Intrapulmonary penetration of linezolid

David Honeybourne1,*, Caroline Tobin2, Gail Jevons3, Jenny Andrews3 and Richard Wise3

1 Department of Respiratory Medicine, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS; 2 Department of Microbiology, Southmead Hospital, Bristol; 3 Department of Medical Microbiology, City Hospital, Birmingham, UK

Received 23 October 2002; returned 6 December 2002; revised 3 March 2003; accepted 19 March 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objective: This study was designed to measure the concentrations of linezolid in bronchial mucosa, pulmonary macrophages and epithelial lining fluid and to compare them with simultaneous blood levels.

Methods: Ten adult patients undergoing bronchoscopy for diagnostic purposes were given oral linezolid at a dosage of 600 mg twice a day for a total of six doses. Patients with active lung infection were excluded from the study. Flexible bronchoscopy was carried out between 2 and 8 h after the last dose of linezolid. Bronchial biopsies and bronchoalveolar lavage were carried out and a simultaneous blood sample obtained. Linezolid levels were measured using high-performance liquid chromatography (HPLC).

Results: Mean concentrations of linezolid were 13.4 mg/L in serum, 10.7 mg/kg in mucosa, 8.1 mg/L in alveolar macrophages and 25.1 mg/L in epithelial lining fluid. The mean site/serum concentration ratios were 0.79 for bronchial mucosa, 0.71 for macrophages and 8.35 for epithelial lining fluid.

Conclusions: The MIC90 (<=4 mg/L) of linezolid for Staphylococcus aureus and Streptococcus pneumoniae was exceeded in serum and bronchial mucosa in all subjects, in epithelial lining fluid in nine subjects and in macrophages in six subjects.

Keywords: linezolid, lung penetration


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Linezolid is an oxazolidinone recently licensed in Europe and the USA. Oxazolidinones uniquely inhibit bacterial protein synthesis at an early stage of translation. Linezolid is active against a variety of Gram-positive organisms, including Staphylococcus spp., Streptococcus pneumoniae and the enterococci. Linezolid has high potency against penicillin-resistant and also macrolide-resistant pneumococci.1 Phase III trials demonstrate efficacy in community and nosocomial pneumonia and infections due to the drug-resistant bacteria methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).1 Linezolid is also active against mycobacteria.1 Linezolid is the first oxazolidinone antibiotic to complete clinical development.

Absorption of linezolid after oral dosing is rapid and Cmax is achieved 1–2 h after dosing.1 The Cmax after multiple doses of 600 mg twice a day is 21 mg/L. Oral bioavailability approaches 100%. Pharmacodynamic studies in rats suggest that time above MIC is the main predictor of efficacy against S. pneumoniae.2 Linezolid has a relatively high volume of distribution and penetrates well into inflammatory fluid.3 In the treatment of infections, the concentration of the anti-infective at the site of infection is generally considered to be important.4 Epithelial lining fluid (ELF), which bathes the alveoli, is considered to be an important site of infection in pneumonia. Equally important are alveolar macrophages (AMs), which represent an important site for intracellular infection.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The aim of this study was to assess the concentrations of linezolid in serum, bronchial mucosa and bronchoalveolar lavage fluid (epithelial lining fluid and macrophages) at various times after drug administration and to compare these tissue levels with serum levels in patients undergoing elective fibre-optic bronchoscopy.

The study was an open-labelled, repeat-dose design in patients undergoing elective flexible bronchoscopy. Ten patients (five male) undergoing diagnostic flexible bronchoscopy were enrolled. All patients were over 18 years of age and all female subjects were post-menopausal. The Hospital Ethics Committee approved the study and all subjects gave fully informed, written consent. Patients were excluded if they had an active respiratory tract infection, significant hepatic or renal disease, severe cardiac failure or if they were taking any drugs known to potentially interact with linezolid. All subjects were assessed within 14 days before bronchoscopy. The assessment included a medical history, physical examination and blood analysis for haematology and biochemistry. This assessment was repeated within 14 days after bronchoscopy.

Indications for bronchoscopy were to investigate either a chest X-ray abnormality or symptoms of haemoptyses. No subjects were subsequently found to have a pulmonary malignancy. Chronic obstructive pulmonary disease was the final diagnosis in seven subjects, and no active pulmonary disease was found in three subjects.

The dose administered was 600 mg of linezolid twice a day orally for a total of six doses. Subjects fasted for at least 4 h beforehand and from fluids for 2 h before bronchoscopy. Bronchoscopy was carried out between 2 and 8 h after the final dose of linezolid to obtain samples of bronchial mucosa, bronchoalveolar lavage (BAL) and macrophages. A total of 10 subjects were recruited.

At bronchoscopy, bronchoalveolar lavage was carried out and mucosal samples obtained along with simultaneous blood specimens. The lavage aspirate was centrifuged and the cell pellet containing mainly macrophages and the supernatant containing the ELF were analysed for linezolid concentrations. All of the cell pellets were predominantly composed of macrophages, as determined by microscopy. Linezolid concentrations in ELF were calculated using the urea diffusion technique. Linezolid concentrations were assayed using isocratic HPLC.5

Sample collection

Bronchoscopy samples were collected as previously de-scribed.6 Briefly, at bronchoscopy bronchial mucosa biopsies and bronchoalveolar lavage were carried out using standard procedures. In the case of the BAL, 200 mL of pre-warmed 0.9% saline was divided into four 50 mL aliquots followed by gentle aspiration. The aspirate from the first aliquot was discarded to avoid contamination of the sample with proximal airway fluids and cells. The remaining aspirates were pooled for analysis.

Processing of BAL and bronchial mucosa

A small amount of lavage from each of the samples was removed, using siliconized glassware, and placed into an improved Neubauer counting chamber so that the number of macrophages in the lavage could be counted under a microscope. The remainder of the lavage was centrifuged immediately in Teflon containers at 400g for 5 min. After centrifugation, the supernatant was separated from the cells without delay. Approximately 2 mL of the supernatant was removed so that the urea level in the lavage sample could be measured. The remainder of the supernatant was then used for linezolid assay. The lavage sample was freeze-dried and then reconstituted with distilled water to one-tenth of the original volume. Macrophages were ultrasonicated once in a known volume of chilled pH 7 phosphate buffered saline before assay. A previous study reported no drug loss after sonication and freeze drying.7

Bronchial biopsies were collected into a humidity chamber to avoid loss of moisture from the tissue, any blood-stained tissue was discarded. Tissue was weighed and then ultrasonicated in a known volume of chilled pH 7 buffer before assay.

Serum and tissue specimens were prepared for assay by the addition (50:50) of acetonitrile (Chromanorm-grade, Prolabs Fontenay, France). The samples were mixed, allowed to rest at room temperature for 10 min and centrifuged for 5 min (2000 rpm). Twenty microlitres of the supernatant was injected. Samples were frozen at –70°C and transported for assay.

Calculation of linezolid in epithelial lining fluid (ELF), alveolar macrophages (AM) and bronchial mucosa (BM)

For the calculation of drug concentration in the epithelial lining fluid (ELF), the following formula was used, where [ ] indicates concentration:

[antibiotic] ELF = [antibiotic] BAL x [urea] serum/[urea] BAL

The concentration of urea in BAL was determined using a modified Sigma Diagnostic Kit (UV-66, Sigma Chemicals, Poole, UK).

The concentration of antibiotic in bronchial biopsy was calculated according to the following formula:

Ct = [Ca x (Vb + W)]/W

where Ca is the assayed concentration (mg/L), Vb is the volume of buffer added to the tissue (µL) and W is the weight of the biopsy (mg).

Antibiotic concentrations in macrophages were determined assuming a mean cell volume of an alveolar macrophage of 2.48 µL/106 cells.6

Assay

Linezolid was assayed using isocratic HPLC as described in detail elsewhere.5 The lower limit of quantification is 0.1 mg/L. The intra-day reproducibility was <6.0% and the inter-day reproducibility <12.5%. This method using HPLC has been used successfully to quantify linezolid in bone, fat and muscle.8

We assayed three sets of drug-free samples to investigate for any potential chromatographic interference with the linezolid peak. None was found.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The 10 subjects had a mean age of 59 (range 41–75 years). Levels of linezolid in serum, macrophages, ELF and bronchial mucosa in individual subjects are shown in Table 1. The individual site/serum concentration ratios are listed in Table 2.


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Table 1.  Levels of linezolid in individual subjects
 

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Table 2.  Lung site/serum ratios of linezolid in individual subjects
 
These results confirm good penetration of linezolid into pulmonary tissues with concentrations in bronchial mucosa in all subjects, ELF in nine subjects and alveolar macrophages in six subjects exceeding the MIC90 values (<=4 mg/L) of linezolid for S. aureus (including MRSA) and S. pneumoniae for at least 8 h after the last dose. The subjects had received six doses of linezolid over the 72 h before bronchoscopy and taking into account the T1/2 (5.5 h)1 steady-state tissue concentrations are likely to have been achieved. The suggested MIC breakpoint for S. aureus is 4 mg/L or less and 2 mg/L or less for penicillin-susceptible and -resistant pneumococci.9 Linezolid has some in vitro activity against Chlamydia and Mycoplasma spp.,1 but there are limited data on clinical efficacy against these respiratory pathogens.

The mean concentration in alveolar macrophages was higher than reported in another study7 despite an unexplained low concentration of 0.5 mg/L in one of our subjects. Many of our subjects had chronic obstructive lung disease and were current or recent ex-smokers of cigarettes at the time of the study. We cannot exclude the possibility that penetration of linezolid into lung sites including alveolar macrophages may have been influenced in our subjects due to underlying lung disease or because some subjects were smokers. The previously reported study only included healthy non-smokers.7 Interestingly, concentrations in ELF in that study were slightly higher than in our own study although both studies showed ELF concentrations to exceed serum concentrations of linezolid in the time window of 4–8 h. Diffusion of antibiotic may occur between macrophages and ELF after collection of the lavage samples. We immediately centrifuged and separated the cell pellet from the BAL supernatant containing the ELF in the bronchoscopy suite, thereby minimizing any potential error caused by antibiotic diffusion ex vivo. Penetration of linezolid into macrophages is also predicted by the findings of efficacy against Mycobacterium tuberculosis in a murine model.10 Our study is the first to report on the concentrations of linezolid in human bronchial mucosa.

A rat model of pneumococcal pneumonia used oral linezolid at doses of 25 or 50 mg/kg given 18 h after the intrapulmonary instillation of penicillin-susceptible S. pneumoniae. The results showed that the strongest indication of successful outcome was the percentage of time that linezolid remained above the MIC for greater than 45% of the dosing interval.2 Our results indicate that lung tissue levels remain high for up to 8 h after the last dose of 600 mg of linezolid suggesting the likelihood of clinical efficacy based upon the pharmacodynamic results in the rat model.

Linezolid is an important alternative antibiotic for the treatment of multiresistant Gram-positive bacteria.1 Its use in lung infections due to such organisms is supported by our findings that the penetration of linezolid in bronchial mucosa and epithelial lining fluid is excellent for up to 8 h after the last 600 mg oral dose. Further clinical studies are required in humans to assess the efficacy of linezolid in patients with community-acquired pneumonia due to Mycoplasma pneumoniae, Chlamydia pneumonia and Legionella spp.


    Acknowledgements
 
We thank Professor A. McGowan for his help in arranging the assays of linezolid in his department, and Dr R. Wiltshire for his assistance in designing the protocol. This study was supported by an academic grant from Pharmacia & Upjohn plc.


    Footnotes
 
* Corresponding author. Tel: +44-121-424-3731; Fax: +44-121-424-3147; E-mail: david.honeybourne{at}heartsol.wmids.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Diekema, D. J. & Jones, R. N. (2001). Oxazolidinone antibiotics. Lancet 358, 1975–82.[CrossRef][ISI][Medline]

2 . Olsen, K. M., Preheim, L. C. & Gentry-Nielsen, M. J. (2000). The pharmacodynamic activity and efficacy of linezolid in a rat model of pneumococcal pneumonia. In Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 2251, p. 34. American Society for Microbiology, Washington, DC, USA.

3 . Gee, T., Ellis, R., Marshall, G., Andrews, J., Ashby, J. & Wise, R. (2001). Pharmacokinetics and tissue penetration of linezolid following multiple oral doses. Antimicrobial Agents and Chemotherapy 45, 1843–6.[Abstract/Free Full Text]

4 . Honeybourne, D. (1997). Antibiotic penetration in the respiratory tract and implications for the selection of antimicrobial therapy. Current Opinion in Pulmonary Medicine 3, 170–4.[Medline]

5 . Tobin, C. M., Sunderland, J., White, L. O. & MacGowan, A. P. (2001). A simple isocratic high-performance liquid chromatography assay for linezolid in human serum. Journal of Antimicrobial Chemotherapy 48, 605–8.[Abstract/Free Full Text]

6 . Andrews, J. M., Honeybourne, D., Brenwald, N. P., Banerjee, 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, 797–802.[Abstract]

7 . Conte, J. E., Golden, J. A., Kipps, J. & Zurlinden, E. (2002). Intrapulmonary pharmacokinetics of linezolid. Antimicrobial Agents and Chemotherapy 46, 1475–80.[Abstract/Free Full Text]

8 . Lovering, A. M., Zhang, J., Bannister, G. C., Lankester, B. J. A., Brown, J. H. M., Narendra, G. et al. (2002). Penetration of linezolid into bone, fat, muscle and haematoma of patients undergoing routine hip replacement. Journal of Antimicrobial Chemotherapy 50, 73–7.[Abstract/Free Full Text]

9 . Livermore, D. M., Mushtaq, S. & Warner, M. (2001). Susceptibility testing with linezolid by different methods, in relation to published general breakpoints. Journal of Antimicrobial Chemotherapy 48, 452–4.[Free Full Text]

10 . Cynamon, M. H., Klemens, S. P., Sharpe, C. A. & Chase, S. (1999). Activities of several novel oxazolidinones against Mycobacterium tuberculosis in a murine model. Antimicrobial Agents and Chemotherapy 43, 1189–91.[Abstract/Free Full Text]