JAC Advance Access originally published online on February 19, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1110-1119; doi:10.1093/jac/dkn047
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Original research |
A new combined flow-cytometry-based assay reveals excellent activity against Toxoplasma gondii and low toxicity of new bisphosphonates in vitro and in vivo
1 Institut für Mikrobiologie und Hygiene, Charité Universtätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin, Germany 2 Institut für Biometrie und klinische Epidemiologie, Charité Universtätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 20, 12200 Berlin, Germany
Received 16 January 2007; returned 24 April 2007; revised 12 December 2007; accepted 20 January 2008
* Corresponding author. Tel: +49-30-8445-3630; Fax: +49-30-8445-3830; E-mail: oliver.liesenfeld{at}charite.de
Objectives: The aim of this study was to investigate the antiparasitic activity and toxicity of bisphosphonates using a new combined flow cytometry assay.
Methods: Using Toxoplasma gondii tachyzoites carrying the green-fluorescent protein (GFP), we established a new flow cytometry assay combining testing of in vitro and in vivo activity plus toxicity of newly synthesized bisphosphonates against T. gondii. Toxicity as determined by this assay was compared with toxicity as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test.
Results: In vivo, therapeutic efficacy was 100% for bisphosphonates 2F, 3B, 18A, 22A and 30B at 490, 1000, 512, 44.05 and 47.6 µM concentrations, respectively. Toxicity at 100% inhibitory concentrations was 20% for 2F and 3B, 60% for 22A and 30B, and 75% for 18A. In vitro, 6 (91A, 203A, 200C, 210A, 204A and 282A) of 15 newly synthesized bisphosphonates (12 nitrogen-containing and 3 n-alkyl) inhibited parasite replication by >50% at a concentration of 100 µM. Whereas substances 91A and 282A (high efficacy) showed moderate and low toxicity (cell viability between 70% and 100%), respectively, toxicities of 203A, 200C, 210A and 204A were 70%, 65%, 80% and 70%, respectively, as determined by flow cytometry. Compounds 290A, 218A, 214A, 266A and 219A inhibited parasite replication by between 20% and 50% at a concentration of 100 µM.
Conclusions: Newly synthesized bisphosphonates 2F, 3B, 91A and 282A showed excellent therapeutic activity and low toxicity. These antiparasitic drugs may therefore be promising compounds for use in patients with acute and reactivated toxoplasmosis. The new flow cytometry assay allowed simultaneous determination of therapeutic efficacy and toxicity.
Keywords: infection , treatment , antiparasitic drugs , toxoplasmosis , cell viability