a Department of Thoracic Medicine, City Hospital, Birmingham, UK b Department of Medical Microbiology, City Hospital, Birmingham, UK
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Abstract |
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Introduction |
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Materials and methods |
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Sample collection and processing
Samples of bronchial mucosa (BM) were taken from macroscopically normal areas of the lung, in addition to diagnostic samples. Standard bronchoalveolar lavage (BAL) was performed using 200 mL of prewarmed 0.9% saline divided into four 50 mL aliquots, followed by gentle aspiration. Aspirate from the first 50 mL was discarded to avoid contamination of the sample with larger airway fluids and cells. The remaining three aspirates were pooled and divided into two Teflon-coated containers for analysis. A small volume of lavage fluid was removed from each sample and the number of macrophages was counted, using an improved Neubauer counting chamber. The remaining lavage aspirate was immediately centrifuged at 400g for 5 min and the supernatant and cells separated. Approximately 2 mL of the supernatant was removed for estimation of urea content. The remaining fluid was used to measure the concentration of moxifloxacin present, by freeze drying followed by the addition of distilled water to reconstitute to one-tenth of the original volume. Other samples were prepared for assay as follows. Cell pellets were ultrasonicated on ice using a known volume of chilled phosphate buffer pH 7 before assay. BM biopsies from each patient were pooled in a humidity chamber, to avoid loss of moisture from tissue before weighing. Samples were weighed (heavily blood-stained tissue was discarded) and ultrasonicated as previously described.3 Immediately after bronchoscopy, serum samples were taken for measurement of urea and moxifloxacin levels. All samples were stored at 4°C and protected from light before assay. All assays were performed within 2 h of collection, with the exception of those on the lavage samples.
Microbiological assay
Concentrations of moxifloxacin were measured using a microbiological assay. Briefly, assay plates (Mast Diagnostics, Bootle, UK) containing IsoSensitest agar (Oxoid, Basingstoke, UK) were flooded with an organism suspension (Escherichia coli 4004, Bayer Wuppertal, AG) adjusted to an optical density of 0.004 at 630 nm. Antibiotic standards, prepared in human serum (range 0.1252 mg/L), phosphate buffer pH 7 (range 0.061 mg/L) and 9% sodium chloride (range 0.061 mg/L), internal controls and tests were applied to the plate (in triplicate following a random pattern), by filling 5 mm wells which had been cut from the agar with a cork borer. After overnight incubation at 30°C, zones were measured using an image analyser (Imaging Associates, Theme, UK) and the concentration calculated using Bennet's calculation.4
Calculation of antibiotic concentrations
Bronchial mucosa (BM). Moxifloxacin concentration was calculated from the formula described below.
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Alveolar macrophages (AM). Antibiotic concentration in macrophages was determined assuming a mean cell volume of an alveolar macrophage of 2.48 µL/106 cells.5
Epithelial lining fluid (ELF). BAL fluid urea concentration was determined using a modified Sigma Diagnostic Kit (UV-66, Sigma Chemicals, Poole, UK). The ELF moxifloxacin level was calculated using the method described by Renard et al.6
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where ACL is the antibiotic concentration in the lavage fluid (mg/L), BU is the blood urea concentration (mmol/L) and LU is the lavage fluid urea concentration (mmol/L).
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Results |
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Discussion |
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Compared with trovafloxacin,9 mean site:serum ratios (at 12 and 24 h) were greater for moxifloxacin. Higher site: serum ratios have been reported after multiple dosing with quinolones.9 The AUC/MIC ratio (AUIC) indicates the relationship between the pharmacokinetics of an antibiotic and its pharmacodynamic interaction with pulmonary pathogens. The AUIC has been used as a surrogate marker for predicting clinical outcomes. A threshold value of 125 for the AUIC has been proposed as a marker for efficacy.10 As shown in Table II this threshold was exceeded in all sites at the time of measurement.
Alveolar macrophage penetration by moxifloxacin was, as with other quinolones, excellent, suggesting clinical efficacy against atypical organisms associated with lower respiratory tract infection such as mycoplasma and chlamydia.
Overall, moxifloxacin should be effective against a wide variety of respiratory tract pathogens. In view of recently increasing penicillin resistance of the pneumococcus, it is of importance that moxifloxacin should demonstrate efficacy against this pathogen.
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Acknowledgments |
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Notes |
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Tel: +44-121-766-6611;
Fax:
+44-121-772-0292; E-mail: davidhoneybourne{at}compuserve.com
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References |
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2 . Wise, R. & Honeybourne, D. (1996). A review of the penetration of sparfloxacin into the lower respiratory tract and sinuses. Journal of Antimicrobial Chemotherapy 37, Suppl. A, 5763.[ISI][Medline]
3 . Honeybourne, D., Andrews, J. M., Ashby, J. P., Lodwick, R. & Wise, R. (1988). Evaluation of the penetration of ciprofloxacin and amoxycillin into the bronchial mucosa. Thorax 43, 7159.[Abstract]
4 . Bennet, J. V., Brodie, J. L., Benner, E. J. & Kirby, W. M. (1966). Simplified, accurate method for antibiotic assay of clinical specimens. Applied Microbiology 14, 1707.[ISI][Medline]
5 . Johnson, J. D., Hand, W. L., Francis, J. B., King-Thompson, N. L. & Corwin, R. W. (1980). Antibiotic uptake by alveolar macrophages. Journal of Laboratory and Clinical Medicine 95, 42939.[ISI][Medline]
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Rennard, S. I., Basset, G., Lecossier, D.,
O'Donnell,
K. M., Pinkston, P., Martin, P. et al. (1986). Estimation of volume of
epithelial
lining fluid recovered by lavage using urea as a marker of dilution. Journal of Applied
Physiology 60, 5328.
7 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In-vitro activity of BAY 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 1016.[Abstract]
8 . Renaudin, H., Bebear, D. & Boudjadja, A. (1996). In-vitro activity of BAY 12-8039, a new fluoroquinolone, against mycoplasmas. In Programme and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy. Poster, Abstract F9.
9 . Andrews, J. M., Honeybourne, D., Brenwald, N. P., Bannerjee, D., Iredale, M., Cunningham, B. et al. (1997). Concentrations of trovafloxacin in bronchial mucosa, epithelial lining fluid, alveolar macrophages and serum after administration of single or multiple oral doses to patients undergoing fibre-optic bronchoscopy. Journal of Antimicrobial Chemotherapy 39, 797802.[Abstract]
10 . Forrest, A., Nix, D. E., Ballow, C. H., Goss, T. F., Birmingham, M. C. & Schentag, J. J. (1993). The pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrobial Agents and Chemotherapy 37, 107381.[Abstract]
Received 24 March 1999; returned 15 June 1999; revised 9 August 1999; accepted 31 August 1999