intracellular - uclouvain
TRANSCRIPT
10/09/2004Magic Bullets ...1
Intracellular antibiotics
Paul M. Tulkens, MD, PhDFrançoise Van Bambeke, Pharm., PhD
in collaboration with Cristina Seral, Pharm., PhD
Stéphane Carryn, Pharm., PhDHugues Chanteux, Pharm., PhD
Sandrine Lemaire, BSc.
www.www.facmfacm..uclucl.ac.be.ac.beCellular and Molecular Pharmacology
Catholic University of Louvain, Brussels, Belgium
D DD*
10/09/2004Magic Bullets ...2
Why intracellular antibiotics ?
The Cellantibiotic bacteria
Black Box...
10/09/2004Magic Bullets ...3
Which bacteria … and which diseases ...
� Obligatory or mainly intracellular:� respiratory infections (pneumopathies):
Chlamydia pneumoniae: 10% in childrenLegionella pneumophila: frequent if
immunosuppressionMycobacterium spp.: frequent if immunosuppression
� sexually transmitted diseasesChlamydia trachomatis: most common pathogen
� CNS infections + other sites:Listeria monocytogenes: pregnant women;
immunosuppression
� Facultative or mainly extracellular:� digestive tract infections
Salmonella spp., Shigella spp.� respiratory, cutaneous, etc…tract infections
Streptococcus spp., Staphylococcus spp. etc...
10/09/2004Magic Bullets ...4
Subcellular localization of antibiotics ?
cytosol
• fluoroquinolones• beta-lactams• ansamycins• macrolides (1/3)
lysososomes• macrolides (2/3)• aminoglycosides ?
?endosomes
phagosomes
phago-lysososomes
?
10/09/2004Magic Bullets ...5
Mechanisms of localisation and accumulation ...
cytosol
• fluoroquinolones
• Mechanism unknown(loose binding to lipids / lipoproteins ? …)
• Efflux demonstrated (MRP ?)
10/09/2004Magic Bullets ...6
Mechanisms of localisation and accumulation ...
lysososomes• macrolides • aminoglycosides
� proton trapping� binding to phospholipids� for aminoglycosides:
inability to cross membranes
10/09/2004Magic Bullets ...7
Subcellular bioavailability of antibiotics ?
High Fair Nil
FQ / Ansamyc. / cytosol. ML lysosom. ML / AG
10/09/2004Magic Bullets ...8
Subcellular bioavailability of antibiotics ?
FQ
Fluoroquinolonesmove easilyacrossmembranes
10/09/2004Magic Bullets ...9
Subcellular bioavailability of antibiotics ?
aminoglycosidesand lysosomal macrolidesreamain largely if not totally sequesteredin an acidic environment ...
10/09/2004Magic Bullets ...10
Listeria monocytogeneshly+
antibiotics:
• ampicillin/meropenem• azithromycin• sparfloxacin/moxifloxcin• pivampicillin
10/09/2004Magic Bullets ...11
Intracellular infection cycle of Listeria monocytogenes hly+
from Portnoy et al.
10/09/2004Magic Bullets ...12
phagocytosis
Following the intracellular fate of Listeria m. by EM
escape from vacuole
in cytosol
10/09/2004Magic Bullets ...13
1st question: is there a simple relation between MIC, accumulation and intracellular activity (5 h model)
Accumulation
AMPI AZ SP0
25
50
75
100
MIC
AMPI AZ SP0.0
0.5
1.0
1.5
Activity *
AMPI AZ SP0.0
0.5
1.0
1.5
* log CFU 5hCe = 10 x MICOuadhriri et al., AAC,1999
10/09/2004Magic Bullets ...14
Listeria m. and ampicillin
Ampicillin is poorly active against intracellularListeria m. in spite of its favourable MIC;
lack of accumulation ...
Why do you keep ampicillin ?extracellular bacteriaget intracellular activity with very large doses ?? (but β-lactams are NOT dose-dependent…)but may be you just have to wait ...
10/09/2004Magic Bullets ...15
β-lactams become bactericidal intracellularly after 24h and if you give a dose high enough
0 5 10 15 20 25104
105
106
107
108
109
1010
controlampicillin
Time (h)
CFU
/ m
g pr
otei
n
(Carryn et al., 2003, JAC 51:1051-52
-2 -1 0 1 2 3
ampicillineméropénème
-2
-1
0
1
2
Multiples of MIC (log10)
Change from
original inoculum (log
10 )
(Lemaire et al, 2004, unpublished
10/09/2004Magic Bullets ...16
Listeria m. and azithromycin
Azitromycin is poorly active against intracellular Listeria m. in spite of its exceptionally large intracellular concentration
most azithromycin is trapped in lysosomes
azithromycin is poorly bactericidal
Is there a future for macrolides ?
10/09/2004Magic Bullets ...17
In a pharmacological model*, fluoroquinolones appear most active in spite of relatively unfavourable MICsand modest cellular accumulation compared to macrolides …
Fluoroquinoloneshave a large subcellular bioavailabilityare highly bactericidal
Listeria m. and fluoroquinolones
* all Ce = 10 X the MIC
10/09/2004Magic Bullets ...18
Comparative intracellular activities at multiples of Cmax
% of Cmax
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
25 50 75 100
ampicillinmoxifloxacin
Cha
nge
from
orig
inal
inoc
ulum
(∆lo
g)
(Carryn et al., 2002, AAC 43:2095-103)
Moxifloxacin is profoundly bactericidal at clinically achievable concentrations
10/09/2004Magic Bullets ...19
However, intracellularmoxifloxacin is NOT moreactiveintracellularlythan extracellularly when using the extracellular concentration as comparison basis ...
broth
cells
Carryn et al., AAC, 2002
10/09/2004Magic Bullets ...20
And this is obvious if you compare intracellular and extracellular activities at the same
apparent concentration * / MIC ratio ….
0.1 1 10 100
-2
-1
0
1
concentration / MIC
log
CFU
(5 h
-0h
)
extracellular
intracellular
levofloxacinemoxifloxacine
levofloxacinemoxifloxacine
extracellular = actualintracellular = calculated
Seral et al., unpublished
10/09/2004Magic Bullets ...21
A basic prodrug of a β-lactam ?
ProD
DDD
ProDH+H+
ProDH+
H+ProD ProD
pH 7.4 pH 7.0 pH 5.4
10/09/2004Magic Bullets ...22
N
S
C
CH3O
CH3
HNC
O
OO O
OH2N
N
S
C
CH3O
CH3
HNC
O
OO N
O
O
H2N
Phthalimidomethylampicillin (PIMA)
Pivaloyloxymethylampicillin (PIVA)
• regenerate ampicillin
• accumulate in J774 macrophages
Paternotte et al, Biorg. Med. Chem. 2001
Fan et al, Bioorg. Med. Chem. Let.1997
Basic compounds that
H2N
H2N
10/09/2004Magic Bullets ...23
Intracellular activity for extracellular concentrations of ...
0 1 2 3 4 5
106
107
0.5X MIC
Only PIVA is active
0 1 2 3 4 5
106
107
10X MIC
CFU
/mg
prot
Time (h)Same activity for PIVA, PIMA and AMPI
Chanteux et al, JAC, 2003
10/09/2004Magic Bullets ...24
0 5 10 15 2010 4
10 6
10 8
10 10
CTRL
PIVA
But maintains it if the medium is renewed every 5 h
Time (h)
CFU
/mg
prot
At low extracellular concentration, PIVA loses its activity after 5 h if the medium is not renewed
0.5X MIC
Extracellularconcentration
Chanteux et al, JAC, 2003
10/09/2004Magic Bullets ...25
0 5 10 15 20 250
5
10
15
25
30
AMPI from PIVA
AMPI from AMPI
AMPI from PIVA with changes of medium)
Time (h)
Cc/
Ce
PIVA releases large amount of intracellular ampicillin
Chanteux et al, JAC, 2003
10/09/2004Magic Bullets ...26
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
The seven pillars of intracellular / intratissular activity ?
10/09/2004Magic Bullets ...27
D D2 D5
D*
The 6 pillars of intracellular / intratissularaccumulation and activity of antibiotics...
4
Françoise Van Bambeke, Stéphane Carryn, Cristina Seral, Hugues Chanteux, Sandrine Lemaire ...
Françoise Van Bambeke
Hugues Chanteux
Stéphane Carryn
Sandrine Lemaire
Christina Seral