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Are intracellular drug concentrations relevant for efficacy ?
A discussion about accumulation, efflux and activity …
Paul M. Tulkens, MD, PhDFrançoise Van Bambeke, PharmD, PhD
Cellular and Molecular PharmacologyCatholic University of Louvain
Brussels, Belgium
Najaarsvergadering – Amsterdam – 16 november 2006
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A simple figure …
Carryn et al. Infect Dis Clin North Am. 2003 Sep;17(3):615-34.
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First statements …
• If a drug does not accumulate, it cannot be active …
Quick answer: this is correct if you mean "it does not get in cells at all…"
More elaborate answer: no "accumulation" does not mean that the drug is not present, and if present, it may be active if above the critical concentration for sufficient time …
Experimental evidence: β-lactams, known for "no accumulation" are active against intraphagocyticL. monocytogenes and S. aureus if their extracellular concentration is large enough… and if you let them enough time to act…
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Intraphagocytic S. aureus and β-lactams
24 h model
Cmax= 63 mg/L (total)
Barcia-Macay et al. M, Antimicrob Agents Chemother. 2006 Mar;50(3):841-51.
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Intraphagocytic L. monocytogenes and β-lactams
Lemaire et al. J Antimicrob Chemother. 2005;55(6):897-904.
Cmax
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Observation …
• The activity of β-lactams is larger than anticipated…
Quick answer: Their concentration may simply be large enough
More elaborate answer: The intracellular milieu may favor their activity …
Experimental evidence for a potential explanation:• Acid pH increases the activity of β-lactams against intraphagocytic
S. aureus…• We do not have it (yet) for L.monocytogenes …
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Acid pH favors the activity of β-lactams …
Baudoux et al., J. Antimicrob. Chemother., in press.
extracellular activity
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And acidity compensates for poor intracellular accumulation …
Baudoux et al., J. Antimicrob. Chemother., in press.
intracellular activity
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What about other antibiotics …• Aminoglycosides accumulate (slowly) in phagolysosomes
but their activity is defeated by the acid pH for phagolysosomal organisms (S. aureus...) >< β-lactams… !
They are inactive against L. monocytogenes (not present in the cytosol …)
• Macrolides accumulate … but their activity is severely defeated by the acid pH … and they are only bacteriostatic…
• Quinolones accumulate modestly, but their activity is maintained at acid pH … and they have access to most intracellular compartments… Yet, their intracellular activity is at most similar to their extracellular activity …
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A not so simple figure …
Carryn et al. Infect Dis Clin North Am. 2003 Sep;17(3):615-34.
Activity = accumulation x bioavailability x favorable conditions
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A few words about bacterial response: the case of telavancin and VRSA…
lipophilicside chain
polar group
Higgins et al., Antimicrob Agents Chemother. (2005) 49:1127-34
classical pharmacophoreof glycopeptides
this causes increase in bacterial membrane permeability at larger
concentrations …
membrane permeability assay
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Televancin dual mode of action ?
3 h kill curvesextracellular
bacteriaBarcia-Macay et al., JAC, Oct 24; [Epub ahead of print]
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Intracellular activity of telavancinvs.vancomycin:
MSSAMRSA
24h CFU at Cmax: • vanco: ∼ 0.5 log• TLV: ∼ 2 log
Barcia-Macay et al., JAC, Oct 24; [Epub ahead of print]
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24h CFU at Cmax: • vanco: static• TLV: ∼ 1.2 log
Intracellular activity of telavancinvs.vancomycin:
VISAVRSA
Barcia-Macay et al., JAC, Oct 24; [Epub ahead of print]
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The picture gets a bit more complex …
Activity = accumulation x bioavailability x favorable conditions x bacterial responsiveness
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Efflux from eucaryotic cells and intracellular activity
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The story of the eucaryotic ABC transporters
ATP ADP
MDR-1 (P-glycoprotein) MRP1-10
cationicamphiphiles
anionicamphiphiles
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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 is cationic
Ciprofloxacin is zwitterionic
N
O
OH
O
HNN
F
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How to inhibit ABC transporters ?
ATP ADP
MDR-1 (P-glycoprotein) MRP1-10
cationicamphiphiles
anionicamphiphiles
deoxyglucoseNaN3
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How to inhibit ABC transporters ?
OCH3
H3CO
CNCH(CH3)2
N
CH3
OCH3
OCH3
NH
C
OHN
H3CO
O
N
H3CO
H3CO
ATP
MDR-1 (P-glycoprotein)
ADP
cationicamphiphiles
verapamil
GF120918
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How to inhibit ABC transporters ?
COOHSN
O
OC3H7
C3H7
NCl
S
S
HOOC
N(CH3)2
O MRP1-10
anionicamphiphilesprobenecid
MK571
(CH2)3 C
CH3
CH3
COOHH3C
CH3gemfibrozil
ATP ADP
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Differential recognition by MDR pumps
azithromycin&
P-glycoprotein
ciprofloxacin&
MRP
Influence of ATP-depletion and pump inhibitorson accumulation at equilibrium
Michot et al. AAC (2004) 48:2673-82
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Models of intracellular infection
L. monocytogenes S. aureus
cytosol phagolysosomes
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Influence of pump inhibitorson intracellular activity
azithromycin and L. monocytogenes
L. monocytogenes
AZMAZM
Seral et al (2003) JAC 51:1167-73
Δlo
g C
FU (5
h –
0 h)
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azithromycin and S. aureus
AZMAZM
S. aureus
Influence of pump inhibitorson intracellular activity
Seral et al (2003) JAC 51:1167-73
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ciprofloxacin and L. monocytogenes
CIP
Δlo
g C
FU (5
h –
0 h)
L. monocytogenes
Influence of pump inhibitorson intracellular activity
Seral et al (2003) JAC 51:1167-73
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ciprofloxacin and S. aureus
CIP
S. aureus
Influence of pump inhibitorson intracellular activity
Seral et al (2003) JAC 51:1167-73
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Influence of pump inhibitors on antibiotic distribution
L. monocytogenes
AZMAZM
S. aureus
verapamil enhances azithromycin concentrationin cytosol and vacuoles
Seral et al (2003) JAC 51:1167-73
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L. monocytogenes
CIP
S. aureus
gemfibrozil increases ciprofloxacin cytosolic content ONLY
Influence of pump inhibitors on antibiotic distribution
Seral et al (2003) JAC 51:1167-73
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in eucaryotic cells …
Over-expression of efflux pumps as mechanism of « resistance »
Michot et al, AAC (2006) 50:1689-95
in prokaryotic cells …
wild-type macrophages
resistantmacrophages
wild-type L. monocytogenes
resistantL. monocytogenes
Godreuil et al, AAC (2003) 47:704-8
probenecid reserpine
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Cooperation between procaryoticand eucaryotic efflux pumps
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Cooperation between procaryoticand eucaryotic efflux pumps
Lismond et al, ICAAC (2006) A1108
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To conclude …
accumulation x bioavailability x favorable conditions x bacterial responsiveness
effluxActivity =