title: In vitro 'time-to-kill' assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum.
abstract: Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented.
  
  
  
  An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50,2 × IC50and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay.
  
  
  
  While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC50was ≥240 h for pentavalent antimony (77 μg eq./mL), 96 h for trivalent antimony (44 μg eq./mL), 168 to >240 h for miltefosine (10 μM), 168 h for paromomycin (100 μM) and >240 h for amphotericin B (2 μM). No differences were noted between both Leishmania species.
  
  
  
  Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire.
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium louis.maes@uantwerpen.be.
authors.forename: L
authors.initials: L
authors.lastname: Maes
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Beyers
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Mondelaers
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Van den Kerkhof
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Eberhardt
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: G
authors.initials: G
authors.lastname: Caljon
authors.affiliation: Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Hendrickx
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007893:Leishmania donovani / Q000187:drug effects* / Q000502:physiology*; D018314:Leishmania infantum / Q000187:drug effects* / Q000502:physiology*; D021261:Parasitic Sensitivity Tests; D013997:Time Factors
chemical_list: D000981:Antiprotozoal Agents