved for the treatment

of gastric and duodenal ulceration caused by Helicobacterpylori.

Clarithromycin is a macrolide antimicrobial agent which achieves low plasma

concentrations relative to the minimum inhibitory concentration (MIC) of the pathogens

it is used to treat. The physiochemical properties of clarithromycin, including its

lipophilicity indicate that the drug concentrations at a peripheral site would be greater

than concurrent serum concentrations (Drusano, 2005). The disposition of clarithromycin

in pulmonary epithelial lining fluid (PELF) and alveolar macrophages (AM) has been

investigated extensively in healthy human volunteers. In humans, clarithromycin

achieves considerably greater concentrations in pulmonary epithelial lining fluid and

alveolar macrophages than either erythromycin or azithromycin (Conte et al., 1995;Conte

et al., 1996; Patel et al., 1996; Rodvold et al., 1997). However, the half-life of

clarithromycin at these sites is much shorter than that of azithromycin.

A recent preliminary study confirmed that therapeutic concentrations are achieved

in serum following oral administration of clarithromycin to foals (Jacks et al., 2002). In a

retrospective study of foals presented to a referral institution, the combination of

clarithromycin-rifampin was found to be superior to azithromycin-rifampin or

erythromycin-rifampin for the treatment of pneumonia caused by R. equi (Giguere et al.,

2004). However, concentrations of the drug in body fluids, pulmonary epithelial lining

fluid (PELF) and bronchoalveolar (BAL) cells have not been measured. Recent studies

demonstrate that the concentration of macrolides at the site of infection may be a better

indicator of clinical efficacy than serum concentrations alone (Drusano, 2005).









Tilmicosin

Tilmicosin is a semi-synthetic derivative of tylosin. Typical of macrolides, it

inhibits Gram-positive bacteria including Clostridium spp., Staphylococcus spp., and

Streptococcus spp., some Gram-negative bacteria including Actinobacillus spp.,

Campylobacter spp., Histophilus spp., and Mannheimia/Pasteurella spp. (Prescott, 2000).

All Enterobacteriaceae are resistant to tilmicosin. Mycoplasma susceptibility can be quite

variable because of resistance (Vicca et al., 2004). Mannheimia/Pasteurella spp. isolated

from cattle are regarded as susceptible if their MIC is < 8 jig/ml, intermediate if MIC is

16 ig/ml, and resistant if their MIC is > 32 tg/ml (Shryock et al., 1996).

The pharmacokinetic properties of tilmicosin are similar to that of macrolides in

general, and are characterized by low serum concentrations but large volumes of

distribution (> 2 L/kg), with accumulation and persistence in tissues including the lung,

which may concentrate drug 20-60 fold compared to serum (Ziv et al., 1995; Clark et al.,

2004). Intracellular concentrations have been shown to be 40 times greater than that of

serum (Ziv et al., 1995; Scorneaux and Shryock, 1999).

Tilmicosin has been developed as a long-acting formulation for use in bovine and

ovine respiratory disease. A single SC dose of 10 mg/kg results in lung concentrations

exceeding the MIC ofM haemolytica for 72 hours (Ziv et al., 1995; Scorneaux and

Shryock, 1999). Experimental and field data support the value of a single-dose SC

prophylaxis on arrival of cattle in feedlots and in the treatment in pneumonia of cattle

(Ose and Tonkinson, 1988; Musser et al., 1996; Morck et al., 1997; Hoar et al., 1998).

Repeat injections after three days are necessary in some animals (Laven and Andrews,

1991; Scott, 1994). Tilmicosin is not approved for use in lactating cattle because of the

prolonged period (two to three weeks) during which milk residues can be detected.









Intramammary tilmicosin at drying-off have been shown to be efficacious in curing some

existing S. aureus infection (Dingwell et al., 2003).

Tilmicosin is also approved as an oral medication for the control ofActinobacillus

spp. or P. multocida pneumonia in swine (Paradis et al., 2004). It may also be useful in

the control of atrophic rhinitis. In-feed, treatment with 400 ppm of tilmicosin phosphate

significantly reduced the presence of A. pleuropneumoniae on the surface of tonsils but

was unable to completely eliminate the organism from deeper tonsillar tissues and to

prevent bacterial shedding by carrier animals (Fittipaldi et al., 2005).

Macrolides have immunomodulatory effects that are beneficial for humans

suffering from many inflammatory pulmonary diseases. These effects are independent of

the antibacterial activity of these drugs. Neutrophils play an important role in the

destruction and elimination of bacterial invaders. However, they also release lipid

mediators such as leukotriene B4 (LTB4) which induce a local inflammatory response.

Lesions of the lung contain viable as well as necrotic neutrophils which add to the tissue

damage caused by invading microorganisms. Apoptotic cell death is less damaging

because the cells maintain cellular membranes, preventing further release of damaging

LTB4 (Nerland et al., 2005). Tilmicosin has been shown to induce apoptosis and reduce

LTB4 as well as prostaglandin E2 concentrations in pulmonary fluid of cattle and swine

with pneumonia (Lakritz et al., 2002; Nerland et al., 2005). These anti-inflammatory

effects may contribute to the therapeutic efficacy of tilmicosin.

Tilmicosin is potentially toxic to the cardiovascular system, which varies to some

extent with species. According to the product insert, the drug is fatal to swine when

administered by IM injection at doses ranging between 10-20 mg/kg. The toxic dose for









goats is only about 30 mg/kg SC. The toxic effects of tilmicosin are mediated through its

effects on the heart, possibly by causing rapid depletion of calcium (Main et al., 1996).

There are no published reports on the safety of tilmicosin in horses. The product insert

suggests that the currently available tilmicosin formulation may be fatal in the equine

species. This formulation is also toxic when used in cats. A tilmicosin-fatty acid salt has

been developed as a safe and convenient formulation for cats (Kordick et al., 2003).

Tilmicosin may be a useful alternative to the current antimicrobial agents used in horses

owing to its accumulation in lung tissue and phagocytic cells, as well as in vitro activity

against many Gram-positive and Gram-negative bacterial species. In addition,

availability of a long acting antimicrobial agent providing sustained therapeutic

concentrations at the site of infection would result in less frequent administration, which

in turn may improve client compliance.






16


Table 1.1 MIC90 (tlg/mL) of azithromycin, clarithromycin, and erythromycin against
common equine bacterial pathogens.
Organism (n) Azithromycin Clarithromycin Erythromycin
Rhodococcus equi (60) 1.0 0.12 0.25
Streptococci (45) <0.12 <0.06 <0.25
Staphylococcus spp. (18) 0.5 0.25 0.25
Pasteurella spp. (10) 0.25 1.0 1.0
Klebsiella spp. (9) >8.0 >4.0 >4.0
Escherichia coli (16) >8.0 >4.0 >4.0
Salmonella enterica (11) 4.0 >4.0 >4.0
Adapted from Jacks and Giguere 2003











Macrolide antibiotics
I


nbered


Id g
14-membe Ied ring


id ring


I
16-membered ring


:ural Semisynth

1 1


Figure 1.1 Classification of macrolides according to the number of atoms comprising
the lactone ring.














CHAPTER 3
PHARMACOKINETICS OF CLARITHROMYCIN AND CONCENTRATION IN
BODY FLUIDS AND BRONCHOALVEOLAR CELLS IN FOALS

Abstract

The objective of this research was to determine pharmacokinetics of clarithromycin

and the concentrations achieved in body fluids and bronchoalveolar cells in foals. Six

healthy 2- to 3-week-old foals were used in this project. Clarithromycin (7.5 mg/kg of

body weight) was administered to each foal via intravenous (IV) and intragastric (IG)

routes, in a cross-over design. After the first IG dose, 5 additional doses were

administered at 12-hour intervals. Concentrations of clarithromycin and its 14-hydroxy

metabolite in serum were measured by HPLC. A microbiologic assay was used to

measure clarithromycin activity in serum, urine, peritoneal fluid, synovial fluid,

cerebrospinal (CSF), pulmonary epithelial lining fluid pelfF), and bronchoalveolar

(BAL) cells. Following IV administration, clarithromycin had a ti/ of 5.4 hours, a body

clearance of 1.27 L/h/kg, and an apparent volume of distribution at steady state of 10.4 +

2.1 L/kg. Detection of 14-hydroxy-clarithromycin was achieved in all 6 foals by 1 h

post-administration. Oral bioavailability of clarithromycin was 57.3 12.0 %. Peak

serum clarithromycin concentration following multiple IG administration was 0.88 + 0.19

[tg/mL. After multiple IG doses, peritoneal fluid, CSF, and synovial fluid clarithromycin

concentrations were similar to or lower than serum concentrations whereas urine, PELF,

and BAL cell concentrations were significantly higher than concurrent serum

concentrations. Oral administration at a dosage of 7.5 mg/kg every 12 hours would









maintain serum, PELF, and BAL cell concentrations above the minimum inhibitory

concentrations ofRhodococcus equi isolates for the entire dosing interval.

Introduction

Clarithromycin is a semi-synthetic macrolide antimicrobial agent chemically

derived from erythromycin A. It differs from erythromycin A by having an O-methyl

ether substitution at position 6 of the macrolide ring. This change provides greater

stability in gastric acid resulting in enhanced absorption by the oral route. This structural

difference also results in a longer elimination half life, a larger volume of distribution, as

well as improved tissue and phagocytic cell uptake compared to erythromycin (Conte et

al., 1995; Rodvold, 1999). Clarithromycin undergoes extensive hepatic metabolism in

people and is primarily metabolized to 14-hydroxy-clarithromycin (Ferrero et al., 1990).

This metabolite is responsible for approximately 50% of the total biological activity of

clarithromycin and has an additive or synergistic effect with the parent compound

(Fernandes et al., 1988; Martin et al., 2001).

Macrolide antimicrobial agents in combination with rifampin are commonly used

in equine medicine for treatment of Rhodococcus equi infections in foals. R. equi, a

Gram-positive facultative intracellular pathogen surviving in macrophages, is a common

cause of pneumonia in foals between 3 weeks and 5 months of age. Combined therapy

with erythromycin and rifampin has dramatically improved the historical survival rate of

affected foals (Hillidge, 1987). However, recent evidences indicate that clarithromycin

may be superior to erythromycin for the treatment ofR. equi pneumonia in foals.

Clarithromycin is more active against R. equi in vitro than either erythromycin or

azithromycin (Jacks et al., 2003). In addition, in a retrospective study of foals presented

to a referral institution, the combination of clarithromycin-rifampin was found to be









superior to azithromycin-rifampin or erythromycin-rifampin for the treatment of

pneumonia caused by R. equi (Giguere et al., 2004).

A recent preliminary study confirmed that therapeutic concentrations are achieved

in serum following oral administration of clarithromycin to foals (Jacks et al., 2002).

However, a single oral dose was given precluding accurate determination of steady state

drug concentrations and calculation of important pharmacokinetic parameters such as oral

bioavailability, clearance, and apparent volume of distribution. In addition,

concentrations of the drug in body fluids, pulmonary epithelial lining fluid and

bronchoalveolar (BAL) cells were not measured. Recent studies demonstrate that the

concentration of macrolides at the site of infection may be a better indicator of clinical

efficacy than serum concentrations alone (Drusano, 2005). Finally, the methodology

used to measure drug concentration in the preliminary study did not allow detection of

metabolites such as 14-hydroxy-clarithromycin.

The objectives of the present study were to determine the pharmacokinetics and

oral bioavailability of clarithromycin in foals as well as to measure drug concentrations in

body fluids and BAL cells after a multi-dose intragastric (IG) regimen. An additional

objective was to determine if clarithromycin is converted to the 14-hydroxy metabolite in

foals.

Materials and Methods

Horses and Experimental Design

Four male and two female foals (5 Thoroughbred and 1 Quarter Horse) between 2

and 3 weeks of age and weighing between 71 and 100 kg were selected for use in the

study. The foals were considered healthy on the basis of history, physical examination,

complete blood count and plasma biochemical profile. The foals were kept with their









dams in individual stalls during the experiment with ad libitum access to grass hay and

water.

Clarithromycin was administered at a dose of 7.5 mg/kg of body weight via the IV

and the IG routes, using a cross-over design. For the IV study, purified clarithromycin

powder (Courtesy of Franks Pharmacy, Ocala, FL, USA) was dissolved in sterile water

(100mg/ml) and administered as a single bolus through a catheter placed into the left

jugular vein. Blood samples were obtained from a catheter placed in the right jugular vein

at 0 (prior to administration), 3, 6, 10, 20, 30, 60, 90 minutes and at 2, 3, 4, 6, 8, 12, and

24 hours after the drug was administered.

For the IG route, clarithromycin tablets (250 mg tablets; Biaxin, Abbott

Laboratories, Chicago, IL, USA) were dissolved in 50 ml of water and administered by

nasogastric tube. For the first 24 hours, blood samples were collected as described for

the IV study. Afterwards, 5 additional doses were administered at 12 hour intervals (24,

36, 48, 60, 72 hours after the initial dose). Blood samples were collected immediately

before each additional dose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after dose 2, 4,

and 6.

Bronchoalveolar lavage was performed and samples of synovial fluid, peritoneal

fluid, cerebrospinal fluid (CSF), and urine were collected aseptically 2 and 12 hours after

administration of the last IG dose. Foals were sedated by administration of xylazine

hydrochloride (1.0 mg/kg, IV), and butorphanol tartrate (0.07 mg/kg, IV). Immediately

after collection of BAL fluid (see below), general anesthesia was induced by IV

administration of diazepam (0.1 mg/kg, IV) and ketamine (2.5 mg/kg). Samples of

synovial fluid were obtained from the intercarpal or radiocarpal joint by use of a 20-









gauge needle. Samples of CSF were collected from the atlantooccipital space by use of a

3.5-inch, 20-gauge spinal needle. Abdominal fluid was collected by use of an 18-gauge

needle. A flexible 8-F Foley catheter was used to collect urine directly from the bladder.

Samples were centrifuged and the supernatants were stored at -800C until analysis.

Bronchoalveolar Lavage

A 10 mm diameter, 1.8 m bronchoscope (Pentax, Welch Allen, Orangeburg, NY,

USA) was passed via nasal approach into either the left or right lung until wedged in a

fourth to sixth generation bronchus. The lavage solution consisted of 4 aliquots of 50 ml

physiologic saline (0.9% NaC1) solution infused and aspirated immediately. The

bronchoscope was passed alternating into either the left or right lung to prevent the effect

of repeated bronchoalveolar lavages on differential cell counts. Total nucleated cell count

in BAL fluid was determined by use of a hemacytometer. Bronchoalveolar fluid was

centrifuged at 200 X g for 10 minutes. Bronchoalveolar cells were washed, re-suspended

in 1 mL of phosphate-buffered solution, vortexed and frozen at -800C until assayed.

Supernatant BAL fluid was also frozen at -800C until assayed. Before assaying, the cell

pellet samples were thawed, vortexed vigorously, and sonicated for 2 minutes to ensure

complete cell lysis. The resulting suspension was centrifuged at 500 g for 10 minutes and

the supernatant fluid was used for determination of intracellular clarithromycin

concentrations.

Drug Analysis by High Performance Liquid Chromatography (HPLC)

Serum samples underwent a two-step extraction procedure prior to analysis by

HPLC. Samples (500 pl) of serum were thawed and mixed with an equal volume of

internal standard roxithromycin (Sigma, St-Louis, MO, USA) (4 pg/ml in 10 mM

NaH2PO4 buffer, pH 3) and acidified with 2 N HC1 to a final pH of 3. Each acidified









sample was mixed briefly and transferred quantitatively onto a solid phase extraction

column (Varian Bond Elut C18, 500 mg. ovarian, Inc. Palo Alto, CA, USA) that had been

pre-conditioned with 5 ml of methanol and 10 mM phosphate buffer (pH 3). Following

sample loading, each column was rinsed with 5 ml of 10 mM phosphate buffer (pH 3)

prior to elution of drugs with 5 ml of alkalinized methanol (99:1 mix of methanol: 1 N

NaOH). Methanolic eluates were collected and evaporated to dryness in a vacuum

concentrator at ambient temperature. Dried samples were reconstituted in 4 N NaOH

(500 pl) by incubation at room temperature for 30 min with intermittent vortex mixing.

Thereafter, 3 ml of hexane:ethyl acetate (50:50) was added and samples mixed

vigorously. Aqueous and organic layers were separated by centrifugation (4 min at 4000

g) and a portion of the organic layer was removed and evaporated to dryness. Dried

samples were reconstituted in mobile phase and analyzed immediately by HPLC

(Beckman System Gold; Beckman Coulter, Inc. Fullerton, CA, USA) with

electrochemical detection (HPLC-EC). Samples were injected and separated on a

reversed phase column (Supelco Discovery C18, 150 x 5.6 mm, 5 [m particle size) using

a filtered (0.2 [tm) degassed mobile phase containing a 55:45 mixture (v/v) of 1 mM

sodium phosphate (pH 7.0) and acetonitrile (final adjusted pH 7.5) at a flow rate of

lml/min. Concentrations of the three macrolide antibiotics were measured by

amperometric detection using an LC-4C electrochemical detector (BAS, Lafayette, IN,

USA) with a platinum electrode set at +1100mV potential (InA full scale). Peak areas for

all three compounds exhibited a linear relationship versus drug concentration over the

ranges of 0.25 5.00 [tg/ml for 14-hydroxy-clarithromycin (Courtesy of Abbott

Laboratories, Abbott Park, IL, USA) and 0.50 -5.00 [tg/ml for clarithromycin (US









Pharmacopeia, Rockville, MD, USA) and roxithromycin with a correlation coefficient (r)

value > 0.99. Each sample was run in duplicate and drug concentrations were estimated

by comparison of peak areas against linear standard curves for each analyte. Therefore,

0.5 tlg/ml was used as the lowest limit of quantification for clarithromycin. Average

retention times were 3.8 (14-hydroxy-clarithromycin), 9.0 (clarithromycin) and 11.0 min

(roxithromycin). In spiked serum samples, drug extraction yields of clarithromycin (76

+ 3.2 %) and the internal standard roxithromycin (77 3.9 %) were highly correlated (r =

0.98). In contrast, extraction yield for 14-hydroxy-clarithromycin was greater but more

variable 92 11.4 % and was poorly correlated (r = 0.78) with internal standard and, for

that reason, exact concentrations of the metabolite are not reported here.

Measurement of Clarithromycin Activity Using a Microbiologic Assay

Concentrations of clarithromycin were determined in serum, synovial fluid,

peritoneal fluid, CSF, BAL fluid, and BAL cells, using an agar well diffusion

microbiologic assay with Micrococcus luteus (ATCC 9341, American Type Culture

Collection, Rockville, MA, USA) as the assay organism. One milliliter of a bacterial

suspension was grown overnight in trypticase soy broth and adjusted to an optical density

of 0.5 at 550 nm. This suspension was added to tempered neomycin assay agar

(Neomycin assay agar, Fischer Scientific Inc, Pittsburgh, PA, USA) and distributed

evenly over the assay plates. The plates were allowed to solidify for 45 minutes, and 0.5

mm wells were punched and filled with 50 [tl of samples or clarithromycin standards (US

Pharmacopeia, Rockville, MD, USA) ranging in concentrations from 0.02 to 5.0 [tg/ml.

Known amount of purified clarithromycin were added to equine serum, synovial fluid,

and urine to produce standard curves for each type of substrate. Bronchoalveolar cells,









BAL fluid, CSF, and peritoneal fluid were assayed with standards diluted in phosphate-

buffered saline. The agar plates were incubated for 36 hours at 30 C. Zones of bacterial

inhibition were measured to the nearest 0.1 cm. Each sample or standard was assayed in

triplicate and mean values for 3 measurements of the zone diameters were determined.

The lower limit of quantification of the assay was 0.02 tlg/ml for serum, BAL cells, and

body fluid samples. Negative control samples did not cause bacterial inhibition, which

indicated no antibacterial activity of equine serum, or body fluids, or BAL cell

supernatants. Plots of zone diameters versus standard clarithromycin concentrations were

linear between 0.02 and 5 tlg/ml with r values ranging between 0.993 and 0.998. The

coefficients of variation for repeatedly assayed samples at concentrations > 0.1 tlg/ml and

< 0.1 [tg/ml were < 5% and < 10%, respectively.

Estimation of PELF and BAL Cell Volumes and Determination of Clarithromycin
Concentrations in PELF and BAL Cells

Pulmonary distribution of clarithromycin was determined as reported (Baldwin et

al., 1992). Estimation of the volume of PELF was done by urea dilution method (Conte et

al., 1996; Jacks et al., 2001). Serum urea nitrogen concentrations (UreasERUM) were

determined by use of enzymatic methodology (Labsco Laboratory Supply Company;

Louisville, KY, USA) on a chemistry analyzer (Hitachi 911 analyzer, Boehringer

Mannheim Inc, Indianapolis, IN, USA).

For measurement of urea concentration in BAL fluid (UreaBAL), the proportion of

reagents to specimen was changed from 300 [tl/3 [l in serum to 225 [tl/50 [tl. The

volume of PELF (VPELF) in BAL fluid was derived from the following equation: VPELF

VBAL X (UreaBAL/UreaSERUM), where VBAL is the volume of recovered BAL fluid. The

concentration of clarithromycin in PELF (CLRPELF) was derived form the following









relationship: CLRPELF =CLRBAL X (VBAL/ VPELF), where CLRBAL is the measured

concentration of clarithromycin in BAL fluid.

The concentration of clarithromycin in BAL cells (CLRBAL) was calculated using

the following relationship: CLRBAL = (CLRPELLET/VBALC) where CLRPELLET is

the concentration of antimicrobial in the BAL cell pellet supernatant and VBALC is the

mean volume of foal BAL cells. A VBALC of 1.20 [tl/106 cells was used for calculations

based on a previous study in foals (Jacks et al., 2001).

Pharmacokinetic Analysis

For each foal, the plasma concentration versus time data were analyzed based on

noncompartmental pharmacokinetics using computer software. (PK Solutions 2.0,

Summit Research Services, Montrose, CO, USA). The elimination rate constant (Kei) was

determined by linear regression of the terminal phase of the logarithmic plasma

concentration versus time curve using a minimum of 3 data points. Elimination half-life

(ti/) was calculated as the natural logarithm of 2 divided by Kel. Pharmacokinetic values

were calculated as reported by Gibaldi and Perrier (1982). The area under the

concentration-time curve (AUC) and the area under the first moment of the

concentration-time curve (AUMC) were calculated using the trapezoidal rule, with

extrapolation to infinity using Cmin/ Kel, where Cmin was the final measurable plasma

concentration. Mean residence time (MRT) was calculated as: AUMC/AUC. Apparent

volume of distribution based on the AUC (Vdarea) was calculated as: dose /AUC* Kel,

apparent volume of distribution at steady state (VDss) was calculated as

(dose/AUC)/(AUMC/AUC), and systemic clearance (CL) was calculated from:

dose/AUC. Bioavailability was calculated as (AUCIG/AUCiv) x (doseiv/doselG).









Statistical Analysis

Pharmacokinetic-derived data are presented as mean SD unless otherwise

specified. The paired t-test was used to compare differences in Kel between IV and IG

administration as well as peak serum concentration after the first dose (Cmax 0-24h) and

peak serum concentrations after the last dose (Cmax 72-84h). The Friedman repeated

measures ANOVA on ranks was used to compare clarithromycin concentrations between

sampling sites (serum, synovial fluid, peritoneal fluid, CSF, urine, PELF, BAL cells).

When indicated, multiple pairwise comparisons were done using the Student-Newman-

Keuls test. Differences were considered significant at P < 0.05.

Results

Following IV administration of clarithromycin (7.5 mg/kg), serum drug

concentrations were similar when measured by HPLC or the microbiologic assay;

although, HPLC-based measurements tended to be higher immediately following drug

administration (Figure 2.1). Pharmacokinetic parameters were calculated based on data

obtained with the microbiologic assay because the high limit of quantification of the

HPLC method did not allow accurate evaluation of the terminal elimination phase of the

drug. Clarithromycin had a ti, of 5.4 hours (harmonic mean), a body clearance of 1.27

L/h/kg, and a Vdss of 10.4 + 2.1 L/kg (Table 1). Detection of 14-hydroxy-clarithromycin

was first achieved 0.5 h after IV administration in 3 foals and by 1 h post-administration

in all 6 foals. Time to maximum concentration (Tmax) of 14-hydroxy-clarithromycin

following IV administration was 1.7 1.2 h.

After IG administration, quantifiable clarithromycin activity was found in 4 of 6

foals at 10 minutes and in all 6 foals at 15 minutes. The time to peak serum

clarithromycin concentration (Tmax) was 1.6 0.4 h and F was 57.3 12.0 % (Table 1).









Peak serum clarithromycin concentration following multiple IG administration (Cmax 72-

84h: 0.88 + 0.19 tlg/mL) was significantly higher (P = 0.011) that that achieved after the

first IG dose (Cmax 0-24h: 0.52 0.17 [tg/mL) (Figure 2). Differences between Kei after IV

and IG administration were not significant (P = 0.617). After multiple IG doses,

peritoneal fluid, CSF, and synovial fluid clarithromycin concentrations were similar to or

lower than serum concentrations whereas urine, PELF, and BAL cell concentrations were

significantly higher than concurrent serum concentrations (Table 2). Detection of 14-

hydroxy-clarithromycin was first achieved 0.5 h after IG administration in 2 foals and by

2 h post-administration in all 6 foals with a Tmax of 1.7 1.2 h.

One foal developed transient tachypnea and profuse sweating 5 min after

administration of the IV bolus. One foal developed diarrhea after the third IG dose and

another foal developed diarrhea after the last intragastric dose. In both foals, the diarrhea

resolved without therapy within 36 h.

Discussion

Clarithromycin undergoes extensive metabolism in people. Of the 8 metabolites

that have been identified, 14-hydroxy-clarithromycin is the most abundant and the only

one with substantial antimicrobial activity (Fernandes et al., 1988; Ferrero et al., 1990).

The metabolism of clarithromycin is unique in people since it is the only 14-membered

macrolide to demonstrate 14-hydroxylation. The 14-hydroxy metabolite of

clarithromycin is also produced in monkeys but not in rats, mice, or desert tortoises

(Ferrero et al., 1990; Bedos et al., 1992; Wimsatt et al., 1999). The present study

confirms the production of 14-hydroxy-clarithromycin in foals with peak concentrations

detected approximately 1.7 h following IV or IG administration. In people, peak 14-









hydroxy-clarithromycin concentrations at approximately 1.3 tlg/ml were detected 3 h

following oral administration of a dose of 7.5 mg/kg of body weight (Gan et al., 1992).

Unfortunately, exact concentrations of 14-hydroxy-clarithromycin could not be

determined in the present study due to the inability to find an internal standard exhibiting

parallel recovery efficiency.

The microbiological assay used to calculate pharmacokinetic parameters in the

present study only allows an approximation of the drug disposition because it does not

differentiate between clarithromycin and its 14-hydroxy metabolite. However, in a

clinical situation, the total antimicrobial activity measured by the microbiological assay is

adequate to determine a dosage regimen. The oral bioavailability of clarithromycin in the

present study (57%) is similar to that reported in people (55%) but lower than the 70-75%

reported in dogs (Chu et al., 1992; Vilmanyi et al., 1996). The oral bioavailability of

clarithromycin in the present study is similar to that of azithromycin (38-56%) and much

higher than that of erythromycin (14%) in foals (Lakritz et al., 2000:1011-1015; Jacks et

al., 2001; Davis et al., 2002). Clarithromycin elimination half-life in the present study

(5.4 h) was slightly longer than that reported after oral administration to dogs (3.9 h)

(Vilmanyi et al., 1996). Elimination half-lives reported in people range between 3 to 5 h

for clarithromycin and 4 to 9 h for 14-hydroxy-clarithromycin (Rodvold, 1999). The

elimination half-life of clarithromycin in the present study is longer than that reported for

erythromycin (1 h) but considerably shorter than that of azithromycin (16-20 h) in foals

(Prescott et al., 1983; Lakritz et al., 1999; Lakritz et al., 2000:914-919; Jacks et al., 2001;

Davis et al., 2002).









The optimal dosing of antimicrobial agents is dependent not only on the

pharmacokinetics, but also on the pharmacodynamics of the drug. The

pharmacodynamic properties of a drug address the relationship between drug

concentration and antimicrobial activity. Much confusion exists over the

pharmacodynamics of macrolides and azalides because their concentration-time profile is

low relative to the minimum inhibitory concentrations of the pathogens for which they

are used typically. An important factor in determining the efficacy of many macrolides

in animal models of infection with extracellular bacteria is the length of time that serum

concentrations exceed the MIC of the pathogen (T > MIC) (Rodvold, 1999). In a mouse

thigh model of Streptococcus pneumoniae infection, T > MIC for at least 60% of the

dosage interval with clarithromycin was the best predictor of efficacy (Craig, 1997). In a

murine model of pneumococcal pneumonia, T > MIC of 50-70%, Cmax/MIC of 3-7, and

AUCo-24/MIC 40-100 were all comparable in predicting efficacy (Tessier et al., 2002).

Recent data suggest that traditional pharmacodynamic parameters based on plasma

concentrations of macrolides may not best apply to the treatment of pulmonary infections

and infections caused by facultative intracellular pathogens such as R. equi (Drusano,

2005).

While drug concentration in plasma is clearly a driving force for penetration to the

site of infection, the actual drug-concentration time profile in a peripheral site may be

quite different from that of plasma (Drusano, 2005). Macrolides cross the cellular

membranes primarily by passive diffusion (Fietta et al., 1997). They are potent weak

bases that become ion-trapped within acidic intracellular compartments such as

lysosomes and phagosomes. A number of in vitro and in vivo studies support the notion









that white blood cells act as carriers for the delivery of macrolides to the site of infection

(Retsema et al., 1993; Mandell & Coleman, 2001). However, the white blood cell

delivery theory does not explain the very high concentrations of these drugs in PELF, as

this was demonstrated in healthy subjects where trafficking of white blood cells to the

PELF should have been minimal (Conte et al., 1995; Rodvold et al., 1997). A high

concentration of macrolides in PELF has long been proposed as a key factor in their

efficacy against respiratory pathogens in people. The preferential activity of

clarithromycin in the lung was recently demonstrated in mice infected with S.

pneumoniae isolates with efflux-mediated macrolide resistance. Consistent bacterial kill

was observed in the lung model whereas no drug effect was seen in the thigh model

(Maglio et al., 2004). These differences in bacterial activity between sites were

explained by the higher concentrations in PELF than in serum (Maglio et al., 2004).

In the present study, administration of clarithromycin at 7.5 mg/kg every 12 h

resulted in serum concentrations above the MIC inhibiting 90% of R. equi isolates (MIC90

=0.12 [tg/ml) throughout the entire dosing interval, a mean Cmax/MIC90 ratio of 7, and

mean AUC0-24/MIC90 ratio of 57. Because serum concentrations alone should not be

used to determine the likelihood of clinical efficacy in the treatment of R. equi

pneumonia of foals, clarithromycin concentrations were also measured in PELF and BAL

cells. Estimation of PELF volume by use of the urea dilution method may result in falsely

increased BAL fluid urea concentration by diffusion of urea from the interstitium and

blood if BAL fluid dwell-time is prolonged (Baldwin et al., 1992). Prolonged BAL fluid

dwell-time was minimized in our study by use of rapid infusion of 100 ml of saline

solution followed by immediate aspiration. Overestimation of urea concentrations in









BAL fluid would falsely increase the volume of PELF, which would in turn result in an

underestimation of clarithromycin concentrations in PELF (Baldwin et al., 1992).

Concentrations of clarithromycin in PELF and BAL cells in the present study

considerably exceeded the MIC90 of R. equi isolates obtained from foals with pneumonia.

Clarithromycin concentrations in PELF and BAL cells in the present study were also

considerably higher than concentrations reported following multiple daily administration

of azithromycin to foals. In the present study, clarithromycin concentrations in BAL

cells and PELF had decreased considerably 12 h following administration. This is in

contrast to azithromycin concentrations in PELF and BAL cells which do not decrease

for at least 48 h following administration to foals (Jacks et al., 2001). Collectively, these

findings in foals are consistent with studies in people showing much higher peak

clarithromycin concentrations in PELF and BAL cells compared to azithromycin, but

much longer persistence of azithromycin than clarithromycin at these sites (Conte et al.,

1996; Patel et al., 1996; Rodvold et al., 1997). Following administration of a single oral

dose to people, clarithromycin is no longer detectable in PELF after 24 h and in BAL

cells after 48 h (Conte et al., 1996). The release of azithromycin from cells is much

slower than that of erythromycin and clarithromycin, resulting in sustained

concentrations of azithromycin in tissues for days following discontinuation of therapy

(Fietta et al., 1997). Clarithromycin concentrations in peritoneal fluid, synovial fluid,

and CSF were significantly lower than PELF concentrations in the present study

indicating preferential diffusion of clarithromycin into pulmonary fluid.

Adverse effects in humans receiving clarithromycin are rare and usually related to

the gastrointestinal tract with diarrhea, nausea, and abdominal pain being the most









frequently reported (Alvarez-Elcoro & Enzler, 1999). Two of 6 foals in the present study

developed mild self-limiting diarrhea. The incidence of diarrhea in the present study

was similar to that of a retrospective study in which 5 of 18 foals (28%) with R. equi

pneumonia treated with clarithromycin and rifampin also developed diarrhea (Giguere et

al., 2004). This is similar to the incidence of diarrhea reported in foals being treated with

erythromycin-rifampin (17 to 36%) (Stratton-Phelps et al., 2000; Giguere et al., 2004).

In contrast, the incidence of gastrointestinal adverse effects in people is significantly

lower during clarithromycin (4%) than during erythromycin (19%) therapy (Anderson et

al., 1991).









Table 3.1 Pharmacokinetic variables (mean SD unless otherwise specified) for
clarithromycin after IV or intragastric administration to 6 foals at dose of 7.5
mg/kg of body weight.
Variable IV Intragastric
Kel (h-) 0.129 0.022 0.141 0.05
AUCo-,o (lgh/mL) 6.2 1.5 3.4 1.1
AUMCo, (lg.h2/mL) 51.1 + 16.2 24.4 9.7
MRT (h) 8.25+0.989 7.1+1.70
t/, (h) 5.4* NA
Vdrea (L/kg) 9.9 + 1.8 NA
Vdss (L/kg) 10.4 + 2.1 NA
Clearance (L/h/kg) 1.27 + 0.25 NA
Tmax (h) NA 1.6 0.4
Cmax 0-24h (lg/mL) NA 0.52 + 0.17
Cmax 72-84h (tg/mL) NA 0.88 0.19
C84h NA 0.20 + 0.06
F (%) NA 57.3 + 12.0
NA = Not applicable
*Harmonic mean.
Kei = Elimination rate constant. ti/ = Elimination half-life. AUC = Area under the serum
concentration versus time curve. AUMC = Area under the first moment of the
concentration versus time curve. MRT = Mean residence time. Vdarea = Apparent volume
of distribution (area) Vdss = Apparent volume of distribution (steady-state) tl2abs =
Absorption half-life. Tmax = Time to peak serum concentration. Cmax 0-24h = Peak serum
concentration after the first dose. Cmax 72-84h = Peak serum concentration after repeated
doses. C84h = Minimum serum concentration 12 h after the last dose. F = Oral
bioavailability.


Table 3.2 Mean SD clarithromycin activity in body fluids and BAL cells of six foals
after 6 intragastric administrations (7.5 mg /kg every 12 hours).
Time after administration (h)
Sample 2 12
Serum ([lg/mL) 0.83 + 0.18a 0.20 + 0.06a
Synovial fluid (tg/mL) 0.27 0.06b 0.08 0.02a
Peritoneal fluid (btg/mL)* 0.43 0.32b 0.11 0.06a
Urine (tg/mL) 36.8 + 46.4c 2.53 0.82b
CSF (tg/mL) 0.22 0.09b 0.13 0.09 a
Pulmonary epithelial lining fluid (|tg/mL) 76.2 59.4 c 21.4 20.5c
Bronchoalveolar cells (ltg/mL)t 269 232d 117 107d
*n=3 at 2 h and n=5 at 12 h
tDrug concentrations are in pg/ml of bronchoalveolar cell volume
abcdDifferent letters within a column indicate statistically significant difference in
clarithromycin concentrations (P < 0.05)










10


-- HPLC
-m- Microbiologic

E 1
0



E

0.1






0.01
0 4 8 12 16 20 24

Time (h)
Figure 3.1-Mean (+ SD) serum clarithromycin concentration as measured by HPLC
method or microbiologic assay in 6 foals administered a single IV dose of 7.5
mg/kg.











10


-1
E





0.1







0.001
0 12 24 36 48 60 72 84

Time (h)

Figure 3.2-Mean (+ SD) serum clarithromycin activity ([tg/ml) in 6 foals following
intragastric clarithromycin (7.5 mg/kg) administration at 0, 24, 36, 48, 60, and
72 hours. Results are based on measurements with the microbiologic assay.














CHAPTER 4
PULMONARY DISPOSITION OF TILMICOSIN IN FOALS AND IN VITRO
ACTIVITY AGAINST RHODOCOCCUS EQUI AND OTHER COMMON EQUINE
BACTERIAL PATHOGENS

Abstract

Tilmicosin is a long-acting macrolide currently approved for treatment of

respiratory disease in cattle, sheep and swine. The objectives of this study were to

determine the serum and pulmonary disposition of tilmicosin in foals and to investigate

the in vitro activity of the drug against R. equi and other common bacterial pathogens of

horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of

body weight) was administered to 7 healthy 5- to 8-week-old foals by the intramuscular

route. Concentrations oftilmicosin in serum were measured by HPLC and

concentrations in lung tissue, pulmonary epithelial lining fluid pelfF), and

bronchoalveolar (BAL) cells were measured by mass spectrometry. Mean peak

tilmicosin concentrations were significantly higher in BAL cells (20.1 + 5.1 [tg/mL) than

in lung tissue (1.90 + 0.65 [tg/mL), PELF (2.91 + 1.15 [tg/mL), and serum (0.19 0.09

[tg/mL). Harmonic mean elimination half life in lung tissue (193.3 h) was significantly

longer than that of serum (18.4 h). Elimination half lives in BAL cells and PELF were

62.2 h and 73.3 h, respectively. The MIC90 of 56 R. equi isolates was 32 [tg/mL.

Tilmicosin was active in vitro against most streptococci, Staphylococcus spp.,

Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus

spp., Pseudomonas spp., and Enterobacteraceae. In conclusion, the formulation of









tilmicosin investigated in the present study resulted in high and sustained concentrations

of tilmicosin in the lung, PELF, and BAL cells of foals.

Introduction

Tilmicosin is a semi-synthetic 16-membered lactone ring macrolide chemically

derived from tylosin (Prescott, 2000). Tilmicosin is approved as a suspension for

subcutaneous administration in the therapy or control of pneumonia caused by

Mannheimia haemolytica in cattle and sheep. It is also approved for use in feed for the

control of swine respiratory disease associated with Actinobacilluspleuropneumoniae

and Pasteurella multocida. In addition, the drug is active in vitro against a variety of

pathogens of cattle and swine including Histophilus somni, Haemophilus parasuis,

Actinobacillus suis, Arcanobacterium pyogenes, Erysipelothrix rhusiopathiae,

Staphylococcus spp., some Streptococcus spp., and many Mycoplasma spp. (Watts et al.,

1994; DeRosa et al., 2000; Prescott, 2000). The pharmacokinetic properties of tilmicosin

are similar to that of macrolides in general, and are characterized by low serum

concentrations but large volumes of distribution, with accumulation and persistence in

many tissues including the lung, which may concentrate the drug 60-fold compared to

serum (Ziv et al., 1995; Scorneaux and Shryock, 1999; Clark et al., 2004). Despite low

extracellular concentrations, tilmicosin accumulates substantially in phagocytic cells of

cattle and swine (Scorneaux and Shryock, 1998; Scorneaux and Shryock, 1999).

Pneumonia is a leading cause of morbidity and mortality in foals (Cohen, 1994).

Gram-positive bacteria such as Streptococcus equi subspecies zooepidemicus and

Rhodococcus equi are the most common causes of pneumonia in foals between 1 and 6

months of age (Hoffman et al., 1993; Giguere et al., 2002). Gram-negative bacteria such

as Pasteurella spp., Actinobacillus spp., Bordetella bronchiseptica, Escherichia coli,









Klebsiellapneumoniae, and Salmonella enterica may also be cultured from

tracheobronchial aspirates of affected foals (Wilson, 1992). Macrolide antimicrobial

agents are commonly used in equine medicine for treatment of foal pneumonia,

particularly when infection with Rhodococcus equi is suspected or confirmed. Tilmicosin

may be a useful alternative to currently used antimicrobial agents owing to its

accumulation in lung tissue and phagocytic cells, as well as in vitro activity against many

Gram-positive and Gram negative bacterial species. In addition, availability of a long

acting antimicrobial agent providing sustained therapeutic concentrations at the site of

infection would result in less frequent administration, which in turn may improve client

compliance. However, the lack of pharmacokinetic studies and in vitro susceptibility

data with bacterial pathogens of horses precludes the rational use of this antimicrobial

agent in foals.

The objectives of the study reported here were to determine the pulmonary

disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against

R. equi and other common bacterial pathogens of horses.

Material and Methods

Horses and experimental design

Four male and three female Thoroughbred foals between 5 and 8 weeks of age and

weighing between 80 and 135 kg were selected for this study. The foals were considered

healthy on the basis of history, physical examination, complete blood count and plasma

biochemical profile. The foals were kept with their dams in individual stalls during the

experiment with ad libitum access to grass hay and water. The study was approved by

the Institutional Animal Care and Use Committee at the University of Florida.









Experimental design and sample collection

A proprietary fatty acid salt formulation of tilmicosin (250 mg/mL; Idexx

Pharmaceuticals, Durham, NC) was administered as a single dose of 10 mg/kg of body

weight via the intramuscular route in the semimembranosus/semitendinosus muscles.

Blood samples (8 mL) were obtained from a jugular catheter at 3, 6, 10, 20, 30, 60, 90

minutes and at 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 168, and 288 hours

after the drug was administered. Bronchoalveolar lavage (BAL) was performed 24, 48,

72, 168, and 288 hours and samples of cerebrospinal fluid (CSF) were collected

aseptically 4, 24, and 72 hours after administration oftilmicosin. Lung tissue was

obtained 24, 72, 168, and 288 hours after administration of the drug. Prior to collection

of BAL, lung tissue, and CSF, foals were sedated by administration of xylazine

hydrochloride (1 mg/kg, IV) and butorphanol tartrate (0.07 mg/kg, IV). Immediately after

collection of BAL fluid, general anesthesia was induced by IV administration of

diazepam (0.1 mg/kg) and ketamine (2.5 mg/kg) for collection of lung tissue and CSF

fluid. Using sterile techniques, CSF was collected from the atlantoocciptal space by use

of a 3.5 inch, 20-gauge spinal needle. Blood and CSF samples were centrifuged and

serum and CSF supernatants were stored at -800C until analysis. Lung tissue was

obtained aseptically from the 8th intercostal space at the level of the point of the shoulder

using a 16 gauge spring activated biopsy instrument with a 20 mm specimen notch

(J528a, Jorgenson laboratories, Loveland, CO).

Bronchoalveolar lavage

A 10 mm diameter, 2.4 m bronchoalveolar lavage catheter (Jorgenson laboratories,

Loveland, CO) was passed via nasal approach until wedged into a bronchus. The lavage

solution consisted of 4 aliquots of 50 mL physiologic saline (0.9% NaC1) solution infused









and aspirated immediately. Total nucleated cell count in BAL fluid was determined by

use of a hemacytometer. Bronchoalveolar fluid was centrifuged at 200 X g for 10

minutes. Bronchoalveolar cells were washed, re-suspended in 500 pl of phosphate-

buffered solution, vortexed and frozen at -800C until assayed. Supernatant BAL fluid was

also frozen at -800C until assayed. Before assaying, the cell pellet samples were thawed,

vortexed vigorously and sonicated for 3 minutes to ensure complete cell lysis. The

resulting suspension was centrifuged at 500 X g for 10 minutes and the supernatant fluid

was used to determine the intracellular concentrations of tilmicosin.

Drug analysis

The serum and other tissue samples were analyzed by validated methods at Idexx

Pharmaceuticals (Durham, NC). Serum concentrations of tilmicosin were determined by

HPLC analysis. The extraction efficiency from serum was 98%. The limit of

quantification (LOQ) was 0.08 Og/mL. The tilmicosin concentrations in all other body

fluids or tissues were determined by mass spectrometry. The LOQ were 0.5 ng/mL, 1.44

ng/mL, 1.9 ng/mL, and 0.6 ng/mL for lung tissue, BAL fluid, CSF, and BAL cells,

respectively.

Estimation of PELF and BAL Cell Volumes and Determination of Tilmicosin
Concentrations in PELF and BAL Cells

Pulmonary distribution of tilmicosin was determined as reported (Baldwin et al.,

1992). Estimation of the volume of PELF was done by urea dilution method (Rennard et

al., 1986; Conte et al., 1996). Serum urea nitrogen concentrations (UreasERUM) were

determined by use of enzymatic methodology (Labsco Laboratory Supply Company;

Louisville, KY, USA) on a chemistry analyzer (Hitachi 911 analyzer, Boehringer

Mannheim Inc, Indianapolis, IN, USA). For measurement of urea concentration in BAL









fluid (UreaBAL), the proportion of reagents to specimen was changed from 300 [tl/3 [tl in

serum to 225 [tl/50 [tl. The volume of PELF (VPELF) in BAL fluid was derived from the

following equation: VPELF = VBAL X (UreaBAL/UreasERUM), where VBAL is the volume of

recovered BAL fluid. The concentration of tilmicosin PELF (TILPELF) was derived form

the following relationship: TILPELF =TILBAL X (VBAL/ VPELF), where TILBAL is the

measured concentration of tilmicosin in BAL fluid.

The concentration of tilmicosin in BAL cells (TILBAL) was calculated using the

following relationship: TILBAL = (TILPELLET/VBALC) where TILPELLET is the concentration

of antimicrobial in the BAL cell pellet supernatant and VBALC is the mean volume of foal

BAL cells. A VBALC of 1.20 [tl/106 cells was used for calculations based on a previous

study in foals (Jacks et al., 2001).

Pharmacokinetic Analysis

For each foal, serum, lung tissue, PELF, or BAL cells tilmicosin concentration

versus time data were analyzed based on noncompartmental pharmacokinetics using

computer software (PK Solutions 2.0, Summit Research Services, Montrose, CO, USA).

The elimination rate constant (Kel) was determined by linear regression of the terminal

phase of the logarithmic concentration versus time curve using a minimum of 3 data

points. Elimination half-life (ti2) was calculated as the natural logarithm of 2 divided by

Kei. Pharmacokinetic values were calculated as reported by Gibaldi and Perrier (1982).

The area under the concentration-time curve (AUC) and the area under the first moment

of the concentration-time curve (AUMC) were calculated using the trapezoidal rule, with

extrapolation to infinity using Cmin/ Kel, where Cmin was the final measurable tilmicosin

concentration. Mean residence time (MRT) was calculated as: AUMC/AUC.









Statistical Analysis

Normality of the data and equality of variances were assessed using the

Kolmogorov-Smimov and Levene's tests, respectively. A one way repeated measure

ANOVA was used to compare each pharmacokinetic parameter between sampling sites

(serum, lung tissue, PELF, BAL cells). In rare instances when the assumptions of the

ANOVA were not met, a Friedman repeated measure ANOVA on ranks was used. When

indicated, multiple pairwise comparisons were done using the Student-Newman-Keuls

test. Differences were considered significant at P < 0.05.

Determination of minimum inhibitory concentration (MIC) and minimum
bactericidal concentrations (MBC) of tilmicosin against R. equi

R. equi isolates (n =56) were obtained from tracheobronchial aspirates or post-

mortem specimens from pneumonic foals. For each isolate, MIC and MBC were

determined by a macrodilution broth dilution technique in glass tubes in accordance to

the guidelines established by the Clinical and Laboratory Standard Institute (formerly

NCCLS) (NCCLS, 1999a; NCCLS, 1999b; NCCLS, 2000) A standard inoculum of 5 x

105 was used for each isolate. Concentrations of tilmicosin tested ranged between 256

and 0.03 tlg/mL. All MIC and MBC determinations were performed in triplicate for each

isolate. MIC was determined as the first dilution with no bacterial growth after 24 h of

incubation at 370C (National Committee for Clinical Laboratory Standards, 2000). MBC

was calculated as the lower concentration of drug resulting in a 99.9% reduction of the

original inoculum(National Committee for Clinical Laboratory Standards, 1999a).

Control strains used to validate the assay were Staphylococcus aureus ATCC 29213,

Escherichia coli ATCC 25922, and Enterococcusfaecalis ATCC 29212 (Odland et al.,









2000). The MIC required to inhibit growth of 50% of isolates (MIC5o) and the MIC

required to inhibit growth of 90% of isolates (MIC90) were determined.

Checkerboard assay

Activity of tilmicosin in combination with rifampin, gentamicin, amikacin,

doxycycline, enrofloxacin, trimethoprim-sulfa, vancomycin, imipenem, or ceftiofur

against R. equi was assessed using the modified checkerboard technique as previously

described (Pillai et al., 2005). Three isolates ofR. equi were randomly selected for this

assay. All experiments were performed in triplicate for each of the isolate. For each

antimicrobial agent, concentrations of 64-, 16-, 4-, 1-, and 0.5-times the MIC were used

to study antibiotic combinations. An inoculum of 5 x 105 was used for each R. equi

isolate. For each combination, the fractional inhibitory concentration (FIC) index after

24 h of incubation was calculated using the following formula: FIC index = FIC A + FIC

B = (MIC of A in combination/MIC of A alone) + MIC of B in combination/MIC of B

alone). A FIC index of < 0.5 indicates synergism, a FIC index > 0.5-4 indicates

indifference and a FIC index > 4 indicates antagonism (Pillai et al., 2005).

Time kill curve assay

A time kill curve assay was used to evaluate the effect of time and tilmicosin

concentration on in vitro survival ofR. equi. All experiments were performed in triplicate

using the same 3 R. equi isolates as for the checkerboard assay. An inoculum of 5 X 105

CFU/mL was used for each isolate. All experiments were performed with 4 mL of

Mueller-Hinton broth in glass tubes. After 0, 2, 6 and 24 hours of incubation, aliquots

were collected from each tube. The aliquots were centrifuged, the bacterial pellets were

washed twice to prevent antimicrobial carry over, and the CFU was counted.









In vitro activity of tilmicosin against equine bacterial pathogens

A total of 183 bacterial isolates from various equine clinical samples were

examined. Isolates were obtained from clinical samples submitted to the microbiology

laboratory at the University of Florida Veterinary Medical Center from July 2005 to

January 2006. Susceptibility testing was performed using the disk diffusion method.

Briefly, fresh isolates were grown on blood agar plates, and colonies were suspended in

sterile water to achieve turbidity equal to that of a 0.5 McFarland standard (final bacterial

concentration of approximately 1 X 105 CFU/mL). A sterile swab was dipped into the

inoculum suspension and used to inoculate the entire surface of 100 mm Mueller-Hinton

plates 3 times by rotating the plate approximately 600 for each inoculation to ensure an

even distribution. After allowing the excess moisture to dry (approx 10 to 15 minutes), 15

|tg tilmicosin disks (BBL Sensi-Disc, Hardy Diagnostics, Santa Maria, CA) were applied

to the agar. The plates were incubated for 18 to 24 hours at 370C. A test was considered

valid only when there was adequate growth on the plate. The zone diameter was

measured to the nearest millimeter. Control strains used weekly to validate the assay

were Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and

Enterococcusfaecalis ATCC 29212. Results were considered valid only when zone

diameters obtained with the control stains were within the reference range proposed

(Odland et al., 2000). According to CLSI guidelines, isolates with a zone diameter > 14

mm (corresponding to a MIC < 8 [tg/mL) were considered susceptible (Shryock et al.,

1996).









Results

Serum and pulmonary disposition of tilmicosin in foals

Quantifiable tilmicosin concentrations were found in 2 of 7 foals at 3 minutes after

IM injection and in 5 of 7 foals at 10 minutes post-injection. Serum concentrations

remained below the limit of quantification throughout the sampling period in 2 foals.

Concentrations below the limit of quantification were reported as 0 for calculation of

mean + SD (Figure 3.1). Serum pharmacokinetic parameters were derived from the 5

foals with quantifiable serum concentrations (Table 3.1). Maximum tilmicosin

concentrations (Cmax) and AUC were significantly higher in BAL cells than in serum,

lung tissue, and PELF (Table 3.1). Similarly, Cmax and AUC were significantly higher in

PELF and lung tissue than in serum. Elimination half life in lung tissue (193.3 h) was

significantly longer than that of serum (18.4 h).

One foal died as a result of hemothorax within minutes of collection of the 72 h

lung biopsy. One foal developed tachypnea and profuse sweating approximately 2 h after

injection. The clinical signs persisted for approximately 45 min. Two foals developed a

10-15 cm in diameter area of painful swelling at the injection site within 12-24 h of

injection. In one foal, the lesion was associated with hind limb lameness that persisted

for 48 h. Three foals developed a small 1-2 cm in diameter hard nodule at the injection

site. Four foals developed watery diarrhea 36-48 h after administration of tilmicosin.

Diarrhea resolved without therapy within 48 h of onset.

In vitro susceptibility testing and antimicrobial drug combinations

Both the MIC5o and MIC90 of 56 R. equi isolates were 32 [tg/mL (range 16-64

[tg/mL). Tilmicosin was not bactericidal against R. equi at concentrations up to 256

[tg/mL. Combination of tilmicosin with rifampin, gentamicin, amikacin, doxycycline,









enrofloxacin, trimethoprim-sulfa, vancomycin, imipenem, or ceftiofur did not result in

synergistic or antagonistic activity with median FIC indices ranging between 0.53 and

1.5. The time-kill experiment revealed that tilmicosin is a time dependent antimicrobial

agent with no benefit from increasing drug concentrations above 4 times the MIC (Figure

3.2). Tilmicosin was active in vitro against most streptococci, Staphylococcus spp.,

Actinobacillus spp., and Pasteurella spp. (Table 3.2).

Discussion

A safe antimicrobial agent providing high and sustained drug concentrations in the

lungs would be a useful addition to currently available antimicrobial agents for the

treatment or prevention of pneumonia in foals. Tilmicosin has been approved for the

control and treatment of respiratory disease in cattle, sheep, and swine. Tilmicosin has

also been shown to be effective for the treatment of mastitis in cattle and sheep,

pasteurellosis in rabbits, and Mycoplasma gallisepticum infections in chicken (McKay et

al., 1996; Kempfet al., 1997; Croft et al., 2000; Dingwell et al., 2003). The currently

available injectable tilmicosin formulation has been advocated as potentially fatal when

administered to horses, swine, and goats (Micotil 300 package insert, 1995). The

cardiovascular system is the target of toxicity in laboratory and domestic animals with

tachycardia and decreased cardiac contractility being reported following parenteral

administration of tilmicosin (Main et al., 1996). To minimize the risk of toxicity, a fatty

acid salt formulation of tilmicosin newly developed as a safer and convenient formulation

for use in cats (Kordick et al., 2003) was used in the present study.

Mean peak serum concentrations and AUC achieved in the present study (0.19

[tg/mL) were considerably lower that that achieved after subcutaneous administration of









the same dose to cattle (0.87 tlg/mL), sheep (0.82 tlg/mL), and goats (1.56 tlg/mL)

(Ramadan, 1997; Modric et al., 1998). Peak serum concentrations in foals were also

lower that that observed after administration of the same fatty acid salt formulation

administered at a dose of 10 mg/kg SC to cats (0.73 tlg/mL) (Kordick et al., 2003).

Tilmicosin serum elimination half-life in the present study (18.4 h) was slightly shorter

than that reported after SC administration to cattle (29.4 h), sheep (34.6 h), and goats

(29.3 h) (Ramadan, 1997; Modric et al., 1998).

Recent data suggest that traditional pharmacodynamic parameters based on plasma

concentrations of macrolides may not best apply to the treatment of pulmonary infections

and infections caused by facultative intracellular pathogens such as R. equi (Drusano,

2005). Serum concentrations of tilmicosin in cattle and swine are much lower than its

MICs for common respiratory tract pathogens. Nevertheless, multiple studies have

demonstrated the efficacy of tilmicosin in the treatment of respiratory disease in these

species (Musser et al., 1996; Paradis et al., 2004). Lung concentrations of tilmicosin

remain above the MIC ofMannheimia haemolytica (3.15 [tg/mL) for at least 72 hours

following a single SC injection at a dose of 10 mg/kg (Micotil 300 package insert, 1995).

In cats, maximum lung concentrations of tilmicosin of 5.62 [tg/mL are achieved on day

two following administration of the fatty acid salt formulation and measurable

concentrations are still present in the lungs on day 21 (Kordick et al., 2003).

While drug concentration in plasma is clearly a driving force for penetration to the

site of infection, the actual drug-concentration time profile at a peripheral site may be

quite different from that of plasma. Macrolides cross the cellular membranes primarily

by passive diffusion (Fietta et al., 1997). Tilmicosin, like other macrolides, is a potent









weak base that becomes ion-trapped within acidic intracellular compartments such as

lysosomes (Scorneaux and Shryock, 1999). The ratio of cellular to extracellular

concentration oftilmicosin is 193, 43, and 13, respectively, in bovine alveolar

macrophages, monocyte-derived macrophages, and mammary epithelial cells (Scorneaux

and Shryock, 1999). Consistent with these findings, peak tilmicosin concentrations in

BAL cells of foals were approximately 107 times higher than peak serum concentrations.

A number of in vitro and in vivo studies support the notion that white blood cells act as

carriers for the delivery of macrolides to the site of infection (Retsema et al., 1993;

Mandell and Coleman, 2001). Studies with tilmicosin in rats support this concept as drug

concentrations in the lung of rats inoculated with Mycoplasmapulmonis were

significantly higher that those of noninfected controls (Modric et al., 1999).

Macrolides inhibit protein synthesis by reversibly binding to 50S subunits of the

ribosome. Macrolides are generally bacteriostatic agents but they may be bactericidal at

high concentrations (Prescott, 2000). In the present study, tilmicosin was only

bacteriostatic against R. equi at concentrations up to 256 [tg/mL. The MIC90 of

tilmicosin against foal isolates of R. equi (32 [tg/mL) in the present study was similar to

that of a previous study looking at a combination of human and equine isolates (> 32

[tg/mL) (Bowersock et al., 2000). Consistent with a bacteriostatic antimicrobial agent,

tilmicosin exerted time dependent activity against R. equi in vitro. Even if tilmicosin

concentrated more than 100-fold in BAL cells of foals, drug concentrations achieved in

lung tissue, PELF, and BAL cells were consistently below the MIC90 ofR. equi.

Tilmicosin was active in vitro against all P-hemolytic streptococci and Pasteurella spp.,

and most ca-hemolytic streptococci, Staphylococcus spp., and Actinobacillus spp.









Additional studies will be required to determine the clinical efficacy of this fatty acid salt

formulation of tilmicosin against these pathogens in foals.

Adverse effects observed in the present study consisted mainly of swelling at the

injection site in 5 foals and self limiting diarrhea in 4 foals. One foal developed

tachypnea and profuse sweating approximately 2 h after injection. In swine, IM

administration of the commercially available formulation at a dose of 10 mg/kg has

resulted in tachypnea, and convulsions, and death occurs with dosages > 20 mg/kg

(Micotil 300 package insert, 1995). Tilmicosin included in the diet of horses at

concentrations of 400, 1200, and 2000 ppm has resulted in gastrointestinal disturbance in

all groups and death of 1 horse consuming the 2000 ppm diet (Pulmotil 90 package insert,

1995). In another study, SC administration of the commercially available formulation of

tilmicosin to foals at a dose of 10 mg/kg resulted in immediate loss of the normal fecal

streptococcal population and a corresponding massive overgrowth of coliform bacteria

(Clark and Dowling, 2004). The fecal flora slowly recovered over the next 7 days. Mild

self-limiting diarrhea was observed in one foal (Clark and Dowling, 2004).

In conclusion, the fatty acid salt formulation of tilmicosin investigated in the

present study resulted in high and sustained concentrations of tilmicosin in the lung,

PELF, and BAL cells of foals following a single IM administration. The drug was active

in vitro against a variety of bacterial pathogens. These data warrant further investigations

into the clinical efficacy of this formulation of tilmicosin in foals with respiratory disease.









Table 4.1 Serum and pulmonary pharmacokinetic variables (mean SD unless
otherwise specified) for tilmicosin after IM administration to seven foals at a
dose of 10 mg/kg of body weight.
Variable Serum' Lung2 PELF BAL cells
Kel (h-1) 0.04 0.02a 0.004 0.001b 0.009 0.004b 0.01 + 0.003
AUCo-0 (|pg*h/mL) 5.76 1.87a 711 351b 461 + 115b 2342 1006C
MRT (h) 34.5 18.0a 323 91.0b 180 48.9c 117 + 29.6c


tl/2 (h)* 18.4a 193.3b 73.1ab 62.2a'b
Tmax (h) 5.50 + 3.43a 30.8 18.1a b 52.0 18.1b 54.9 33.
Cmax (tg/mL or tg/g) 0.19 0.09a 1.90 + 0.65b 2.91 + 1.15b 20.1 5.1'
'n=5 because 2 foals had serum tilmicosin concentrations below the limit of
quantification.
2n=5 because one foal died after the 72 h sample and lung samples were too small for
drug analysis in one foal.
*harmonic mean.
abcdDifferent letters within a row indicate a statistically significant difference between
sampling sites (P < 0.05).
Kei = Elimination rate constant. AUC = Area under the serum concentration versus time
curve. MRT = Mean residence time. t/ = Elimination half-life. Tmax = Time to peak
serum concentration. Cmax = peak serum concentration.


Table 4.2 Tilmicosin in vitro susceptibility of 183 bacterial isolates obtained from
horses.
Microorganism (n) Zone diameter (mm) Susceptibility
Median 25th Range (%)
percentile
Gram positives
0-hemolytic streptococci (7) 19 11 0-20 71
0-hemolytic streptococci (37) 19 18 15-26 100
Enterococcus spp. (5) 0 0 0 0
Rhodococcus equi (9) 0 0 0 0
Staphylococcus spp. (25) 18 16 0-28 96
Gram-negatives
Actinobacillus spp. (9) 16 12 0-19 67
Enterobacter spp. (9) 0 0 0
Escherichia coli (11) 0 0 0-14 9
Klebsiella spp. (5) 0 0 0 0
Pasteurella spp. (6) 23 18 18-34 100
Pseudomonas spp. (12) 0 0 0-13 0
Salmonella enterica (48) 0 0 0-11 0


I b













10


1


0.1


0.01


0.001 .I4
0 48 96 144 192 240 288
Time (h)

Figure 4.1 Mean + SD tilmicosin concentrations in serum, BAL cells, PELF ([tg/mL),
and lung tissue ([tg/g) of 7 foals following a single IM dose of tilmicosin (10
mg/kg of body weight).




8

7 -*- Control
S.-- ------ 0.25 X MIC
SA- 1 X MIC
u. 6 -
S --- 4 X MIC
--. -u-16 X MIC
J5 ------64XMIC
--------- 64 XMIC


6 12 18 24


Time (h)



Figure 4.2 Effect of time and tilmicosin concentration on in vitro survival of a clinical
isolate of R. equi. Identical results were obtained with 2 additional isolates.














CHAPTER 5
SUMMARY AND CONCLUSIONS

The present study investigated the pharmacokinetics and pulmonary disposition of

clarithromycin and tilmicosin in foals. The optimal dosing of antimicrobial agents is

dependent not only on their pharmacokinetics, but also on the pharmacodynamics of the

drug. The pharmacodynamic properties of a drug address the relationship between drug

concentration and antimicrobial activity. Much confusion exists over the

pharmacodynamics of macrolides. An important factor in determining the efficacy of

many macrolides in animal models of infection with extracellular bacteria is the length of

time that serum concentrations exceed the MIC of a given pathogen (T > MIC) (Rodvold,

1999). However, recent data suggest that traditional pharmacodynamic parameters based

on plasma concentrations of macrolides may not best apply to the treatment of pulmonary

infections and infections caused by facultative intracellular pathogens such as R. equi and

that concentrations at the site of infection are more important in predicting efficacy

(Drusano, 2005). Macrolides enter phagocytic cells by passive diffusion and they

accumulate in acidic intracellular compartments such as lysosomes and phagosomes. A

number of in vitro and in vivo studies support the notion that white blood cells act as

carriers for the delivery of macrolides to the site of infection (Mandell et al., 2001).

To provide a better assessment of the potential usefulness of clarithromycin and

tilmicosin for the treatment of bronchopneumonia in foals, we measured drug

concentrations in PELF, BAL cells, and lung tissue. Oral clarithromycin was

wellabsorbed in foals and resulted in mean PELF concentrations approximately 95 times









higher and mean BAL cell concentrations approximately 335 times higher than

concurrent serum concentrations. Clarithromycin undergoes extensive metabolism in

people. Of the 8 metabolites that have been identified, 14-hydroxy-clarithromycin is the

most abundant and the only one with substantial antimicrobial activity (Fernandes et al.,

1988; Ferrero et al., 1990). Although clarithromycin is not converted tol4-hydroxy

clarithromycin in rodents and reptiles, the present study confirmed production of 14-

hydroxy-clarithromycin in foals.

A new fatty acid salt formulation of tilmicosin, developed as a safer and convenient

formulation for use in cats (Kordick et al., 2003), was investigated in the present study.

Serum concentrations of tilmicosin in cattle and swine are much lower than its MICs for

common respiratory tract pathogens. Nevertheless, multiple studies have demonstrated

the efficacy of tilmicosin in the treatment of respiratory disease in these species because

the drug concentrates in lung tissue and phagocytic cells (Musser et al., 1996; Paradis et

al., 2004). Tilmicosin accumulated in the lungs of foals. BAL cells achieved the highest

concentrations with Cmax approximately 100 times higher than that achieved in plasma.

Tilmicosin concentrations in PELF were almost identical to that of lung concentrations,

indicating that measurement of drugs in PELF may represent a less invasive alternative to

lung biopsies. Lung tissue and PELF tilmicosin concentrations were approximately 10-

15 times higher than peak serum concentrations. However, tilmicosin concentrations at

all of the times sampled remained considerably below the MIC90 of R. equi. Therefore,

tilmicosin, at the dose used in the present study, would not be adequate for the treatment

of R. equi infections in foals.









In conclusion, oral administration of clarithromycin at a dosage of 7.5 mg/kg every

12 hours would maintain serum, PELF, and BAL cell concentrations above the minimum

inhibitory concentration for R. equi and S. zooepidemicus isolates for the entire dosing

interval. The formulation of tilmicosin investigated in the present study resulted in high

and sustained concentrations in the lung, PELF, and BAL cells of foals and may be

appropriate for the treatment of susceptible bacterial infections. Additional studies will be

required to establish the safety and determine the efficacy of these drugs in a clinical

setting.















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BIOGRAPHICAL SKETCH

Ariel Womble was born in Charlotte, N.C., to David Womble and Connie Harris.

She moved to Palm Harbor, Florida when she was eight years old where she lived and

attended high school. While in high school she earned a scholarship to attend the

University of Florida where she earned a Bachelor of Science in animal science. During

her years as an undergraduate she worked part-time at the Veterinary Teaching Hospital

where she met Dr. Steeve Giguere. Through this meeting she became interested in

research and pursuing a Master of Science degree in veterinary medical science. After

graduation she began work in graduate studies.