Journal of Antimicrobial Chemotherapy (1990) 25, Suppl. ^,61-71
Correlation of the extravascular pharmacokioetics of azithromycin with in-vivo efficacy in models of localized infection Arthur £ . Girard*, Dennis Girard and James A. Retsema
Infection models were used to clarify the roles of serum and extravascular concentrations in the in-vivo efficacy observed with azithromycin. In-vivo experiments were designed to give serum concentrations well below the MIC and tissue levels generally above the MIC at time of challenge and during the course of infection. The efficacy of azithromycin against a Salmonella entcritidis oral challenge (a tissue-associated infection model) in mice correlated directly with azithromycin liver levels, but not serum concentrations. The significance of extravascular pharmacokinetics was observed in a comparative study of azithromycin and ciprofloxacin against the salmonella challenge. Ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this organism, but azithromycin (Smg/kg) produced a greater reduction in cfu than ciprofloxacin (100 mg/kg) at the primary site of infection (liver). In another model, extravascular fluid levels, measured by bioassay of implanted paper discs, were compared with plasma levels in relation to control of a localized Staphylococcus aweus infection in rats. Extravascular fluid levels of azithromycin were greater than the MIC of the strain used for five days after a 100 mg/kg dose, while erythromycin levels were less than 20% of the MIC at 30 h after a 200 mg/kg dose. Serum concentrations of both compounds were less than 20% of the MIC at the time of challenge. The antibiotic levels at the site of infection correlated with the reduction of Staph. aureus cfu (99% with azithromycin compared with controls, P < 0-01; 0% with erythromycin) recovered from inoculated discs. The significance of extravascular concentrations of azithromycin was further supported in other models of localized infections induced with Escherichia coli or a mixture of Staph. aureus and Bacteroides fragilis.
Introduction The importance of extravascular pharmacokinetics in in-vivo efficacy is not a new observation. Eagle (1948) noted it was the concentration at the site of infection that was the important therapeutic consideration, and that the plasma level was of significance only in providing a measure of the tissue concentration. Later Chabbert et al. (1957) reported on the potential clinical significance of tissue levels as described in the spiramycin paradox, i.e. the efficacy associated with peak serum levels at or below the MIC. Recently Smith (1988) reviewed the possible explanation of this apparent paradox, focusing again on the tissue and intracellular concentrations of spiramycin. Orally administered azithromycin has been shown to be well absorbed in animals (Girard etal., 1987; Shepard & Falkner, 1990, this Volume) and man (Foulds, Shepard & Johnson, 1990, this Volume). Furthermore, rapid distribution to and concentration •Corresponding author 61 0305-7453/90/25A061 +11 S02.00/0
© 1990 The British Society for Antimicrobial Chemotherapy
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Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA
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in tissues occurs to a much greater extent than with erythromycin. The resulting high and sustained tissue levels of azithromycin were considered to lead to increased efficacy compared with crythromycin and other antibiotics in a series of mouse tissue infection models (Girard etal., 1987). However, it was possible that blood levels contributed significantly to the therapeutic outcome. In this investigation, we have evaluated the relative importance of azithromycin extravascular levels in efficacy, using treatment regimens and models of localized infection that differentiate between the contributions made by the tissue and the blood concentrations. Additionally we have investigated the potential of azithromycin to treat localized polymicrobial (aerobe/anaerobe) and urinary infections.
Azithromycin was obtained from the Process Research Department of Pfizer Central Research. All other antimicrobials were obtained from their respective pharmaceutical manufacturers. Bacterial strains Salmonella enteritidis NCTC 5694 was used in oral challenge of mice. Staphylococcus aureus ATCC 21351 was employed in the rat disc model. Escherichia coli Pfizer 51A266 was used in acute and suture models of infection; Pfizer 51A028 was used in the urinary tract infection model. Staph. aureus Pfizer 01A052 and Bacteroides fragilis ATCC 25285 were used in the polymicrobial infection. Animals Mice (CF1; 11 to 13 g; both sexes) and CD male rats (250 or 90 g) were obtained from Charles River Breeding Laboratories Inc., Kingston, NY, USA. Media and growth conditions Challenge inocula of Staph. aureus, Salm. enteritidis and E. coli were grown on Brain Heart Infusion (BHI; Scott Laboratories, Fiskeville, RI, USA) agar. Bact. fragilis was grown in chopped-meat medium (Scott Labs) inside an anaerobic chamber. All cultures were incubated overnight at 37°C. Determinations of colony forming units (cfu) After the animals were killed, tissue samples (liver, kidney, suture site) were aseptically removed from individual animals, placed in pH 7-4 sterile buffered saline, and homogenized in a Stomacher 80 (Tekmar Co., Cincinnati, OH, USA). Serial ten-fold dilutions were prepared and plated on appropriate media. Material from the polymicrobial infection was plated on BHI agar enriched with vitamin K plus haemin and incubated in an anaerobic chamber. Differential counts of Bact. fragilis and Staph. aureus were possible because of the characteristic colonial morphologies of these organisms. Counts of Salm. enteritidis and E. coli were determined by plating on Brilliant Green and MacConkey agar, respectively (Difco Laboratories, Detroit, MI, USA). Staph. aureus was recovered from implanted discs, as described by Retsema etal. (1990, this Volume).
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Materials and methods Antimicrobial agents
Phannacokinetics andto-yivoefficacy
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Bioassay for azithromycin Samples were analysed by a modified agar well diffusion bioassay procedure using Micrococcus luteus ATCC9341 as the bioassay organism. Standard curves were prepared as appropriate in tissue homogenate, serum, or plasma from non-dosed animals. Models of infection used to examine the importance of azithromycin extravascular levels in efficacy
Models of localized infection to investigate the potential of azithromycin's extravascular phannacokinetics Models o/E. coli infection. An acute infection was produced in mice as described by Girard, et al. (1987). A urinary tract infection was established in anaesthetized CD rats (250 g) (sodium pentobarbital) by temporarily clamping the ureter with a haemostat. Challenge was then given iv. Animals were treated (50 mg/kg/dose) daily for three consecutive days. Mortality was not observed in untreated controls. Animals were killed on day 4, when kidneys were removed and cfu/kidney determined. A localized infection was induced by contaminating surgical silk with E. coli and placing a single suture (one square knot with loose ends trimmed) into the upper left back muscle of anaesthetized mice (sodium pentobarbital). The incision was closed with wound clips to prevent removal of the suture. Mice were treated (100 mg/kg/dose)
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Generally regimens were designed to provide very low blood levels (below the MIC) at the time of and after challenge. For clarity, the exact protocol used in each infection model is described below and presented with the data. Salmonella enteritidis oral challenge. This model has been described previously (Girard et al., 1987). Mice challenged by gavage developed systemic salmonellosis, with mortality occurring between days 7 and 11; the liver and spleen were usually involved by days 2-5. Blood and liver samples were taken for determination of drug concentrations and presence of salmonellae at the times indicated in Tables I and II. Survival results were also recorded at the end of the study. The first studies examined the effect of azithromycin administered prophylactically. Mice were treated with azithromycin at 50 mg/kg/dose tid injected subcutaneously for one day only, 48 h before challenge. Extravascular azithromycin levels produced by a multiple low dose therapeutic regimen were also studied. The efficacy of azithromycin was compared with that of ciprofloxacin. Animals were challenged orally and oral drug therapy was initiated 24 h later, continuing once daily for the next three days. The survivors and the cfu recovered from their livers were recorded seven days after challenge. Staph. aureus disc infection in rats. Rats (90 g) were given a single oral dose of azithromycin (lOOmg/kg) 72 h before challenge. At challenge, two paper discs (those commonly used in bioassay), one containing Staph. aureus and the other sterile, were implanted into two different sites on the back of anaesthetized rats (ketamine HC1/ xylazine). There was no mortality observed in this model. Blood samples were taken and discs were removed at various times. The cfu and extravascular concentrations of azithromycin in the inoculated and sterile discs were determined; plasma concentrations were determined from the blood samples.
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Statistical analysis Where indicated, the statistical significance (P value) was determined by the Student's f-test. The 50% protective dose (PT>X) was expressed in mg/kg/dose and calculated by the probit method as described by English, Retsema & Lynch (1976). Results Importance of azithromycin tissue concentrations in efficacy Oral salmonella challenge. All animals were free of salmonellae and untreated animals were also free of any antimicrobial agent just before oral introduction of the organism (Table I). Animals treated with azithromycin tid at 50 mg/kg 48 h earlier had serum concentrations one-tenth or less of the MIC for Salm. enteritidis at the time of and following challenge. These concentrations decreased with time. However, liver concentrations exceeded the MIC by two to four-fold for 48 h after challenge. At 24 h after challenge, no salmonellae were recovered from blood and liver samples taken Table I. The effect of serum and tissue concentration of azithromycin in determining efficacy in a Salm. enteritidis" infection model
Time after challenge (h) 0 24 48
Azithromycin concentration1 serum liver (mg/1) (mg/kg) 018 007 100
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both organisms and did not differ significantly from azithromycin. Cefoxitin effectively reduced the Bact. fragilis population, but was unimpressive against Staph. aureus. Erythromycin was not effective against either organism. Discussion
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Initial experiments were specifically designed to provide azithromycin plasma concentrations below the MIC at the time of challenge, while tissue concentrations generally exceeded the MIC. Our interpretation assumes that plasma concentrations below the MIC would have an insignificant influence on the response. By a parenteral prophylactic approach, azithromycin was shown to be effective against Saint, enteritidis, even though the azithromycin plasma concentrations were below the MIC at all times during and after challenge. Since tissue concentrations were maintained above the MIC while plasma concentrations were well below the MIC, and treated animals showed increased survival, it is clear that tissue concentrations correlated with a decrease in the progression and mortality from the infection. In addition to the prophylactic approach, therapeutic regimens that provided serum concentrations well below the MIC for the duration of the studies were evaluated. In these studies, ciprofloxacin was selected as the comparative agent, since it has been reported (Easmon & Crane, 1985a, b) to be concentrated in phagocytes (two- to fourfold in macrophages; four- to seven-fold in neutrophils) and effectively to kill engulfed Staph. aureus. Ciprofloxacin was also shown to be effective against systemic salmonella infection (Easmon & Blower, 1985). Since salmonellae are generally considered intracellular pathogens, successful therapy was attributed to the inherent in-vitro activity of ciprofloxacin (MIC 0-006 mg/1) and its good penetration into phagocytic cells. All azithromycin dose levels were effective in significantly reducing salmonella populations. Although mice given azithromycin at 5 mg/kg had peak serum concentrations one-tenth the MIC and peak liver concentrations only three to five times the MIC, this dose expressed greater in-vivo activity than ciprofloxacin given at 100 mg/kg. This is particularly striking, since ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this pathogen. These studies provide evidence that the bioavailability of azithromycin in the tissue site (liver) is significant. The lowest dose level used (5 mg/kg) gave peak liver concentrations only slightly higher than the MIC (3-5 x MIC). As this dose was efficacious, the bioavailability of azithromycin in the tissue site must be significant. In addition to tissue concentrations, the invivo performance of azithromycin may be related to its intracellular concentration and release at sites of infection (Gladue etal., 1989). These studies were not conducted to provide data to support the clinical use of azithromycin against salmonellosis in humans. However, the MIC^ of azithromycin for 16 isolates of Salm. enteritidis was reported to be 4mg/1 (Retsema etal., 1987). Furthermore, Metchock (1990, this Volume) reported a MIQm of 8 mg/1 azithromycin for 60 isolates of Salm. typhi; 20 of these isolates were resistant to chloramphenicol and tetracycline. Except for rosaramicin which showed a M I Q Q of 32 mg/1, none of the other macrolides (erythromycin, roxithromycin, clarithromycin, spiramycin, josamycin) evaluated against Salm. typhi had any activity (i.e. MIQoS > 128 mg/I). Jones, Felmingham & Ridgway (1988) also reported azithromycin MIC^s of 4 mg/1 for Salm. enteritidis and Salm. typhi. As plasmid-mediated resistance to chloramphenicol, ampicillin and other agents used to treat systemic salmonellosis is an ever present problem
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(Easmon & Blower, 1985), clinical trials of azithromycin against this disease may be considered. In a rat model using Staph. aureus, control of this Gram-positive localized infection correlated with extravascular concentrations rather than plasma concentrations. Throughout the study, plasma concentrations of azithromycin were well below, and extravascular concentrations were above, the MIC. In fact, while plasma concentrations were steadily declining from the time of challenge and during the entire course of the experiment, extravascular concentrations increased, with the highest concentration observed at 24 h after challenge (96 h after dosage). Since an acute cellular inflammatory response was observed in and about the implanted disc containing Staph. aureus, and azithromycin has been shown to achieve high intracellular concentrations in phagocytes (Gladue etal., 1989), the observations noted here are consistent with the possibility that host phagocytic cells may participate in delivery of azithromycin to the site. A variety of infection models, acute and chronic, were used to show the effect of the different pharmacokinetic characteristics of antibiotics. Antibiotics that rapidly develop high blood levels tend to perform well in acute models of infection, but those that do not develop high and sustained concentrations in tissue perform poorly in localized infection systems. In sites where drugs develop concentrations through elimination, such as kidney, many antibiotics perform well. It is clear that azithromycin exhibited exceptional activity in the localized E. coli tissue infection (suture), while not performing well against the acute infection. This was in direct contrast to the in-vivo activities of amoxycillin and cefaclor, both of which were quite effective against the acute infection in which they rapidly achieved high concentrations in serum, although their tissue distribution is poor. The in-vitro activities of azithromycin, amoxycillin, and cefaclor are all reflected in the UTI model, in which all develop effective levels of drug at the site of infection (Edberg & Sabath, 1980; Shepard & Falkner, 1990, this Volume). Similar comparisons were made in acute and more chronic tissue-associated infections utilizing Salm. enteritidis. Although the intraperitoneal challenge is described as acute because of the rapid onset of mortality (1-3 days compared with 7-11 days with the oral challenge), this infection is considerably less acute than those induced by E. coli or Staph. aureus (mortality in 8-24 h). Therefore agents that rapidly achieve inhibitory serum concentrations, or temporary inhibitory and tissue concentrations, can prevent the spread of salmonellae from the peritoneal cavity, if antibiotic is administered shortly after challenge. Salm. enteritidis is rapidly distributed to tissues following introduction into the peritoneal cavity. Azithromycin, amoxycillin, and cefaclor all achieve early circulating inhibitory concentrations of antibiotic. Presumably, these concentrations have a positive effect in preventing the distribution of the pathogen to tissues, and therefore give low PDJQS. In contrast, when the organism is given orally, a slowly developing infection is produced by initial infiltration of the Peyer's patches and then progression to liver and spleen by two to five days. Delaying initiation of treatment until 18 h after challenge further ensures that control of the disease is largely dependent on tissue concentrations. Consequently amoxycillin and cefaclor failed (PDM > 200 mg/kg) when administered after the establishment of infection, while azithromycin was highly effective (PDM 12-4 mg/kg). The evaluation of azithromycin against a polymicrobial localized infection further
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emphasizes the importance of tissue pharmacokinetics. Azithromycin performed particularly well in this model and was as effective as clindamycin, a recognized effective agent in this setting. Azithromycin has been shown to be effective in several other models of localized infection, including those induced by: Fusobacterwm necrophorum (Girard etal., 1987), Borrelia burgdorferi (Johnson et aL, 1990, this Volume), Legionella pneumophUa (Fitzgeorge, Featherstone & Baskerville, 1990, this Volume) and Treponema pallidum (Lukehart, Fohn & Baker-Zander, 1990, this Volume). These studies uniformly show that the outstanding in-vivo activity of azithromycin is due to its extravascular pharmacokinetics.
We acknowledge the significant technical contributions made by Ms Caroline R. Cimochowski, Mr James A. Faiella, and Mr William U. Schelkly in administration of doses and bacterial challenges, collection of samples, and bacteriological analysis and bioassay of specimens. References Boon, R. J. (1986). Experimental anaerobic localized tissue infection. In Experimental Models in Antimicrobial.Chemotherapy. Vol.2 (Zak, O. & Sande, M.A., Eds), pp. 122-7. Academic Press, London. Brook, I. (1987). Metronidazole and spiramycin in abscesses caused by Bacteroides spp. and Staphylococcus aureus in mice. Journal of Antimicrobial Chemotherapy 20, 713-8. Chabbert, Y., Boyer, F., Saviard, M., Boulingre, H. & Herve, J. (1957). Determination de Faction bactericide in vivo des antibiotiques dans la staphylococde renale de la souris. Annales de I'Institut Pasteur 92, 760-77. Eagle, H. (1948). Speculation as to the therapeutic significance of the penicillin blood level. Annals of Internal Medicine 28, 260-78. Easmon, C. S. F. & Blower, A. (1985). Ciprofloxacin treatment of systemic salmonella infection in sensitive and resistance mice. Journal of Antimicrobial Chemotherapy 16, 615-9. Easmon, C. S. F. & Crane, J. P. (1985a). Uptake of ciprofloxacin by macrophages. Journal of Clinical Pathology 38, 442-4. Easmon, C. S. F. & Crane, J. P. (1985ft). Uptake of ciprofloxacin by human neutrophils. Journal of Antimicrobial Chemotherapy 16, 67-73. Edberg, S. C. & Sabath, L. D. (1980). Antibiotic levels in body fluids. In Antibiotics in Laboratory Medicine, lstedn (Lorian, V., Ed.), pp.259. Williams & Wilkins, Baltimore, MD. English, A. R., Retsema, J. A. & Lynch, J. E. (1976). Laboratory evaluation of 3-(5tetrazoly)penam, a new semisynthetic beta-lactam antibacterial agent with extended broadspectrum activity. Antimicrobial Agents and Chemotherapy 10, 132-8. Fitzgeorge, R. B., Featherstone, A. S. R. & Baskerville, A. (1990). Efficacy of azithromycin in the treatment of guinea pigs infected with Legionella pneumophUa by aerosol. Journal of Antimicrobial Chemotherapy 25, Suppl.A, 101-8. Foulds, G. Shepard, R. M. & Johnson, R. B. (1990). The pharmacokinetics of azithromycin in human serum and tissues. Journal of Antimicrobial Chemotherapy 25, Suppl. A. 73-82. Girard, A. E., Girard, D., English, A. R., Gootz, T. D., Cimochowski, C. R., Faiella, J. A. et al. (1987). Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrobial Agents and Chemotherapy 31, 1948-54. Gladue, R. P., Bright, G. M., Isaacson, R. E. & Newborg, M. F. (1989). In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrobial Agents and Chemotherapy 33, 277-82.
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Acknowledgements
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Jones, K., Febningham, D. & Rklgway, G. L. (1988). In-vitro activity of azithromycin (CP62,993). A novel macrolide, against enteric pathogens. Drugs Under Experimental and Clinical Research XTV, 613-5. Johnson, R. G, Kodner, C , Russell, M. & Girard, D. (1990). In-vitro and in-vivo susceptibility of Borrelia burgdorferi to azithromycin. Journal of Antimicrobial Chemotherapy 25, Suppl. A. 33-8. Lukehart, S. A., Fohn, M. J. & Baker-Zander, S. A. (1990). Efficacy of azithromycin for therapy of active syphilis in the rabbit model. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 91-9. Metchock, B. (1990). In-vitro activity of azithromycin compared with other macrolides and oral antibiotics against Salmonella typhi. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 29-31. Retsema, J., Girard, A. Schelkly, W., Manousos, M., Anderson, M., Bright, G., etal. (1987). Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against Gram-negative organisms. Antimicrobial Agents and Chemotherapy 31, 1939-47. Retsema, J. A., Girard, A. E., Girard, D. & Milisen, W. B. (1990). Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 83-9. Shepard, R. M. & Falkner, F. C. (1990). Phannacokinetics of azithromycin in rats and dogs. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 49-60. Smith, C. R. (1988). The spiramycin paradox. Journal of Antimicrobial Chemotherapy 22, Suppl. B. 141-4.
Correlation of the extravascular pharmacokioetics of azithromycin with in-vivo efficacy in models of localized infection Arthur £ . Girard*, Dennis Girard and James A. Retsema
Infection models were used to clarify the roles of serum and extravascular concentrations in the in-vivo efficacy observed with azithromycin. In-vivo experiments were designed to give serum concentrations well below the MIC and tissue levels generally above the MIC at time of challenge and during the course of infection. The efficacy of azithromycin against a Salmonella entcritidis oral challenge (a tissue-associated infection model) in mice correlated directly with azithromycin liver levels, but not serum concentrations. The significance of extravascular pharmacokinetics was observed in a comparative study of azithromycin and ciprofloxacin against the salmonella challenge. Ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this organism, but azithromycin (Smg/kg) produced a greater reduction in cfu than ciprofloxacin (100 mg/kg) at the primary site of infection (liver). In another model, extravascular fluid levels, measured by bioassay of implanted paper discs, were compared with plasma levels in relation to control of a localized Staphylococcus aweus infection in rats. Extravascular fluid levels of azithromycin were greater than the MIC of the strain used for five days after a 100 mg/kg dose, while erythromycin levels were less than 20% of the MIC at 30 h after a 200 mg/kg dose. Serum concentrations of both compounds were less than 20% of the MIC at the time of challenge. The antibiotic levels at the site of infection correlated with the reduction of Staph. aureus cfu (99% with azithromycin compared with controls, P < 0-01; 0% with erythromycin) recovered from inoculated discs. The significance of extravascular concentrations of azithromycin was further supported in other models of localized infections induced with Escherichia coli or a mixture of Staph. aureus and Bacteroides fragilis.
Introduction The importance of extravascular pharmacokinetics in in-vivo efficacy is not a new observation. Eagle (1948) noted it was the concentration at the site of infection that was the important therapeutic consideration, and that the plasma level was of significance only in providing a measure of the tissue concentration. Later Chabbert et al. (1957) reported on the potential clinical significance of tissue levels as described in the spiramycin paradox, i.e. the efficacy associated with peak serum levels at or below the MIC. Recently Smith (1988) reviewed the possible explanation of this apparent paradox, focusing again on the tissue and intracellular concentrations of spiramycin. Orally administered azithromycin has been shown to be well absorbed in animals (Girard etal., 1987; Shepard & Falkner, 1990, this Volume) and man (Foulds, Shepard & Johnson, 1990, this Volume). Furthermore, rapid distribution to and concentration •Corresponding author 61 0305-7453/90/25A061 +11 S02.00/0
© 1990 The British Society for Antimicrobial Chemotherapy
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Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA
62
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in tissues occurs to a much greater extent than with erythromycin. The resulting high and sustained tissue levels of azithromycin were considered to lead to increased efficacy compared with crythromycin and other antibiotics in a series of mouse tissue infection models (Girard etal., 1987). However, it was possible that blood levels contributed significantly to the therapeutic outcome. In this investigation, we have evaluated the relative importance of azithromycin extravascular levels in efficacy, using treatment regimens and models of localized infection that differentiate between the contributions made by the tissue and the blood concentrations. Additionally we have investigated the potential of azithromycin to treat localized polymicrobial (aerobe/anaerobe) and urinary infections.
Azithromycin was obtained from the Process Research Department of Pfizer Central Research. All other antimicrobials were obtained from their respective pharmaceutical manufacturers. Bacterial strains Salmonella enteritidis NCTC 5694 was used in oral challenge of mice. Staphylococcus aureus ATCC 21351 was employed in the rat disc model. Escherichia coli Pfizer 51A266 was used in acute and suture models of infection; Pfizer 51A028 was used in the urinary tract infection model. Staph. aureus Pfizer 01A052 and Bacteroides fragilis ATCC 25285 were used in the polymicrobial infection. Animals Mice (CF1; 11 to 13 g; both sexes) and CD male rats (250 or 90 g) were obtained from Charles River Breeding Laboratories Inc., Kingston, NY, USA. Media and growth conditions Challenge inocula of Staph. aureus, Salm. enteritidis and E. coli were grown on Brain Heart Infusion (BHI; Scott Laboratories, Fiskeville, RI, USA) agar. Bact. fragilis was grown in chopped-meat medium (Scott Labs) inside an anaerobic chamber. All cultures were incubated overnight at 37°C. Determinations of colony forming units (cfu) After the animals were killed, tissue samples (liver, kidney, suture site) were aseptically removed from individual animals, placed in pH 7-4 sterile buffered saline, and homogenized in a Stomacher 80 (Tekmar Co., Cincinnati, OH, USA). Serial ten-fold dilutions were prepared and plated on appropriate media. Material from the polymicrobial infection was plated on BHI agar enriched with vitamin K plus haemin and incubated in an anaerobic chamber. Differential counts of Bact. fragilis and Staph. aureus were possible because of the characteristic colonial morphologies of these organisms. Counts of Salm. enteritidis and E. coli were determined by plating on Brilliant Green and MacConkey agar, respectively (Difco Laboratories, Detroit, MI, USA). Staph. aureus was recovered from implanted discs, as described by Retsema etal. (1990, this Volume).
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Materials and methods Antimicrobial agents
Phannacokinetics andto-yivoefficacy
63
Bioassay for azithromycin Samples were analysed by a modified agar well diffusion bioassay procedure using Micrococcus luteus ATCC9341 as the bioassay organism. Standard curves were prepared as appropriate in tissue homogenate, serum, or plasma from non-dosed animals. Models of infection used to examine the importance of azithromycin extravascular levels in efficacy
Models of localized infection to investigate the potential of azithromycin's extravascular phannacokinetics Models o/E. coli infection. An acute infection was produced in mice as described by Girard, et al. (1987). A urinary tract infection was established in anaesthetized CD rats (250 g) (sodium pentobarbital) by temporarily clamping the ureter with a haemostat. Challenge was then given iv. Animals were treated (50 mg/kg/dose) daily for three consecutive days. Mortality was not observed in untreated controls. Animals were killed on day 4, when kidneys were removed and cfu/kidney determined. A localized infection was induced by contaminating surgical silk with E. coli and placing a single suture (one square knot with loose ends trimmed) into the upper left back muscle of anaesthetized mice (sodium pentobarbital). The incision was closed with wound clips to prevent removal of the suture. Mice were treated (100 mg/kg/dose)
Downloaded from http://jac.oxfordjournals.org/ at University of Western Ontario on May 28, 2014
Generally regimens were designed to provide very low blood levels (below the MIC) at the time of and after challenge. For clarity, the exact protocol used in each infection model is described below and presented with the data. Salmonella enteritidis oral challenge. This model has been described previously (Girard et al., 1987). Mice challenged by gavage developed systemic salmonellosis, with mortality occurring between days 7 and 11; the liver and spleen were usually involved by days 2-5. Blood and liver samples were taken for determination of drug concentrations and presence of salmonellae at the times indicated in Tables I and II. Survival results were also recorded at the end of the study. The first studies examined the effect of azithromycin administered prophylactically. Mice were treated with azithromycin at 50 mg/kg/dose tid injected subcutaneously for one day only, 48 h before challenge. Extravascular azithromycin levels produced by a multiple low dose therapeutic regimen were also studied. The efficacy of azithromycin was compared with that of ciprofloxacin. Animals were challenged orally and oral drug therapy was initiated 24 h later, continuing once daily for the next three days. The survivors and the cfu recovered from their livers were recorded seven days after challenge. Staph. aureus disc infection in rats. Rats (90 g) were given a single oral dose of azithromycin (lOOmg/kg) 72 h before challenge. At challenge, two paper discs (those commonly used in bioassay), one containing Staph. aureus and the other sterile, were implanted into two different sites on the back of anaesthetized rats (ketamine HC1/ xylazine). There was no mortality observed in this model. Blood samples were taken and discs were removed at various times. The cfu and extravascular concentrations of azithromycin in the inoculated and sterile discs were determined; plasma concentrations were determined from the blood samples.
64
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Statistical analysis Where indicated, the statistical significance (P value) was determined by the Student's f-test. The 50% protective dose (PT>X) was expressed in mg/kg/dose and calculated by the probit method as described by English, Retsema & Lynch (1976). Results Importance of azithromycin tissue concentrations in efficacy Oral salmonella challenge. All animals were free of salmonellae and untreated animals were also free of any antimicrobial agent just before oral introduction of the organism (Table I). Animals treated with azithromycin tid at 50 mg/kg 48 h earlier had serum concentrations one-tenth or less of the MIC for Salm. enteritidis at the time of and following challenge. These concentrations decreased with time. However, liver concentrations exceeded the MIC by two to four-fold for 48 h after challenge. At 24 h after challenge, no salmonellae were recovered from blood and liver samples taken Table I. The effect of serum and tissue concentration of azithromycin in determining efficacy in a Salm. enteritidis" infection model
Time after challenge (h) 0 24 48
Azithromycin concentration1 serum liver (mg/1) (mg/kg) 018 007 100
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both organisms and did not differ significantly from azithromycin. Cefoxitin effectively reduced the Bact. fragilis population, but was unimpressive against Staph. aureus. Erythromycin was not effective against either organism. Discussion
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Initial experiments were specifically designed to provide azithromycin plasma concentrations below the MIC at the time of challenge, while tissue concentrations generally exceeded the MIC. Our interpretation assumes that plasma concentrations below the MIC would have an insignificant influence on the response. By a parenteral prophylactic approach, azithromycin was shown to be effective against Saint, enteritidis, even though the azithromycin plasma concentrations were below the MIC at all times during and after challenge. Since tissue concentrations were maintained above the MIC while plasma concentrations were well below the MIC, and treated animals showed increased survival, it is clear that tissue concentrations correlated with a decrease in the progression and mortality from the infection. In addition to the prophylactic approach, therapeutic regimens that provided serum concentrations well below the MIC for the duration of the studies were evaluated. In these studies, ciprofloxacin was selected as the comparative agent, since it has been reported (Easmon & Crane, 1985a, b) to be concentrated in phagocytes (two- to fourfold in macrophages; four- to seven-fold in neutrophils) and effectively to kill engulfed Staph. aureus. Ciprofloxacin was also shown to be effective against systemic salmonella infection (Easmon & Blower, 1985). Since salmonellae are generally considered intracellular pathogens, successful therapy was attributed to the inherent in-vitro activity of ciprofloxacin (MIC 0-006 mg/1) and its good penetration into phagocytic cells. All azithromycin dose levels were effective in significantly reducing salmonella populations. Although mice given azithromycin at 5 mg/kg had peak serum concentrations one-tenth the MIC and peak liver concentrations only three to five times the MIC, this dose expressed greater in-vivo activity than ciprofloxacin given at 100 mg/kg. This is particularly striking, since ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this pathogen. These studies provide evidence that the bioavailability of azithromycin in the tissue site (liver) is significant. The lowest dose level used (5 mg/kg) gave peak liver concentrations only slightly higher than the MIC (3-5 x MIC). As this dose was efficacious, the bioavailability of azithromycin in the tissue site must be significant. In addition to tissue concentrations, the invivo performance of azithromycin may be related to its intracellular concentration and release at sites of infection (Gladue etal., 1989). These studies were not conducted to provide data to support the clinical use of azithromycin against salmonellosis in humans. However, the MIC^ of azithromycin for 16 isolates of Salm. enteritidis was reported to be 4mg/1 (Retsema etal., 1987). Furthermore, Metchock (1990, this Volume) reported a MIQm of 8 mg/1 azithromycin for 60 isolates of Salm. typhi; 20 of these isolates were resistant to chloramphenicol and tetracycline. Except for rosaramicin which showed a M I Q Q of 32 mg/1, none of the other macrolides (erythromycin, roxithromycin, clarithromycin, spiramycin, josamycin) evaluated against Salm. typhi had any activity (i.e. MIQoS > 128 mg/I). Jones, Felmingham & Ridgway (1988) also reported azithromycin MIC^s of 4 mg/1 for Salm. enteritidis and Salm. typhi. As plasmid-mediated resistance to chloramphenicol, ampicillin and other agents used to treat systemic salmonellosis is an ever present problem
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(Easmon & Blower, 1985), clinical trials of azithromycin against this disease may be considered. In a rat model using Staph. aureus, control of this Gram-positive localized infection correlated with extravascular concentrations rather than plasma concentrations. Throughout the study, plasma concentrations of azithromycin were well below, and extravascular concentrations were above, the MIC. In fact, while plasma concentrations were steadily declining from the time of challenge and during the entire course of the experiment, extravascular concentrations increased, with the highest concentration observed at 24 h after challenge (96 h after dosage). Since an acute cellular inflammatory response was observed in and about the implanted disc containing Staph. aureus, and azithromycin has been shown to achieve high intracellular concentrations in phagocytes (Gladue etal., 1989), the observations noted here are consistent with the possibility that host phagocytic cells may participate in delivery of azithromycin to the site. A variety of infection models, acute and chronic, were used to show the effect of the different pharmacokinetic characteristics of antibiotics. Antibiotics that rapidly develop high blood levels tend to perform well in acute models of infection, but those that do not develop high and sustained concentrations in tissue perform poorly in localized infection systems. In sites where drugs develop concentrations through elimination, such as kidney, many antibiotics perform well. It is clear that azithromycin exhibited exceptional activity in the localized E. coli tissue infection (suture), while not performing well against the acute infection. This was in direct contrast to the in-vivo activities of amoxycillin and cefaclor, both of which were quite effective against the acute infection in which they rapidly achieved high concentrations in serum, although their tissue distribution is poor. The in-vitro activities of azithromycin, amoxycillin, and cefaclor are all reflected in the UTI model, in which all develop effective levels of drug at the site of infection (Edberg & Sabath, 1980; Shepard & Falkner, 1990, this Volume). Similar comparisons were made in acute and more chronic tissue-associated infections utilizing Salm. enteritidis. Although the intraperitoneal challenge is described as acute because of the rapid onset of mortality (1-3 days compared with 7-11 days with the oral challenge), this infection is considerably less acute than those induced by E. coli or Staph. aureus (mortality in 8-24 h). Therefore agents that rapidly achieve inhibitory serum concentrations, or temporary inhibitory and tissue concentrations, can prevent the spread of salmonellae from the peritoneal cavity, if antibiotic is administered shortly after challenge. Salm. enteritidis is rapidly distributed to tissues following introduction into the peritoneal cavity. Azithromycin, amoxycillin, and cefaclor all achieve early circulating inhibitory concentrations of antibiotic. Presumably, these concentrations have a positive effect in preventing the distribution of the pathogen to tissues, and therefore give low PDJQS. In contrast, when the organism is given orally, a slowly developing infection is produced by initial infiltration of the Peyer's patches and then progression to liver and spleen by two to five days. Delaying initiation of treatment until 18 h after challenge further ensures that control of the disease is largely dependent on tissue concentrations. Consequently amoxycillin and cefaclor failed (PDM > 200 mg/kg) when administered after the establishment of infection, while azithromycin was highly effective (PDM 12-4 mg/kg). The evaluation of azithromycin against a polymicrobial localized infection further
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emphasizes the importance of tissue pharmacokinetics. Azithromycin performed particularly well in this model and was as effective as clindamycin, a recognized effective agent in this setting. Azithromycin has been shown to be effective in several other models of localized infection, including those induced by: Fusobacterwm necrophorum (Girard etal., 1987), Borrelia burgdorferi (Johnson et aL, 1990, this Volume), Legionella pneumophUa (Fitzgeorge, Featherstone & Baskerville, 1990, this Volume) and Treponema pallidum (Lukehart, Fohn & Baker-Zander, 1990, this Volume). These studies uniformly show that the outstanding in-vivo activity of azithromycin is due to its extravascular pharmacokinetics.
We acknowledge the significant technical contributions made by Ms Caroline R. Cimochowski, Mr James A. Faiella, and Mr William U. Schelkly in administration of doses and bacterial challenges, collection of samples, and bacteriological analysis and bioassay of specimens. References Boon, R. J. (1986). Experimental anaerobic localized tissue infection. In Experimental Models in Antimicrobial.Chemotherapy. Vol.2 (Zak, O. & Sande, M.A., Eds), pp. 122-7. Academic Press, London. Brook, I. (1987). Metronidazole and spiramycin in abscesses caused by Bacteroides spp. and Staphylococcus aureus in mice. Journal of Antimicrobial Chemotherapy 20, 713-8. Chabbert, Y., Boyer, F., Saviard, M., Boulingre, H. & Herve, J. (1957). Determination de Faction bactericide in vivo des antibiotiques dans la staphylococde renale de la souris. Annales de I'Institut Pasteur 92, 760-77. Eagle, H. (1948). Speculation as to the therapeutic significance of the penicillin blood level. Annals of Internal Medicine 28, 260-78. Easmon, C. S. F. & Blower, A. (1985). Ciprofloxacin treatment of systemic salmonella infection in sensitive and resistance mice. Journal of Antimicrobial Chemotherapy 16, 615-9. Easmon, C. S. F. & Crane, J. P. (1985a). Uptake of ciprofloxacin by macrophages. Journal of Clinical Pathology 38, 442-4. Easmon, C. S. F. & Crane, J. P. (1985ft). Uptake of ciprofloxacin by human neutrophils. Journal of Antimicrobial Chemotherapy 16, 67-73. Edberg, S. C. & Sabath, L. D. (1980). Antibiotic levels in body fluids. In Antibiotics in Laboratory Medicine, lstedn (Lorian, V., Ed.), pp.259. Williams & Wilkins, Baltimore, MD. English, A. R., Retsema, J. A. & Lynch, J. E. (1976). Laboratory evaluation of 3-(5tetrazoly)penam, a new semisynthetic beta-lactam antibacterial agent with extended broadspectrum activity. Antimicrobial Agents and Chemotherapy 10, 132-8. Fitzgeorge, R. B., Featherstone, A. S. R. & Baskerville, A. (1990). Efficacy of azithromycin in the treatment of guinea pigs infected with Legionella pneumophUa by aerosol. Journal of Antimicrobial Chemotherapy 25, Suppl.A, 101-8. Foulds, G. Shepard, R. M. & Johnson, R. B. (1990). The pharmacokinetics of azithromycin in human serum and tissues. Journal of Antimicrobial Chemotherapy 25, Suppl. A. 73-82. Girard, A. E., Girard, D., English, A. R., Gootz, T. D., Cimochowski, C. R., Faiella, J. A. et al. (1987). Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrobial Agents and Chemotherapy 31, 1948-54. Gladue, R. P., Bright, G. M., Isaacson, R. E. & Newborg, M. F. (1989). In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrobial Agents and Chemotherapy 33, 277-82.
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Acknowledgements
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Jones, K., Febningham, D. & Rklgway, G. L. (1988). In-vitro activity of azithromycin (CP62,993). A novel macrolide, against enteric pathogens. Drugs Under Experimental and Clinical Research XTV, 613-5. Johnson, R. G, Kodner, C , Russell, M. & Girard, D. (1990). In-vitro and in-vivo susceptibility of Borrelia burgdorferi to azithromycin. Journal of Antimicrobial Chemotherapy 25, Suppl. A. 33-8. Lukehart, S. A., Fohn, M. J. & Baker-Zander, S. A. (1990). Efficacy of azithromycin for therapy of active syphilis in the rabbit model. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 91-9. Metchock, B. (1990). In-vitro activity of azithromycin compared with other macrolides and oral antibiotics against Salmonella typhi. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 29-31. Retsema, J., Girard, A. Schelkly, W., Manousos, M., Anderson, M., Bright, G., etal. (1987). Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against Gram-negative organisms. Antimicrobial Agents and Chemotherapy 31, 1939-47. Retsema, J. A., Girard, A. E., Girard, D. & Milisen, W. B. (1990). Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 83-9. Shepard, R. M. & Falkner, F. C. (1990). Phannacokinetics of azithromycin in rats and dogs. Journal of Antimicrobial Chemotherapy 25, Suppl. A, 49-60. Smith, C. R. (1988). The spiramycin paradox. Journal of Antimicrobial Chemotherapy 22, Suppl. B. 141-4.
