Concentrations of garenoxacin in plasma, bronchial mucosa, alveolar macrophages and epithelial lining fluid following a single oral 600 mg dose in healthy adult subjects

J. Andrews1,*, D. Honeybourne2, G. Jevons1, M. Boyce3, R. Wise1, A. Bello4 and D. Gajjar4

1 Department of Medical Microbiology, City Hospital NHS Trust, Birmingham; 2 Department of Respiratory Medicine, Heartlands NHS Trust, Birmingham; 3 Hammersmith Medicines Research Ltd, Central Middlesex Hospital, London, UK; 4 Bristol-Myers Squibb, USA

Received 27 August 2002; returned 7 November 2002; revised 15 November 2002; accepted 3 December 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A microbiological assay was used to measure concentrations of garenoxacin (BMS-284756) in plasma, bronchial mucosa (BM), alveolar macrophages (AM) and epithelial lining fluid (ELF), following a single 600 mg oral dose. Twenty-four healthy subjects were allocated into four nominal time intervals after the dose, 2.5–3.5, 4.5–5.5, 10.5–11.5 and 23.5–24.5 h. Mean concentrations in plasma, BM, AM and ELF, respectively, for the four nominal time windows were for 2.5–3.5 h 10.0 mg/L (S.D. 2.8), 7.0 mg/kg (S.D. 1.3), 106.1 mg/L (S.D. 60.3) and 9.2 mg/L (S.D. 3.6); 4.5–5.5 h 8.7 mg/L (S.D. 2.2), 6.0 mg/kg (S.D. 1.9), 158.6 mg/L (S.D. 137.4) and 14.3 mg/L (S.D. 8.2); 10.5–11.5 h 6.1 mg/L (S.D. 1.9), 4.0 mg/kg (S.D. 1.4), 76.0 mg/L (S.D. 47.7) and 7.9 mg/L (S.D. 4.6); and 23.5–24.5 h 2.1 mg/L (S.D. 0.5), 1.7 mg/kg (S.D. 0.7), 30.7 mg/L (S.D. 12.9) and 3.3 mg/L (S.D. 2.3). Concentrations at all sites exceeded MIC90s for the common respiratory pathogens Haemophilus influenzae (0.03 mg/L), Moraxella catarrhalis (0.015 mg/L) and Streptococcus pneumoniae (0.06 mg/L). These data suggest that garenoxacin should be effective in the treatment of community-acquired pneumonia and chronic obstructive pulmonary disease.

Keywords: garenoxacin, concentrations, respiratory tree


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Garenoxacin (BMS-284756, T-3811ME) is a novel des-F(6)-quinolone available for oral and parenteral administration. It is currently in Phase III of its development, and has been shown to have broad-spectrum activity against Gram-positive and -negative organisms,1 as well as ‘atypical’ organisms.2 The aim of this study was to measure concentrations of garenoxacin, using a previously validated microbiological assay,3 in plasma and compartments of the respiratory tree following a single 600 mg oral dose. For therapeutic efficacy, it is important that adequate concentrations of antibiotic are found at potential sites of respiratory infection,4 and quinolones have been shown previously to penetrate lung tissues more efficiently than ß-lactam antibiotics.5


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects

Twenty-four healthy subjects, 14 men (mean age 22.4 years, mean weight 81.8 kg) and 10 women (mean age 29.5 years, mean weight 57.5 kg), were enrolled into the study at Hammersmith Medicines Research at the Central Middlesex Hospital in London, UK. Subjects were dosed with 600 mg oral garenoxacin, and the plasma and lung tissue samples were collected at one of four time intervals: interval 1, 2.5–3.5 h; interval 2, 4.5–5.5 h; interval 3, 10.5–11.5 h; and interval 4, 23.5–24.5 h. The Hospital Ethics Committee approved the study and all subjects gave written informed consent. All subjects were screened within 14 days prior to bronchoscopy; screening included a detailed medical history, physical examination, blood samples for haematological and biochemical analysis and, in the case of females, a pregnancy test.

Sample collection

Bronchoscopy samples were collected as described previously.6 Briefly, at bronchoscopy bronchial mucosa (BM) and broncho-alveolar lavage (BAL) were taken using standard procedures.7 In the case of BAL, 200 mL of pre-warmed 0.9% saline was divided into four 50 mL aliquots. 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.

Microbiological assay

Concentrations of garenoxacin were measured using methods based on those described previously.8 Assay plates (Mast Diagnostics, Bootle, UK) containing Iso-Sensitest agar (Oxoid, Basingstoke, UK) were flooded with an Escherichia coli (4004; Bayer, Wuppertal, AG, Germany) suspension adjusted to an optical density of 0.004 at 630 nm. Antibiotic calibrators were prepared in pooled human serum and pH 7 buffer (range 0.06–1 mg/L) and 9% saline (range 0.12–2 mg/L). Internal control samples (0.8 and 0.08 mg/L prepared in human serum and pH 7 buffer; 1.5 and 0.2 mg/L prepared in 9% sodium chloride) and quality assurance samples (range 0.07–0.9 mg/L for human serum and pH 7 buffer; range 0.15–1.8 mg/L for 9% sodium chloride) were included on every assay plate. Five millimetre holes were punched in the agar, and tests, calibrators, control and quality assurance samples were applied in triplicate to the plate. After overnight incubation at 30°C, zones were measured using an image analyser pre-programmed with Bennet’s calculation9 (Imaging Associates, Thame, UK).

Calculation of garenoxacin in epithelial lining fluid (ELF), alveolar macrophages (AM) and BM

Concentrations of antibiotic were calculated using the following formulae:

BM.

AC x (VB + WS)/WS = Concentration (mg/kg tissue)

where AC = assayed concentration (mg/L), VB = volume of buffer added to homogenize the sample (µL) and WS = weight of tissue (mg).

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

ACL x BL/UL = ELF concentration (mg/L)

where ACL = assayed concentration (mg/L), UL = urea concentration in lavage (mmol/L) and BL = blood urea concentration (mmol/L).

AM. Antibiotic concentration in AMs was determined using a mean cell volume of an alveolar macrophage of 2.48 µL/ 106 cells.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The lower limit of quantification of the microbiological assay was 0.03 mg/L. Between-assay coefficients of variation (mean for the two concentrations tested) for internal controls prepared in human serum, pH 7 buffer, and 9% sodium chloride, were 6.4, 6.3 and 6%, respectively. All of the quality assurance samples prepared in human serum and 9% saline were within ±15% of the assigned concentration. In the case of pH 7 buffer, >=96.2% were within ±15% of the assigned concentration, and 100% were within ±20%. Control urea samples included with every batch of urea estimations were within ±10% of the assigned concentration. For two subjects, urea concentrations in the BAL were below the limits of quantification. For the remaining 22 subjects, the mean urea concentration in BAL was 0.038 mmol/L (range 0.013–0.11 mmol/L). The mean macrophage count and bronchial biopsy weight was 1.4 x 105 cells (range 4.3 x 104–3.4 x 105 cells) and 6.5 mg (range 3.5–10.4 mg), respectively. Mean concentrations in plasma, BM, AM and ELF, respectively, for the four nominal time windows were for 2.5–3.5 h 10.0 mg/L (S.D. 2.8), 7.0 mg/kg (S.D. 1.3), 106.1 mg/L (S.D. 60.3) and 9.2 mg/L (S.D. 3.6); 4.5–5.5 h 8.7 mg/L (S.D. 2.2), 6.0 mg/kg (S.D. 1.9), 158.6 mg/L (S.D. 137.4) and 14.3 mg/L (S.D. 8.2); 10.5–11.5 h 6.1 mg/L (S.D. 1.9), 4.0 mg/kg (S.D. 1.4), 76.0 mg/L (S.D. 47.7) and 7.9 mg/L (S.D. 4.6); and 23.5–24.5 h 2.1 mg/L (S.D. 0.5), 1.7 mg/kg (S.D. 0.7), 30.7 mg/L (S.D. 12.9) and 3.3 mg/L (S.D. 2.3). Individual volunteer results are shown in Table 1. The mean site to plasma ratios are shown in Figure 1.


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Table 1.  Concentrations of garenoxacin in ELF, BM and AM, compared with simultaneous plasma concentrations for three nominal time windows of 3, 5, 11 and 24 h, after a single 600 mg oral dose in healthy volunteers
 


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Figure 1. Site-to-plasma ratios of garenoxacin in BM, ELF and AMs following a single 600 mg oral dose in healthy volunteers at mean times after dosing of 3, 5, 12 and 24 h.

 
No drug-related adverse events were observed. However, a total of 14 adverse events (mostly mild in intensity) were noted to have occurred during or immediately after the BAL procedure; these were considered unlikely/unrelated to the study drug, with the BAL procedure being cited in all cases as the most likely cause.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study has demonstrated that concentrations of garenoxacin, at all potential sites of respiratory infection for a 24 h period, exceeded a tentative MIC breakpoint of 0.5 mg/L (MIC90s for Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, including isolates with reduced susceptibility to penicillin of 0.03, 0.015 and 0.06 mg/L, respectively) that has been suggested for this antibiotic.10 In the case of isolates of S. pneumoniae with reduced susceptibility to quinolones (ciprofloxacin MICs >= 4 mg/L and garenoxacin MICs 0.06 and 1 mg/L),1,2 these data confirm that mean concentrations at each of the sites exceed the MICs of garenoxacin. Mean garenoxacin concentrations in BM at the last sampling interval (23.5–24.5 h post-dose) exceeded the garenoxacin MIC90s for S. pneumoniae, H. influenzae and M. catarrhalis, by ~29-, 58- and 115-fold, respectively. These fold increases are greater than corresponding fold increases noted for moxifloxacin (4-, 17- and 17-fold, respectively).11

Like other quinolones, we have shown a high penetration into AMs that would suggest that this drug should exhibit efficacy against Mycoplasma, Legionella and Chlamydia.12

These data suggest that garenoxacin might be effective for the treatment of respiratory infections, including those caused by pneumococci with reduced susceptibility to penicillin.


    Footnotes
 
* Corresponding author. Tel: +44-121-507-5693; E-mail: jenny.Andrews{at}cityhospbham.wmids.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Weller, T. M. A., Andrews, J. M., Jevons, G. & Wise, R. (2002). The in vitro activity of garenoxacin, a new des-fluorinated quinolone. Journal of Antimicrobial Chemotherapy 49, 177–84.[Abstract/Free Full Text]

2 . Fung-Tomc, J. C., Minassian, B. & Kolek, B. (2000). Antibacterial spectrum of a novel des-fluoro(6) quinolone, BMS-284756. Antimicrobial Agents and Chemotherapy 44, 3351–6.[Abstract/Free Full Text]

3 . Wise, R., Gee, T., Marshall, G. & Andrews, J. M. (2002). Single- dose pharmacokinetics and penetration of BMS 284756 into an inflammatory exudate. Antimicrobial Agents and Chemotherapy 46, 242–4.[Abstract/Free Full Text]

4 . 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, 57–63.[ISI][Medline]

5 . 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, 715–9.[Abstract]

6 . Andrews, J. M., Honeybourne, D., Brenwald, N. P., Banergee, 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 . The BAL Cooperative Group Steering Committee. (1990). Bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. American Review of Respiratory Disease 141, 166–202.

8 . Andrews, J. M. (1999). The assay of antimicrobials in tissues and fluids. In Clinical Antimicrobial Assays (Reeves, D. S., Wise, R., Andrews, J. M. & White, L. O., Eds), pp. 65–75. Oxford University Press, New York, NY, USA.

9 . 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, 170–7.[ISI][Medline]

10 . Andrews, J. M. & Wise, R. (2001). In vitro susceptibility testing of garenoxacin by the BSAC standardized disc testing method. Journal of Antimicrobial Chemotherapy 48, 322–34.[Free Full Text]

11 . Zhanel, G. G., Palatnick, L., Weshnoweski, B., Smith, H., Nichol, K. & Hoban, D. J. (2001). BMS-284756 demonstrates potent activity against Canadian lower respiratory tract infection (RTI) pathogens isolated in 1999–2001. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 2001. Abstract E-711, p. 176. American Society for Microbiology, Washington, DC, USA.

12 . Malay, S., Roblin, P. M., Reznik, T., Kutlin, A. & Hammerschlag, M. R. (2002). In vitro activities of BMS-284756 against Chlamydia trachomatis and recent clinical isolates of Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 46, 517–8.[Abstract/Free Full Text]