Pharmacodynamics and cellular accumulation of amphotericin B and miltefosine in Leishmania donovani-infected primary macrophages

Abstract Objectives We examined the in vitro pharmacodynamics and cellular accumulation of the standard anti-leishmanial drugs amphotericin B and miltefosine in intracellular Leishmania donovani amastigote–macrophage drug assays. Methods Primary mouse macrophages were infected with L. donovani amastigotes. In time–kill assays infected macrophages were exposed to at least six different concentrations of serially diluted drugs and the percentage of infected macrophages was determined after 6, 12, 24, 48, 72 and 120 h of exposure. Cellular drug accumulation was measured following exposure to highly effective drug concentrations for 1, 6, 24, 48 and 72 h. Data were analysed through a mathematical model, relating drug concentration to the percentage of infected cells over time. Host cell membrane damage was evaluated through measurement of lactate dehydrogenase release. The effect of varying the serum and albumin concentrations in medium on the cellular accumulation levels of miltefosine was measured. Results Amphotericin B was more potent than miltefosine (EC50 values of 0.65 and 1.26 μM, respectively) and displayed a wider therapeutic window in vitro. The kinetics of the cellular accumulation of amphotericin B was concentration- and formulation-dependent. At an extracellular concentration of 10 μM miltefosine maximum cellular drug levels preceded maximum anti-leishmanial kill. Miltefosine induced membrane damage in a concentration-, time- and serum-dependent manner. Its cellular accumulation levels increased with decreasing amounts of protein in assay medium. Conclusions We have developed a novel approach to investigate the cellular pharmacology of anti-leishmanial drugs that serves as a model for the characterization of new drug candidates.

[1]  S. Croft,et al.  Pharmacodynamics and Biodistribution of Single-Dose Liposomal Amphotericin B at Different Stages of Experimental Visceral Leishmaniasis , 2017, Antimicrobial Agents and Chemotherapy.

[2]  L. Maes,et al.  In vitro ‘time-to-kill’ assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum , 2017, The Journal of antimicrobial chemotherapy.

[3]  E. Suárez,et al.  In vitro pharmacodynamic modelling of anidulafungin against Candida spp. , 2016, International journal of antimicrobial agents.

[4]  T. Dorlo,et al.  Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages , 2016, The Journal of Biological Chemistry.

[5]  A. Descoteaux,et al.  Leishmania survival in the macrophage: where the ends justify the means. , 2015, Current opinion in microbiology.

[6]  V. Yardley,et al.  Antileishmanial Activity, Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex , 2013, Antimicrobial Agents and Chemotherapy.

[7]  P. Kaye,et al.  Functional complexity of the Leishmania granuloma and the potential of in silico modeling , 2013, Front. Immun..

[8]  J. Beijnen,et al.  Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. , 2012, The Journal of antimicrobial chemotherapy.

[9]  Amin R. Mazloom,et al.  Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages , 2012, Nature Immunology.

[10]  R. Kumar,et al.  Immunobiology of visceral leishmaniasis , 2012, Front. Immun..

[11]  J. Cano,et al.  Leishmaniasis Worldwide and Global Estimates of Its Incidence , 2012, PloS one.

[12]  T. Wynn,et al.  Protective and pathogenic functions of macrophage subsets , 2011, Nature Reviews Immunology.

[13]  S. Croft,et al.  Leishmaniasis chemotherapy--challenges and opportunities. , 2011, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[14]  O. Cars,et al.  Protein Binding: Do We Ever Learn? , 2011, Antimicrobial Agents and Chemotherapy.

[15]  K. Seifert.  Structures, Targets and Recent Approaches in Anti-Leishmanial Drug Discovery and Development , 2011, The open medicinal chemistry journal.

[16]  S. Croft,et al.  In vitro activity of anti-leishmanial drugs against Leishmania donovani is host cell dependent. , 2010, The Journal of antimicrobial chemotherapy.

[17]  Leonore A. Herzenberg,et al.  Two physically, functionally, and developmentally distinct peritoneal macrophage subsets , 2010, Proceedings of the National Academy of Sciences.

[18]  P. Kaye,et al.  SIGNR1-negative red pulp macrophages protect against acute streptococcal sepsis after Leishmania donovani-induced loss of marginal zone macrophages. , 2009, The American journal of pathology.

[19]  O. Cars,et al.  Tissue concentrations: do we ever learn? , 2007, The Journal of antimicrobial chemotherapy.

[20]  R. Peeling,et al.  Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? , 2007, Nature Reviews Microbiology.

[21]  Mats O. Karlsson,et al.  Semimechanistic Pharmacokinetic/Pharmacodynamic Model for Assessment of Activity of Antibacterial Agents from Time-Kill Curve Experiments , 2006, Antimicrobial Agents and Chemotherapy.

[22]  P. Tulkens,et al.  Cellular pharmacodynamics and pharmacokinetics of antibiotics: current views and perspectives. , 2006, Current opinion in drug discovery & development.

[23]  N. Malla,et al.  Pathophysiology of visceral leishmaniasis - some recent concepts. , 2006, The Indian journal of medical research.

[24]  P. Loiseau,et al.  Hexadecylphosphocholine interaction with lipid monolayers. , 2004, Biochimica et biophysica acta.

[25]  H. Grunicke,et al.  Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities. , 2001, Biochemical pharmacology.

[26]  J. Bolard,et al.  Carrier effects on biological activity of amphotericin B , 1996, Clinical microbiology reviews.

[27]  D. Leibfritz,et al.  Early stage monitoring of miltefosine induced apoptosis in KB cells by multinuclear NMR spectroscopy. , 1996, Anticancer research.

[28]  P. Legrand,et al.  Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line. , 1996, The Journal of antimicrobial chemotherapy.

[29]  J. Bolard,et al.  The endocytic process in CHO cells, a toxic pathway of the polyene antibiotic amphotericin B , 1994, Antimicrobial Agents and Chemotherapy.

[30]  T. Walsh,et al.  Pharmacokinetics and safety of a unilamellar liposomal formulation of amphotericin B (AmBisome) in rabbits , 1994, Antimicrobial Agents and Chemotherapy.

[31]  W. Reutter,et al.  Uptake, subcellular distribution and metabolism of the phospholipid analogue hexadecylphosphocholine in MDCK cells. , 1994, Biochimica et biophysica acta.

[32]  A. Alonso,et al.  The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins. , 2017, Biochimica et biophysica acta. Biomembranes.

[33]  C. Unger,et al.  Hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine: a comparison of hemolytic activity, serum binding and tissue distribution. , 1992, Progress in experimental tumor research.

[34]  B. Vion,et al.  Cutaneous leishmaniasis , 1990 .