models of antibiotic intracellular transport · 2005-12-26 · 45th icaac december 19th, 2005 1 d d...
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![Page 1: Models of antibiotic intracellular transport · 2005-12-26 · 45th ICAAC December 19th, 2005 1 D D Models of Intracellular D* Antibiotic Transport Paul M. Tulkens, MD, PhD Cellular](https://reader030.vdocuments.site/reader030/viewer/2022011905/5f2fd4c642573e0cc85e187e/html5/thumbnails/1.jpg)
December 19th, 200545th ICAAC1
D DD*Models of Intracellular
Antibiotic Transport
Paul M. Tulkens, MD, PhD
www.facm.ucl.ac.bewww.facm.ucl.ac.be
Cellular and Molecular PharmacologyCatholic University of Louvain, Brussels, Belgium
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December 19th, 200545th ICAAC2
Why intracellular antibiotics ?
The Cellantibiotic bacteria
Black Box...
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December 19th, 200545th ICAAC3
Intracellular antibiotics: the issues
1. which bacteria and where ?2. which antibiotics accumulate ?3. influx vs efflux ?3. where are antibiotics in cells ?4. intracellular expression of activity ?5. bacterial responsiveness ? 6. cooperation with host defenses ?7. any toxicity ?
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December 19th, 200545th ICAAC4
Which antibiotics accumulate in cells ?
• beta-lactams: ≤ 1x• aminoglycosides: <1 to 2 x• ansamycins: 2-3 x• tetracyclines: 2-4 x• fluoroquinolones: 5 - 20 x• macrolides: 4 to > 100 x *• glycopeptides: 1 to 400 x !! **
* azithromycin, ketolides** oritavancin
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December 19th, 200545th ICAAC5
How do antibiotics penetrate in cells ?
• trans-membrane influx diffusioncarrier mediated
• endocytosis
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December 19th, 200545th ICAAC6
Entry by diffusion …
• amphiphilic compounds• fast• non-saturable• no competition byanalogues
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December 19th, 200545th ICAAC7
Entry by diffusion: some examples…
macrolidesfluoroquinolonestetracyclinesansamycinesβ-lactams,...
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December 19th, 200545th ICAAC8
Entry of azithromycin…
diffusion
Tyteca et al., EJCB, 2001, in press
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December 19th, 200545th ICAAC9
Entry of sparfloxacin …
diffusion
Ouadrhiri et al., AAC, 1999
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December 19th, 200545th ICAAC10
Entry of ampicillin …
diffusion
Ouadrhiri et al., AAC, 1999
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December 19th, 200545th ICAAC11
Carrier-meddiated influx ?
• specific structure• (energy-dependent)• saturable• competition byanalogues
only limited evidence for specific transporters is available so far
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December 19th, 200545th ICAAC12
Carrier-mediated influx ?the case of HSR-903
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December 19th, 200545th ICAAC13
Carrier-mediated influx ?the case of HSR-903
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December 19th, 200545th ICAAC14
Endocytosis…
• non-permeant drugs
• slow unlessmembrane-bound, or receptor-mediated
• confined tovacuolarsystem
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December 19th, 200545th ICAAC15
Endocytosis: examples…
pinocytosis
aminoglycosidesglycopeptides (?)
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December 19th, 200545th ICAAC16
Fluid-phase endocytosis…
Slow (days…)ill-effective (2-4 fold)non-saturable
aminoglycosides in fibroblasts or macrophages
Cc/Ce = 1
Ce = 1.3 mg/ml
Ce = 0.65 mg/ml
Ce = 0.35 mg/ml
Tulkens & Trouet, 1978
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December 19th, 200545th ICAAC17
Receptor-mediated endocytosis…
fast (days…)very effective (100 –fold or more)saturable …
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December 19th, 200545th ICAAC18
Receptor-mediated endocytosis…
entry of aminoglycosides in kidney tubular cells
binding to• megalin
(Moeströp et al., 1995)
• acidic phospholipids (Humes et al, 1983) Giuliano et al., J. Pharm. Exp. Ther., 1986
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December 19th, 200545th ICAAC19
Receptor-mediated endocytosis…
Mice deficient in megalin do not
accumulate gentamicin in
kidney
Schmitz et al., J. Biol. Chem. 277:618-622, 2002
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December 19th, 200545th ICAAC20
Membrane-binding-mediated endocytosis ? …
very effective if tight binding(100 –fold or more)continuous over time …
Van Bambeke et al., AAC (2004) 48:2853-2860
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December 19th, 200545th ICAAC21
Antibiotics efflux ?
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December 19th, 200545th ICAAC22
Transporters - data bases
maindrug transporters
http://www-biology.ucsd.edu/~msaier/transport/
Saier, 2000
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December 19th, 200545th ICAAC23
Transporters involved in the efflux of antibiotics from eucaryotic cells
superfamily transporter physiol. antibioticssubstrates
ABC MDR1 phospholipids fluoroquinolonesmacrolidesβ-lactamstetracyclinesstreptogramins
MRP1 phospholipids fluroquinolonesleukotrienes macrolidesconjugates rifamycins
MRP2 conjugates fluoroquinolonesβ-lactams
MFS NPT1 phosphates β-lactams
OAT OATP1 bile salts β-lactamssteroids
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December 19th, 200545th ICAAC24
Most frequent antibiotic-pumps in eucaryotes (1/2)
TOPOLOGY
Multiple Drug Resistance (MDR also known as PgP)
NH2
COOH
MECHANISM
?
ATP ADP + Pi
+
+
+
+
+
ANTIBIOTICS
tetracyclinesfluoroquinoloneserythromycinlincosamidesrifampicin
chloramphenicol
aminoglycosides
+
+
Van Bambeke et al.,Biochem. Pharmacol. 2000
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December 19th, 200545th ICAAC25
Most frequent antibiotic-pumps in eucaryotes (2/2)
Multidrug Resistance Proteins (MRP)
ANTIBIOTICS
fluoroquinolones-
+ tetracyclinesmacrolides
TOPOLOGY
NH2
COOH
MECHANISM
?
ATP ADP + Pi
-
-
-
-
-
GSH
Van Bambeke et al.,Biochem. Pharmacol. 2000
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December 19th, 200545th ICAAC26
Evidencing active efflux ...
non linear accumulationkinetics ...
diffusion
receptormediateduptake
"facilitateduptake"
Cc
Ce
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December 19th, 200545th ICAAC27
Evidencing active efflux ...
non linear accumulationkinetics ...
Ceat low concentrations,most of the drug is rexported …
Cc
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December 19th, 200545th ICAAC28
Evidencing active efflux ...
non linear accumulationkinetics ...
Cc
at large concentrations, efflux becomes saturated
Ce
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December 19th, 200545th ICAAC29
Evidencing efflux of ciprofloxacin
Ciprofloxacin accumulation in J774 macrophages is facilitated upon increase of its extracellular concentration
Michot et al., AAC (2004) 48:2673-2682
4
0 1 2 3 2 4 9 17 34 68136
cellu
lar c
ipro
floxa
cin
12.5
log
(mg/
L)
3
log (mg/L)
10.0 Cc/C
e
2 7.5
5.01
2.5
0 0
mg/Lextracellular [ciprofloxacin] - 2h incubation at 37°C
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December 19th, 200545th ICAAC30
Characterization of the transporter(s)
ATP depletion increases ciprofloxacin accumulation and decreases ciprofloxacin efflux in J774 macrophages
Michot et al., AAC (2004) 48:2673-2682
0 5 10 150
25
50
75
100ControlATP depletion
Time (min)
Res
idua
l fra
ctio
n (%
)
(17µg/ml)Ce = 50 µM
Control ATP depletion0.0
2.5
5.0
7.5
10.0
Cc/
Ce
2h incubation at 37°C
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December 19th, 200545th ICAAC31
Efflux vary among closely related derivatives and may be impaired by (apparently) unrelated substances
Michot et al. AAC (2005) 49:2429-2437
accumulation of quinlones in J774 macrophages and influence of P-gp and MRP inhibitors
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December 19th, 200545th ICAAC32
Evidencing efflux of azithromycin macrolides(through P-gp) …
Kinetics of influx and efflux of azithromycin in J774 murinemacrophages with or without 20 µM verapamil.
Seral et al., AAC (2003) 47:1047-1051
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December 19th, 200545th ICAAC33
Azithromycin follows the a ‘kick-back’ modelGaj et al. (1998) Biochem. Pharmacol. 55:1199-211
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December 19th, 200545th ICAAC34
Ciprofloxacin is follwing the classical modelKolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42
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December 19th, 200545th ICAAC35
D D1
1. Penetration
2
2. No efflux
D
3. Accumulation
3
5
5. Expression of activity
5
6. Bacterial responsivenessand pharmacodynamics
6
6
7
7. Cooper. with host def.
7
D*
4
44
4. Subcell. bioavailability
Any relation to activity ?
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December 19th, 200545th ICAAC36
D D1
2
D3
5
5
6
6
7
7
D*
4
44
Co-workers on all this stuff ...
M.P. Mingeot, D. TytecaJ.M. Michot, C. SeralM.B. Carlier, A. Zenebergh
C. Renard, H. Fan, E. Sonveaux, …S. Carryn, F. Van Bambeke,M. Heremans, N. Caceres, …B. Scorneaux, Y. Ouadrhriri, I. Paternotte, ….Y.Chanteux, M. Bouvier d’Yvoire