Antibiotic Antibiotic accumulation and efflux accumulation and - PowerPoint PPT Presentation

Intracellular activity on S. aureus control oritavancin Oritavancin can destroy intracellular bacteria THP-1 macrophages; extracell. conc. 25 mg/L; 24 h Barcia-Macay et al . submitted

Time-effect for intracellular activity Oritavancin shows time- and concentration-dependent intracellular bactericidal effects control vancomycin 10 7 oritavancin 0.25 µg/ml CFU / mg prot 2.5 µg/ml 10 5 50 µg/ml 10 µg/ml 10 3 25 µg/ml 10 1 0 5 10 15 20 25 time (h) J774 macrophages Seral et al (2003) AAC 47: 2283-92

Dose-effect for extracell. vs intracell. activity intracellular activity < extracellular activity extra intra static 0.3 X MIC 4.8 X MIC conc. max. - 5.55 log - 3.15 log effect THP-1 macrophages; 24 h Barcia-Macay et al . submitted

Dose-effect intracellular activity < extracellular activity, but bactericidal effects reached at clinically-relevant concentrations C max C min Barcia-Macay et al . submitted

Comparison with other antibiotics oritavancin is one of the most active drugs against intracellular S. aureus MIC 10 X MIC Cmax THP-1 macrophages; 24 h Barcia-Macay et al . submitted

Aim of the study • activity against multi-resistant Gram-positive ( S. aureus ) • rapid bactericidal activity • retention in the organism any place for intracellular infections? cellular pharmacokinetics: accumulation and subcellular distribution in eukaryotic cells cellular pharmacodynamics: cellular toxicity: activity against morphological and biochemical intracellular bacteria alterations

Morphological studies polar lipids macrophages fibroblasts Rat embryo fibroblasts; 25 mg/L; 3 days J774 macrophages, 25 mg/L; 1 day Van Bambeke et al . (2005) AAC – in press

Biochemical studies : time-effects accumulation of phospholipids and cholesterol develops in parallel with oritavancin cellular concentration drug-free drug-free drug-free medium medium medium Rat embryo fibroblasts; 25 mg/L Van Bambeke et al . (2005) AAC – in press

Biochemical studies : dose-effects Rat embryo fibroblasts, 3 days Van Bambeke et al . (2005) AAC – in press J774 macrophages, 1 day

Model of the interaction of oritavancin with eukaryotic cells oritavancin S. aureus polar lipids undefined material

Can we dissociate activity from toxicity ? cellular alterations co-exist with destroyed bacteria …. THP-1 macrophages; 25 mg/L; 24 h

Can we dissociate activity from toxicity ? comparison with two other lysosomotropic cationic antibiotics CH 3 gentamicin azithromycin N CH 3 CH 3 OH OH OH CH 3 OH CH 3 O R 1 H 3 C N(CH 3 ) 2 HN H 2 N O CH HO CH 3 OH CH 2 CH 3 O OH O CH 3 O CH 3 O HN O NH 2 H 2 N R 2 O O OCH 3 CH 3 CH 3 O CH 3 OH • polycationic, hydrophilic • dicationic • endocytosis • diffusion/segregation • phospholipidosis • accumulation of phospholipids and cholesterol

Lysosomotropic antibiotics and activity on S. aureus GEN and ORI are both conc.-dependent intracellularly J774 macrophages

Lysosomotropic antibiotics and activity on S. aureus GEN and ORI are both conc.-dependent intracellularly J774 macrophages

Lysosomotropic antibiotics and activity on S. aureus But GEN activity limited at clinically-relevant conditions J774 macrophages

Lysosomotropic antibiotics and phospholipidosis Phospholipidosis developing on a conc.-dependent manner Rat embryo fibroblasts

Lysosomotropic antibiotics and phospholipidosis Toxic potential variable at clinically-relevant conditions Rat embryo fibroblasts

Lysosomotropic antibiotics and phospholipidosis Toxic potential variable at clinically-relevant conditions Rat embryo fibroblasts

Lysosomotropic antibiotics and phospholipidosis Toxic potential variable at clinically-relevant conditions Rat embryo fibroblasts

Can we dissociate activity from toxicity ? � both processes are dependent on cellular concentration … 180 cellular toxicity 160 ORI 140 GEN 120 AZM 100 0 -1 -2 -3 intracellular activity … and develop in parallel

amphiphilic glycopeptides, Conclusion a new type of « magic bullets» pharmacodynamics: bactericidal, conc-dep. pharmacokinetics: activity lysosomotropic accumulation cellular pharmacodynamics: cellular toxicity: conc. and time-dependent conc. and time-dependent bactericidal activity cellular Morphological studies towards toxicity Dose-effect polar lipids macrophages fibroblasts intracellular activity < extracellular activity, but bactericidal effects reached at clinically-relevant concentrations extra AND intra C min C max S. aureus Rat embryo fibroblasts; 25 mg/L; 3 days J774 macrophages, 25 mg/L; 1 day Van Bambeke et al . (2005) AAC – in press Barcia-Macay et al . submitted

Questions for future work reasons for reduction binding site in activity ? intracellularly ? oritavancin S. aureus polar lipids ? undefined ? mechanism material of accumulation in vivo

Take home message cellular accumulation, the best and the worse of properties… drug development toxicity activity

Intracellular “PK-PD” Concentration Pharmacologic versus time or toxicologic in non-infected effect cells Concentration Dosage versus time regimen in cells Concentration Antimicrobial versus time penetration macrolides effect versus at intracellular distribution time quinolones efflux efflux site of infection PHARMACOKINETICS PHARMACODYNAMICS

Intracellular “PK-PD” Concentration Pharmacologic versus time or toxicologic in non-infected effect cells Concentration Dosage versus time regimen in cells Concentration Antimicrobial Antimicrobial versus time penetration macrolides effect versus effect versus at intracellular distribution time time quinolones efflux efflux site of infection PHARMACOKINETICS PHARMACODYNAMICS

macrolides quinolones from eukaryotic cells Efflux of magic bullets

Why efflux transporters ? polar drug lipophilic drug physico-chemical properties are inadequate for reaching an intracellular target ! Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Why efflux transporters ? amphipathic drug most drugs are amphipathic by design, to be able to cross membrane barriers ! Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Why efflux transporters ? But a diffusible compound may have potentially harmful effects ! Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Why efflux transporters ? Extrusion by efflux pumps Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Why efflux transporters ? Extrusion by efflux pumps general mean of protection against cell invasion by diffusible molecules Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Mechanisms of active efflux flip-flop flippase vacuum cleaner ATP ADP+Pi membrane insertion / release Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Antibiotics as substrates of efflux pumps Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes β -lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones Van Bambeke et al . (2000) Biochem. Pharmacol. 60:457-70

Antibiotics as substrates of efflux pumps O O azithromycin F OH CH 3 N N N H 3 C CH 3 HN OH OH ciprofloxacin H 3 C OH CH 3 (H 3 C) 2 N O O H 3 C HO H 3 CH 2 C O O O CH 3 F C OH O O H 3 CO CH 3 N N CH 3 HN O OCH 3 H 3 C OH moxifloxacin

Macrolides and quinolones as cell-associated antibiotics

Aim of the study amphiphilic antibiotics • accumulating in eucaryotic cells • considered as useful for treating intracellular infections • known substrates of efflux pumps in bacteria efflux from macrophages ? macrolides quinolones • phenotypic characterization of the active efflux • consequences for intracellular activity

Aim of the study amphiphilic antibiotics • accumulating in eucaryotic cells • considered as useful for treating intracellular infections • known substrates of efflux pumps in bacteria efflux from macrophages ? macrolides quinolones • phenotypic characterization phenotypic characterization • of the active efflux of the active efflux • consequences for intracellular activity

Efflux pumps expressed in J774 macrophages

ABC multidrug transporters anionic cationic amphiphiles amphiphiles MDR-1 (P-glycoprotein) MRP1-10 ATP ADP

How to inhibit ABC transporters ? anionic cationic amphiphiles amphiphiles MDR-1 (P-glycoprotein) MRP1-10 deoxyglucose ATP ADP NaN 3

How to inhibit ABC transporters ? OCH 3 OCH 3 verapamil H 3 CO OCH 3 H 3 CO cationic N CH(CH 3 ) 2 H 3 CO CN CH 3 amphiphiles H 3 CO H N N O MDR-1 (P-glycoprotein) C N H O GF120918 ATP ADP

How to inhibit ABC transporters ? CH 3 COOH (CH 2 ) 3 C H 3 C O C 3 H 7 CH 3 N S COOH gemfibrozil CH 3 C 3 H 7 O anionic probenecid amphiphiles S N(CH 3 ) 2 MRP1-10 Cl N S O HOOC MK571 ATP ADP

Differential recognition by MDR pumps Influence of ATP-depletion and pump inhibitors on accumulation at equilibrium azithromycin ciprofloxacin & & P-glycoprotein MRP extracell. conc. 5 mg/L; AZM 3 h; CIP 2 h Michot et al . AAC (2004) 48:2673-82

Aim of the study amphiphilic antibiotics • accumulating in eucaryotic cells • considered as useful for treating intracellular infections • known substrates of efflux pumps in bacteria efflux from macrophages ? macrolides quinolones • phenotypic characterization of the active efflux • consequences for consequences for intracellular activity intracellular activity

phagolysosomes S. aureus Models of intracellular infection L. monocytogenes cytosol

Influence of pump inhibitors on intracellular activity azithromycin and L. monocytogenes L. monocytogenes AZM AZM verapamil 20 µM; 24 h Seral et al (2003) JAC 51:1167-73

Influence of pump inhibitors on intracellular activity azithromycin and S. aureus S. aureus AZM AZM verapamil 20 µM; 24 h Seral et al (2003) JAC 51:1167-73

Influence of pump inhibitors on intracellular activity ciprofloxacin and L. monocytogenes L. monocytogenes CIP gemfibrozil 250 µM; 24 h Seral et al (2003) JAC 51:1167-73

Influence of pump inhibitors on intracellular activity ciprofloxacin and S. aureus S. aureus CIP gemfibrozil 250 µM; 24 h Seral et al (2003) JAC 51:1167-73

Influence of pump inhibitors on antibiotic distribution verapamil enhances azithromycin concentration In cytosol and vacuoles L. monocytogenes AZM AZM S. aureus Seral et al (2003) JAC 51:1167-73

Influence of pump inhibitors on antibiotic distribution gemfibrozil enhances ciprofloxacin cytosolic content L. monocytogenes CIP S. aureus Seral et al (2003) JAC 51:1167-73

Are these effects clinically-relevant ? constitutive efflux makes AZM and CIP activity suboptimal in a clinically-meaningful range of concentrations

Aim of the study amphiphilic antibiotics • accumulating in eucaryotic cells • considered as useful for treating intracellular infections • known substrates of efflux pumps in bacteria efflux from macrophages ? macrolides quinolones macrolides quinolones • cellular pharmacokinetics • model of interaction with the transporters

Aim of the study amphiphilic antibiotics • accumulating in eucaryotic cells • considered as useful for treating intracellular infections • known substrates of efflux pumps in bacteria efflux from macrophages ? macrolides quinolones • • cellular pharmacokinetics cellular pharmacokinetics • • model of interaction model of interaction with the transporters with the transporters

Kinetics of accumulation and efflux for azithromycin accumulation markedly increased; efflux marginally affected extracell. conc. 5 mg/L; verapamil 20 µM Seral et al (2003) AAC 47:1047-51

Kinetics of accumulation and efflux for ciprofloxacin both accumulation and efflux markedly affected extracell. conc. 17 mg/L; probenecid 5 mM Michot et al . AAC (2004) 48:2673-82

Kinetics of accumulation and efflux for moxifloxacin neither accumulation nor efflux affected extracell. conc. 17 mg/L; probenecid 5 mM Michot et al . AAC (2005) – in press

Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin Michot et al . AAC (2005) – in press

Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin • moxifloxacin not affected Michot et al . AAC (2005) – in press

Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin CIP MXF CIP Michot et al . AAC (2005) – in press

Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin CIP MXF CIP moxifloxacin also able to interact with the transporter ! Michot et al . AAC (2005) – in press

Comparison of kinetic parameters influx efflux drug half-life (min) half-life (min) flux flux (pmol/mg (pmol/mg prot/min) prot/min) control inhibitor control inhibitor AZM 1 44 71 1 49 53 CIP 5 8 8 6 1.2 7.2 MXF 68 0.2 0.2 66 0.6 0.6

Comparison of kinetic parameters influx efflux drug half-life (min) half-life (min) flux flux (pmol/mg (pmol/mg prot/min) prot/min) control inhibitor control inhibitor AZM 1 44 71 1 49 53 CIP 5 8 8 6 1.2 7.2 MXF 68 0.2 0.2 66 0.6 0.6

Comparison of kinetic parameters influx efflux drug half-life (min) half-life (min) flux flux (pmol/mg (pmol/mg prot/min) prot/min) control inhibitor control inhibitor AZM 1 44 71 1 49 53 CIP 5 8 8 6 1.2 7.2 MXF 68 0.2 0.2 66 0.6 0.6