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Intracellular vs extracellular activity of antibiotics : PK – PD in action

Cellular PK/PD of oxazolidinones: studies with radezolid (RX-1741; RDZ) and linezolid (LZD) Sandrine Lemaire, Paul M. Tulkens, and Françoise Van Bambeke Unité de Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain.

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Intracellular vs extracellular activity of antibiotics : PK – PD in action

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  1. Cellular PK/PD of oxazolidinones: studies with radezolid (RX-1741; RDZ) and linezolid (LZD) Sandrine Lemaire,Paul M. Tulkens, and Françoise Van BambekeUnité de Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain ISAP

  2. Intracellular vs extracellular activity of antibiotics :PK – PD in action cooperation withhost defenses metabolism binding influx physico-chemical conditions accumulation and bioavailability efflux bacterial responsiveness what about oxazolidinones ? Carryn et al., Infect Dis Clin North Am. (2003) 17:615-34 ISAP

  3. Linezolid vs. Radezolid 2 protonable functions Heteroaryl substituant Biaryl spacer linezolid radezolid ISAP

  4. Physico-chemical properties Similar lipophilicity linezolid radezolid ISAP

  5. Physico-chemical properties weak dibasic character radezolid azithromycin ISAP

  6. Let’s start with pharmacokinetics ! Yes, but CELLULARPK ! Munich subway, ECCMID 2007 ISAP

  7. Aim of the study • to investigate the cellular pharmacokinetics of radezolid • to decipher the mechanisms of its cellular accumulation • in comparison with linezolid and azithromycin Oxazolidinone - low accumulation - cellular conc. ~ extracell. conc. Macrolide - dicationic amphiphile - high accumulation by diffusion-segregation in acidic compartments ISAP

  8. General methodology: cellular accumulation scintillation counting drug cell prot. (Lowry) microbiological assay (B. subtilis) accumulation calculated considering a cell volume of 5 µl/mg prot. • cells incubated with linezolid or 14C-radezolid • washed in PBS and collected by low-speed centrifugation • resuspended in water ISAP

  9. Kinetics of accumulation and efflux  phagocytic cells Accumulation and efflux rapid; RDZ >> LZD ISAP

  10. Comparative accumulation level at equilibrium phagocytic cells non phagocytic cells ISAP

  11. Influence of binding to serum proteins  phagocytic cells accumulation varies depending on serum content ISAP

  12. Influence of binding to serum proteins  phagocytic cells accumulation varies depending on serum content ISAP

  13. Importance of pH gradients pH 7.4 pH 7.0 pH 5.4 B B B BH+ BH+ BH+ De Duve. Biochem Pharmacol. (1974) 23:2495-531 ISAP

  14. How to collapse pH gradients ? monensin pH 7.4 H+ pH 7.4 pH 7.4 H+ B B B BH+ BH+ BH+ ISAP

  15. How to collapse pH gradients ? pH 7.4 pH 5.4 pH 7.0 pH 5.4 B B B BH+ BH+ BH+ ISAP

  16. Influence of pH gradients accumulation reduced by 60-80 % for both oxazolidinoneswhen collapsing pH gradients ISAP

  17. Where is the drug ? ICAAC 2003 ISAP

  18. Subcellular localization of radezolid  J774 mouse macrophages low speed centrifugation extract nuclei / unbroken cells high speed centrifugation soluble fraction sedimentable fraction ISAP

  19. Subcellular localization of radezolid  J774 mouse macrophages low speed centrifugation extract nuclei / unbroken cells high speed centrifugation soluble fraction sedimentable fraction • dual subcellular localization • cytosol • organelles ISAP

  20. Subcellular localization of radezolid sedimentable fraction soluble fraction high speed centrifugation on sucrose gradient • dual subcellular localization • cytosol • lysosomes ISAP

  21. Cellular pharmacodynamics Does accumulation translate in activity ? Madison, Craig’s symposium 2008 ISAP

  22. General methodology: intracellular activity • cells infected by pre-opsonized bacteria • extracellular bacteria eliminated by short incubation with gentamicin • incubation with increasing concentrations of antibiotics for 24/48 h • washings • resuspended in water cell prot. (Lowry) plating and CFU counting activity expressed as change from initial inoculum Barcia-Macay et al., Antimicrob. Ag. Chemother. (2006) 50:841-51 ISAP

  23. Activity on different bacteria PHAGOSOMES LYSOSOMES Staphylococcus aureus ENDOPLASMIC RETICULUM- DERIVED VESICLES PHAGOLYSOSOMES ~ pH 5.0 – 5.5 CYTOSOL ~ pH 7.0 Listeria monocytogenes Legionella pneumophila ~ pH 6.1 ISAP

  24. Activity on different bacteria Listeria monocytogenes Legionella pneumophila Staphylococcus aureus radezolid 10-fold more potent that linezolid in the 3 models ISAP

  25. Comparison of static concentrations radezolid 10-fold more potent that linezolid in the 3 modelsbut similar potency at equivalent x MIC ISAP

  26. Comparison of static concentrations radezolid 10-fold more potent that linezolid in the 3 modelsbut similar potency at equivalent x MIC any role for accumulation ? ISAP

  27. Further analysis for S. aureus • at neutral pH, radezolid is ~equipotent against the LZD-R strain ISAP

  28. Further analysis for S. aureus • at neutral pH, radezolid is ~equipotent against the LZD-R strain • at acidic pH, radezolid activity is reduced ISAP

  29. Intracellular activity (clinically-relevant comparison) • Radezolid is • more potent and effective than LZD • as potent and effective against both strains ISAP

  30. Intracellular activity (equipotent concentrations) • RDZ and LZD ~ equipotent against the LZD-S strain • RDZ~ equipotent against both strains ISAP

  31. Intracellular activity (x MIC pH 5.5) • Radezolid is active at lower • multiples of the MIC assumed • in the infected compartment ISAP

  32. Intracellular activity (pharmacological comparison) • cellular accumulation of RDZ compensates for the effect • of acid pH on activity ISAP

  33. Comparison for strains of clinical interest (MRSA and VISA) Cstatic : 2.75 mg/L ~ 1.4 x MIC Cstatic : 0.37 mg/L ~ 0.7 x MIC difference in potency maintained ISAP

  34. Conclusions • oxazolidinones ~ macrolides regarding mechanism of accumulation/distribution • accumulation level depends on basic character • dual distribution cytosol/lysosomes for radezolid • oxazolidinones show intracellular activity in all subcellular compartments examined • lower MICs translate in improved potency • accumulation may conpensate for defeating effect of intracellular milieu on activity ISAP – post ICAAC 2007 & 2008 • what is the cellular bioavailability of antibiotics ? • how do antibiotics express their activity inside the cells ? ISAP

  35. Conclusions Still room for thinking … ISAP – post ICAAC 2007 & 2008 ISAP

  36. have a nice trip back home ... Architectural cruse, Chicago, ICAAC 2007 ISAP

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