1 Nottingham City Hospital, Hucknall Road, Nottingham NG5 1PB; 2 Antibiotic Research Laboratory, City Hospital NHS Trust, Dudley Road, Birmingham B18 7QH, UK
Received 8 April 2002; returned 24 June 2002; revised 2 August 2002; accepted 15 August 2002
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Abstract |
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Introduction |
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For many antimicrobials, it is important to maintain inhibitory serum concentrations during most of the intervals between doses in order to optimize clinical and bacteriological success.1 However, these observations have largely been validated for extracellular bacterial infections. The response of intracellular bacterial infections may be predicted better from tissue to serum ratios,2,3 especially for infected body sites possessing a permeable membrane such as the alveolar epithelium.
Early studies of drug distribution in the respiratory tract examined whole lung tissue, sputum and pleural fluid. However, this can result in unsatisfactory information. Antimicrobial concentrations in whole lung represent an average of several different tissue compartments. Sputum concentrations often vary because of sampling difficulties, sputum pooling and contamination with blood, while drug instability makes conclusions about pharmacokinetic behaviour unreliable. Pleural fluid concentrations are only likely to be useful in predicting efficacy in the treatment of empyema. Sampling the bronchial mucosa has been used to indicate the likely response to infections, such as exacerbations of chronic bronchitis and possibly bronchiectasis, while bronchoalveolar lavage (BAL) has provided the opportunity to study alveolar infections such as pneumonia. Epithelial lining fluid (ELF) and alveolar macrophages (AM) represent extracellular and intracellular sites, respectively, and have been increasingly studied as predictors of drug behaviour. The objective of our study was to determine the distribution of rifampicin in pulmonary tissues in comparison with those in serum after a standard single adult oral dose of 600 mg in order to define optimal concentrations better when treating intracellular or extracellular lung infection.
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Materials and methods |
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Fifteen patients (11 males and four females) undergoing routine fibre-optic bronchoscopy for diagnostic purposes were included in the study. The study was approved by the Hospital Ethics Committee and all patients gave written informed consent before entry into the study. The mean age was 63.7 years (range 3475 years) and the mean weight was 65 kg (range 4891 kg). Patients were excluded if they had an active pulmonary infection, known hypersensitivity or intolerance to rifampicin, or evidence or history of significant pre-existing haematological, renal, cardiovascular or hepatic disease. Exclusion criteria also included: disturbance of hepatic function (serum transaminase more than twice the upper limit of normal and/or bilirubin above the normal range); other medications that could potentially interact with rifampicin; patients with malabsorption or other conditions affecting drug uptake; smoking on a regular basis within 6 months of study entry; lactation or pregnancy. Subjects were not permitted to take any drugs other than those prescribed by a physician in the 2 weeks prior to the study.
Dosage regimens
This was an open-label pharmacokinetic study designed to assess the distribution of rifampicin in bronchial tissues and fluids following the administration of a single oral dose of 600 mg (2 x 300 mg capsules) administered with 120 mL of tap water. The exact time of administration was noted.
Sample collection
Each patient underwent standard bronchoscopic examination between 2 and 5 h post-dose (having fasted for the previous 4 h). BAL was performed on each patient using 200 mL of pre-warmed 0.9% saline divided into four 50 mL aliquots, followed by gentle aspiration. The initial instillation was aspirated and discarded since this is contaminated by cells from the proximal airways.4 The three remaining samples of aspirate were pooled and divided into two Teflon-coated containers for analysis. Lavage time was maintained at <4 min in order to limit the concentration-dependent efflux of solutes into BAL fluid. The total leucocyte count in the lavage fluid was measured within 5 min of sample collection using an improved Neubauer haemocytometer. Within 5 min of lavage, the pooled BAL fluid was centrifuged at 400g for 5 min. After separation of the supernatant from the cells, 2 mL of the supernatant was removed in order to estimate the urea level in the lavage sample. The supernatant as well as the cells were stored at 80°C until assay. Two venous blood samples were collected: the first sample 10 min before the medication was administered and the second within 5 min of bronchoscopy. Both samples were immediately centrifuged at 1500g for 15 min at 4°C. Sera were separated promptly and stored until assay at 80°C. Bronchial biopsies were taken from macroscopically normal subcarinal mucosa in all patients and stored at 80°C until assay. The samples were transported on ice to the City Hospital NHS Trust, Birmingham (CHT), within 1 week of collection and all assays were undertaken within 1 week of receipt.
Sample processing and microbiological assay
Samples were allowed to thaw to room temperature. Macrophages and bronchial biopsies were ultrasonicated on ice in a known volume of chilled pH 6 phosphate buffer prior to assay, using a sonicator (Life Sciences, Luton, UK). Lavage samples were freeze dried (Edwards, Crawley, UK) where necessary and then reconstituted to one-tenth the original volume with sterile distilled water.
Concentrations of rifampicin (Lepetit, Spain) were measured in macrophages, ELF and plasma samples using an adaptation of a microbiological assay5 using Iso-sensitest agar (Oxoid, Basingstoke, UK) supplemented with 30 g/L potassium dihydrogen orthophosphate (Scientific and General, West Midlands, UK) to give a final pH of 5.5. One per cent Bacteriological agar (Oxoid) was added to aid media setting. The assay organism (Staphylococcus aureus ATCC 29219) was grown overnight at 3537°C in Tryptone soya broth (Oxoid) and diluted in sterile distilled water to give an optical density of 0.022 at 630 nm before surface flooding the plate. Five millimetre diameter wells were punched into the agar using a random pattern and the plugs removed and discarded. The agar plates were then stored at 4°C until use. The lower limit of detection of the assay was 0.03 mg/L and the between-assay coefficient of variation was 6.8% over a concentration range of 0.031 mg/L.
Calibrators and internal controls were prepared in human serum (Tissue Culture Services, Botolph Clayton, UK), for the assay of plasma samples, or pH 6 phosphate buffer for the assay of macrophages, BAL and bronchial mucosa, and 0.9 % sodium chloride for any BAL samples requiring concentration. Calibrators used, irrespective of the matrix, were 1.28, 0.64, 0.32, 0.16 and 0.08 mg/L with internal controls of 1.0 and 0.1 mg/L. Human serum calibrators and internal controls were prepared prior to the study, aliquots were made in Sarstedt pots (Sarstedt, Leicester, UK) and stored at 70°C until use (thawed once and then discarded). Phosphate buffer and 0.9% sodium chloride calibrators and internal controls were prepared on the day of assay. Quality assurance samples were prepared on the day of assay in human plasma at concentrations within the calibrator range. The samples were tested in triplicate using a random pattern. The wells were filled to give a convex meniscus. Plates were incubated overnight at 37°C and zone diameters measured using an image analyser (Imaging Associates, Thame, UK), which was pre-programmed with Bennetts calculation6 to give a line of best fit. Urea concentrations in BAL were determined using a commercial diagnostic kit (Sigma BUN endpoint; Sigma, Poole, UK). The urea assay measured blood urea nitrogen (Kit No. UV-66). This was modified by adjusting the sample: reagent ratio, which was adjusted to 1:1 instead of the 1:200 ratio recommended when testing serum samples because of the extremely low concentrations of urea in ELF. Standards were prepared from a single quality control reference standard and diluted in normal saline. The between-assay coefficient of variation was 7.8%.
Calculation of antibiotic concentration
Calculations of the antibiotic concentrations in biopsy, AM, BAL and plasma were calculated as described previously.5 Briefly, for bronchial biopsy,
[assay concentration (mg/L) x (weight of sample + volume of buffer)]/weight of sample (mg) = concentration (mg/kg)
For epithelial lining fluid, a urea dilution method was used as described by Rennard et al.7 The method was applied to the standard lavage (BAL) specimen and the antibiotic concentration was calculated as follows:
(AC x BU)/LU = ELF concentration (mg/L)
where AC = antibiotic concentration in lavage (mg/L), BU = blood urea level (mmol/L) and LU = lavage urea (mmol/L).
Macrophage concentrations were determined using the mean cell volume of an alveolar macrophage of 2.48 µL/106 cells from data derived by Johnson et al.8 The cellular antimicrobial concentration was calculated as follows: (i) total cell count (TCC) = volume of lavage x cell count/mL x fraction of AM in count; (ii) total cell volume (TCV) = TCC x volume of each cell; and (iii) cell antimicrobial concentration = (assayed concentration x volume of resuspended pellet)/TCV.
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Results |
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No adverse events that were possibly drug related were recorded. Following bronchoscopy two subjects experienced self-limited light-headedness of unclear aetiology during the period of observation.
Mean concentrations of rifampicin in serum, ELF, macrophages and biopsies of bronchial mucosa, as well as site:serum ratios, are presented in Table 1. However, this ratio does not take into account the kinetics of tissue distribution, which is best assessed by determination of the ratios of the area under the curve (AUC) values. Both peak tissue levels and the AUC in tissue may provide important information regarding efficacy. However, determination of the AUC in tissue requires serial samples that were not possible in our study population. Figure 1 shows the concentrations of rifampicin measured in serum, ELF, AM and bronchial mucosa plotted against time following administration of the drug in relation to the MIC90s for Mycobacterium tuberculosis.
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Discussion |
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The alveolar epithelial membrane presents a significant barrier by separating ELF from blood and should be considered a pharmacologically protected environment. Clinical efficacy, particularly against pathogens confined to sites separated from blood by natural barriers, may be more closely related to site/serum-specific drug concentrations. This study has shown satisfactory concentrations of rifampicin at all potential sites of respiratory infection in relation to known MIC90s of target pathogens, and compares favourably with data on other rifamycin derivatives.13 Although the levels for ELF are lower than those for bronchial mucosa and serum concentrations, they are still well in excess of the MIC90 of rifampicin for S. aureus (0.02 mg/L), Legionella spp. (0.25 mg/L) and M. tuberculosis (0.5 mg/L),11,12 which is indicated in Figure 1.
Based on these single dose observations in uninfected volunteers, current dosage regimens of an oral dose of 600 mg rifampicin would appear to be pharmacokinetically appropriate for target pathogens. The limitation of single dose studies is recognized. Ideally multiple-dose steady-state information is desirable and is a logical sequence to this study. Because of the fact that rifampicin is a potent inducer of its own metabolism, we expect that concentrations at steady state will usually be lower than those following single dose administration. Although technically challenging it would be valuable to repeat these studies in patients undergoing active treatment for lung infection in order to provide more clinically applicable information.
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Acknowledgements |
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Footnotes |
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References |
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2 . Kroening, U., Liebig, S. & Wundschock, M. (1978). Tobramycin concentration in human lung tissue (authors translation). Infection 6, 2315.[ISI][Medline]
3 . Stewart, S. M., Anderson, I. M., Jones, G. R. & Calder, M. A. (1974). Amoxycillin levels in sputum, serum, and saliva. Thorax 29, 1104.[ISI][Medline]
4 . Kelly, C. A., Kotre, C. J., Ward, C., Hendrick, D. J. & Walters, E. H. (1987). Anatomical distribution of bronchoalveolar lavage fluid as assessed by digital subtraction radiography. Thorax 42, 6248.[Abstract]
5 . 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]
6 . Bennett, 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]
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Rennard, S. I., Basset, G., Lecossier, D., ODonnell, K. M., Pinkston, P., Martin, P. G. et al. (1986). Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. Journal of Applied Physiology 60, 5328.
8 . Johnson, J. D., Hand, W. L., Francis, J. B., King-Thompson, N. & Corwin, R. W. (1980). Antibiotic uptake by alveolar macrophages. Journal of Laboratory and Clinical Medicine 95, 42939.[ISI][Medline]
9 . Sensi, P., Maggi, N., Furesz, S. & Maffii, G. (1966). Chemical modifications and biological properties of rifamycins. Antimicrobial Agents and Chemotherapy 6, 699714.
10 . Hand, W. L., Boozer, R. M. & King-Thompson, N. L. (1985). Antibiotic uptake by alveolar macrophages of smokers. Antimicrobial Agents and Chemotherapy 27, 425.[ISI][Medline]
11 . Dixson, S., Brumfitt, W. & Hamilton-Miller, J. M. (1984). Activity of rifampicin against staphylococci, with special reference to multiresistant strains. Journal of Antimicrobial Chemotherapy 13, Suppl. C, 716.[ISI][Medline]
12 . Thornsberry, C., Hill, B. C., Swenson, J. M. & McDougal, L. K. (1983). Rifampin: spectrum of antibacterial activity. Review of Infectious Disease 5, Suppl. 3, S4127.[ISI][Medline]
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Conte, J. E., Jr, Golden, J. A., McQuitty, M., Kipps, J., Lin, E. T. & Zurlinden, E. (2000). Single-dose intrapulmonary pharmacokinetics of rifapentine in normal subjects. Antimicrobial Agents and Chemotherapy 44, 98590.
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