Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, Helen S. Atkins To cite this version: Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, et al.. Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis. International Journal of Antimicrobial Agents, Elsevier, 2010, 36 (1), pp.87. 10.1016/j.ijantimicag.2010.03.019. hal-00594507 HAL Id: hal-00594507 https://hal.archives-ouvertes.fr/hal-00594507 Submitted on 20 May 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis Authors: Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, Helen S. Atkins PII: S0924-8579(10)00152-4 DOI: doi:10.1016/j.ijantimicag.2010.03.019 Reference: ANTAGE 3292 To appear in: International Journal of Antimicrobial Agents Received date: 17-11-2009 Revised date: 9-3-2010 Accepted date: 11-3-2010 Please cite this article as: Kenny DJ, Sefton AM, Brooks TJG, Laws TR, Simpson AJH, Atkins HS, Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis, International Journal of Antimicrobial Agents (2008), doi:10.1016/j.ijantimicag.2010.03.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis Dermot J. Kenny a, Armine M. Sefton b, Timothy J.G. Brooks a,1, Thomas R. Laws a, Andrew J.H. Simpson a, Helen S. Atkins a,* a Department of Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK b Centre for Infectious Disease, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Whitechapel, London E1 2AD, UK ARTICLE INFO Article history: Received 17 November 2009 Accepted 11 March 2010 Keywords: Burkholderia Macrolide FluoroquinoloneAccepted Manuscript Mouse * Corresponding author. Tel.: +44 1980 614 755; fax: +44 1980 614 307. E-mail address: [email protected] (H.S. Atkins). 1 Page 1 of 14 1 Present address: Health Protection Agency Centre for Emergency Preparedness & Response, Porton Down, Salisbury SP4 0JG, UK. Crown Copyright 2010. Published with the permission of the Defence Science and Technology Laboratory on behalf of the Controller of HMSO. Accepted Manuscript 2 Page 2 of 14 ABSTRACT The efficacies of the azalide azithromycin and the fluoroquinolones trovafloxacin and grepafloxacin for pre- and post-exposure prophylaxis of infection with high or low challenge doses of Burkholderia pseudomallei strain 576 were assessed in an experimental mouse model. Trovafloxacin and grepafloxacin afforded significant levels of protection, whereas azithromycin was ineffective and potentially detrimental. Overall, the data suggest that some fluoroquinolones may have potential utility in prophylaxis of melioidosis and suggest that azithromycin would not be effective in prophylaxis of B. pseudomallei infection. Accepted Manuscript 3 Page 3 of 14 1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a disease that is endemic in Southeast Asia, Northern Australia, and parts of Africa and South and Central America. Whilst generally considered a relatively rare disease, melioidosis is being diagnosed with increasing frequency, probably as a result of greater awareness and increased global travel. Burkholderia pseudomallei is an incidental pathogen in those who come into contact with it in the environment. Melioidosis can present acutely as septicaemia or pulmonary infection, or as a chronic disease that is difficult to treat with relapses over many years. Currently there is no licensed vaccine for melioidosis. Septicaemia is usually rapidly fatal unless promptly treated, which reduces mortality to ca. 50%. The therapeutic regimen of choice is either ceftazidime or a carbapenem given parentally for 10–14 days, followed by oral treatment with co-trimoxazole, either alone or in combination with doxycycline [1]. However, the intrinsic resistance of B. pseudomallei towards many antibiotics means that it is important to find novel effective therapeutics. BurkholderiaAccepted pseudomallei is known to cause Manuscript latent infection that is believed to be associated with the ability of the pathogen to evade host immune responses within host tissues. Moreover, the pathogen can survive within macrophages. Thus, drugs that are active intracellularly are more likely to eradicate infection and prevent relapses of melioidosis. The azalide azithromycin concentrates inside macrophages at high levels and is effective 4 Page 4 of 14 as treatment for intracellular pathogens such as Salmonella spp. even when the minimum inhibitory concentrations (MICs) are relatively high [2]. Additionally, the fluoroquinolones are active against Burkholderia spp. in vitro [3] and show good intracellular penetration, suggesting that they may also be effective against the pathogen. Clinical experience with the early fluoroquinolone ciprofloxacin was disappointing and previous work performed in our laboratory suggested that it was less effective against B. pseudomallei than doxycycline [4]. However, the pharmacokinetic profiles of the newer fluoroquinolones, trovafloxacin and grepafloxacin, are improved. Unfortunately, grepafloxacin has been withdrawn from the market, whilst use of trovafloxacin has been limited due to safety concerns [5]. However, prior to their withdrawal, we initiated studies to evaluate the potential utility of trovafloxacin and grepafloxacin, as well as azithromycin, as prophylaxis or therapy against experimental melioidosis. 2. Materials and methods 2.1. Bacteria Ten B. pseudomallei strains stored on Protect beads (TSC Ltd., Heywood, UK) at –80AcceptedC in the Defence Science and ManuscriptTechnology Laboratory (Salisbury, UK) culture collection were used in a standard Clinical and Laboratory Standards Institute microdilution method to determine MICs of the antibiotics. Bacteria were cultured in 10 mL of nutrient broth (Oxoid, Basingstoke, UK) at 37 C overnight. Burkholderia pseudomallei strain 576 was used for challenge studies. 5 Page 5 of 14 2.2. Antibiotics Antibiotic solutions were prepared freshly each day. Azithromycin (Pfizer, Sandwich, UK) was prepared by dissolving in a minimal volume of 98% ethanol (Sigma, Poole, UK) and subsequently dissolving in 0.5% methylcellulose (Sigma) in distilled water to achieve a dose of 40 mg/kg in a volume of 50 L per mouse as previously described [6,7]. Trovafloxacin (Pfizer) and grepafloxacin (GlaxoSmithKline, Stevenage, UK) were dissolved in 0.5% methylcellulose in distilled water to achieve a dose of 40 mg/kg in a volume of 20 L per mouse as previously described [7]. To determine the efficacy of the antibiotics against B. pseudomallei in vivo, groups of 24 mice were orally administered once daily with 50 L of azithromycin solution, 20 L of trovafloxacin solution or 20 L of grepafloxacin solution. Further groups of five to six control mice were administered 0.5% methylcellulose diluent for 7 days. Two different prophylaxis regimens were studied. Antibiotic treatment was initiated either at 1 h prior to challenge (as pre-exposure prophylaxis) and continued for 7 days, or at 6 h post exposure and continued for 14 days (post-exposure prophylaxis). Accepted Manuscript 2.3. Animals Antibiotic efficacy was evaluated in female 6–7-week-old BALB/c mice (obtained from Charles River Laboratories, UK). All experimental work was conducted in flexible film isolators according to a UK Home Office licence. All 6 Page 6 of 14 animal studies were carried out in accordance with the Animals Scientific Procedures Act 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Mice were challenged with either a low dose [10–4 dilution of a log-phase culture, ca. 104 colony-forming units (CFU)/mL] or a high dose (10–1 dilution of a log-phase culture, ca. 107 CFU/mL) of B. pseudomallei strain 576 by subcutaneous injection (100 L) and antibiotic prophylaxis was evaluated using the dosing regimens described above. Following the last antibiotic treatment, surviving mice were observed for a further 47 days for relapse. At this point the experiment was terminated. A single experiment at each challenge dose was performed in this study. 2.4. Analysis Kaplan–Meier plots were generated