Antibiotic Antibiotic accumulation and efflux accumulation and efflux in eukaryotic cells: in eukaryotic cells: a journey at the frontier a journey at the frontier of pharmacokinetics of pharmacokinetics and and pharmacodynamics “corpora non agunt nisi fixata” Ehrlich’s “magic bullet” theory pharmacodynamics Unité de Pharmacologie cellulaire et moléculaire F. Van Bambeke
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Antibiotic accumulation and efflux in eukaryotic cellsCraig (1998) CID 26:1-10. ISAP classical view of PK/PD. but classical PD predicts concentration-effects for all drugs …. but
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Antibiotic Antibiotic accumulation and effluxaccumulation and effluxin eukaryotic cells:in eukaryotic cells:
a journey at the frontier a journey at the frontier of pharmacokinetics of pharmacokinetics and and pharmacodynamics
“corpora non agunt nisi fixata”
Ehrlich’s “magic bullet” theory
pharmacodynamics
Unité de Pharmacologiecellulaire et moléculaire
F. Van Bambeke
Magic bullets need to reach their target
Magic bullets need to reach their target
glycopeptides
β-lactams
quinolones
macrolides aminoglycosides
for appropriate time and in sufficient concentration …
Birth of antibiotic “PK-PD”
Concentrationversus timein tissues andother body fluids
Pharmacologicor toxicologiceffect
Concentrationversus timein serum
Dosageregimen
Concentrationversus timeat siteof infection
Antimicrobial effect versustime
absorptiondistributionelimination
PHARMACOKINETICS PHARMACODYNAMICS
Craig (1998) CID 26:1-10
ISAP classical view of PK/PD
but classical PD predicts concentration-effectsfor all drugs ….
but classical PD predicts concentration-effectsfor all drugs ….
ampicillingentamicin
S. aureus; 24 h Barcia-Macay et al, submitted; Lemaire et al (2005) JAC in press
Can we conciliate both theories ?
Cmin Cmax
conc.-dependent
Cmin Cmax
PD profile in the clinics
time -dependentampicillingentamicin
S. aureus; 24 h Barcia-Macay et al, submitted; Lemaire et al (2005) JAC in press
Target accessibility becomes critical for intracellular activity
Main routes of drug entry in cells
passive
diffusionchannel
transporter transporterendocytosis
active
Intracellular “PK-PD”
Concentrationversus timein non-infectedcells
Pharmacologicor toxicologiceffect
Concentrationversus timein cells
Dosageregimen
Concentrationversus timeat intracellularsite of infection
Antimicrobial effect versustime
penetrationdistributionefflux
PHARMACOKINETICS PHARMACODYNAMICS
Intracellular “PK-PD”
Concentrationversus timein non-infectedcells
Pharmacologicor toxicologiceffect
Concentrationversus timein cells
Dosageregimen
Concentrationversus timeat intracellular site of infection
Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
Antibiotics as substrates of efflux pumps
O
H3C
OH
OH
OH
CH3
H3CH2C O
O
CH3
CH3
H3C
O
O
OHO(H3C)2N
CH3
OH
N
CH3
CH3
H3C
H3C
H3CO
azithromycin
ciprofloxacin
N
C
O
FOH
O
OCH3
HN N
moxifloxacin
N
O
OH
O
HNN
F
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
• phenotypic characterization of the active efflux
• consequences for intracellular activity
quinolones
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
•• phenotypic characterization phenotypic characterization of the active efflux of the active efflux
• consequences for intracellular activity
quinolones
Efflux pumps expressed in J774 macrophages
ABC multidrug transporters
ATP ADP
MDR-1 (P-glycoprotein) MRP1-10
cationic amphiphiles
anionic amphiphiles
How to inhibit ABC transporters ?
ATP ADP
MDR-1 (P-glycoprotein) MRP1-10
cationic amphiphiles
anionic amphiphiles
deoxyglucoseNaN3
How to inhibit ABC transporters ?OCH3
H3CO
CNCH(CH3)2
N
CH3
OCH3
OCH3
NH
C
OHN
H3CO
O
N
H3CO
H3CO
ATP
verapamil
cationic amphiphiles
MDR-1 (P-glycoprotein)
GF120918
ADP
How to inhibit ABC transporters ?
COOHSN
O
OC3H7
C3H7
NCl
S
S
HOOC
N(CH3)2
O
ATP
MRP1-10
anionic amphiphilesprobenecid
(CH2)3 C
CH3
CH3
COOHH3C
CH3gemfibrozil
MK571
ADP
Differential recognition by MDR pumps
Influence of ATP-depletion and pump inhibitors on accumulation at equilibrium
ciprofloxacin&
MRP
azithromycin&
P-glycoproteinextracell. 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
• phenotypic characterization of the active efflux
• consequences for consequences for intracellular activityintracellular activity
quinolones
Models of intracellular infection
L. monocytogenes S. aureus
phagolysosomescytosol
Influence of pump inhibitors onintracellular activity
azithromycin and L. monocytogenes
L. monocytogenes
verapamil 20 µM; 24 h
AZMAZM
Seral et al (2003) JAC 51:1167-73
Influence of pump inhibitors onintracellular activity
azithromycin and S. aureus
S. aureus
AZMAZM
verapamil 20 µM; 24 h Seral et al (2003) JAC 51:1167-73
Influence of pump inhibitors onintracellular activity
ciprofloxacin and L. monocytogenes
L. monocytogenes
gemfibrozil 250 µM; 24 h
CIP
Seral et al (2003) JAC 51:1167-73
Influence of pump inhibitors onintracellular 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 concentrationIn cytosol and vacuoles
L. monocytogenes
AZMAZM
S. aureus
Seral et al (2003) JAC 51:1167-73
Influence of pump inhibitors on antibiotic distribution
constitutive efflux makes AZM and CIP activity suboptimalin 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 ?
macrolidesmacrolides
• cellular pharmacokinetics
• model of interaction with the transporters
quinolonesquinolones
Aim of the study
amphiphilic antibiotics• accumulating in eucaryotic cells• considered as useful for treating intracellular infections• known substrates of efflux pumps in bacteria
Moxifloxacin, ‘futile-cycle’ modelEytan et al. (1996) JBC 271:12897-902
Conclusion constitutive efflux of antibiotics in macrophages
pharmacokinetics:suboptimal cellular accumulation
suboptimal intracell.activity
pharmacodynamics:
resistance ?
wide spectrum transporters
pharmacokinetics:
drug interactions ?
differences in affinitywithin a AB class
pharmacology:
Questions for future research
?
mechanismof
transport?
identificationof
transporter
cooperation with bacterial efflux pumps
? ?
drug interactions
metabolicalterations?
Take home message
constitutive efflux is part of the game
Take it into account in the choice of your « magic bullets » …for their optimal targeting
Thanks toThanks to …
Thanks toThanks to …
Evaluating magic bulletsEvaluating magic bullets
•• pharmacokineticspharmacokineticsH.H. ChanteuxChanteux, M., M. HeremansHeremans, J.M., J.M. MichotMichot
•• pharmacodynamicspharmacodynamicsM. Barcia, N. M. Barcia, N. BlesBles, S., S. CarrynCarryn, S., S. LemaireLemaire, , A. Olivier, C.A. Olivier, C. SeralSeral, S. Van de, S. Van de VeldeVelde
•• toxicodynamicstoxicodynamicsJ.P.J.P. MontenezMontenez, H., H. ServaisServais, D., D. TytecaTyteca
•• biophysicsbiophysics & molecular biology& molecular biologyN.N. CaceresCaceres, N., N. Fa Fa
Thanks toThanks to …
New magic bulletsNew magic bullets
•• chemistrychemistryE.E. ColacinoColacino, C., C. DaxDax, L., L. EfronEfron, T., T. HappaertsHappaerts, M., M. RenardRenard
•• pharmacologypharmacologyI.I. TytgatTytgat, D. Van, D. Van AckerenAckeren
•• modelingmodelingM.M. PrévostPrévost, M., M. RoomanRooman, S., S. VandevuerVandevuer
Thanks toThanks to …
Resistance to magic bulletsResistance to magic bullets
•• effluxeffluxL.L. AvrainAvrain, N., N. MesarosMesaros
•• glycopeptidesglycopeptidesP.P. CourvalinCourvalin and his teamand his team
Thanks toThanks to …
Clinical use of magic bulletsClinical use of magic bullets
•• clinical pharmacyclinical pharmacyE.E. AmpeAmpe, V., V. BasmaBasma, A., A. SpinewineSpinewine
Thanks toThanks to …
Playing with magic bulletsPlaying with magic bullets
•• technical stafftechnical staffN. Aguilera, M.C.N. Aguilera, M.C. CambierCambier, O., O. MeertMeert, , F.F. RenoirdRenoird, M., M. VergauwenVergauwen
•• secretarysecretaryM.M. BreugelmansBreugelmans
Thanks toThanks to …
Ehrlich’s colleaguesEhrlich’s colleagues
Thanks toThanks to …
Inspiring research on magic bulletsInspiring research on magic bullets
Thanks toThanks to …
Evaluating research on magic bulletsEvaluating research on magic bullets
P. Courvalin, A. Dalhoff, M. Delmée,P. Courvalin, A. Dalhoff, M. Delmée,H. Derendorf,Y. Glupczynski, H. Derendorf,Y. Glupczynski, E. Sonveaux, F. ZechE. Sonveaux, F. Zech
Thanks toThanks to …
Paying for research on magic bulletsPaying for research on magic bullets
Thanks toThanks to …
Managing research on magic bulletsManaging research on magic bullets
M.P. MingeotM.P. Mingeot--LeclercqLeclercq
P.M. TulkensP.M. Tulkens
Thank you Thank you for your attentionfor your attention