1. Intracellular antibioticsD D*
Paul M. Tulkens, MD, PhD
Françoise Van Bambeke, Pharm., PhD in collaboration with
Cristina Seral, Pharm., PhD
Stéphane Carryn, Pharm., PhD
Hugues Chanteux, Pharm., PhD
Sandrine Lemaire, BSc.
Cellular and Molecular Pharmacology
Catholic University of Louvain, Brussels, Belgium
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10/09/2004

2. Why intracellular antibiotics ?
The Cell
antibiotic
bacteria
Black Box...
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10/09/2004

3. Which bacteria … and which diseases ...
Obligatory or mainly intracellular:
respiratory infections (pneumopathies):
Chlamydia pneumoniae: 10% in children
Legionella pneumophila: frequent if immunosuppression
Mycobacterium spp.: frequent if immunosuppression
sexually transmitted diseases
Chlamydia 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...
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10/09/2004

4. Subcellular localization of antibiotics ?
cytosol
fluoroquinolones
beta-lactams
ansamycins
macrolides (1/3) ?
endosomes
phagosomes
phago- lysososomes
lysososomes
macrolides (2/3)
aminoglycosides
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10/09/2004

5. Mechanisms of localisation and accumulation ...
cytosol
fluoroquinolones
Mechanism unknown (loose binding to lipids / lipoproteins ? …)
Efflux demonstrated (MRP ?)
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10/09/2004

6. Mechanisms of localisation and accumulation ...
proton trapping
binding to phospholipids
for aminoglycosides: inability to cross membranes
lysososomes
macrolides
aminoglycosides
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10/09/2004

7. Subcellular bioavailability of antibiotics ?
High Fair Nil
FQ / Ansamyc. / cytosol. ML lysosom. ML / AG
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10/09/2004

8. Subcellular bioavailability of antibiotics ?
Fluoroquinolones
move easily
across
membranes FQ
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10/09/2004

9. Subcellular bioavailability of antibiotics ?
aminoglycosides
and lysosomal macrolides
reamain largely if not totally sequestered
in an acidic environment ...
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10/09/2004

10. Listeria monocytogenes
Illustration:
the Listeria story
antibiotics:
ampicillin/meropenem
azithromycin
sparfloxacin/moxifloxcin
pivampicillin
Listeria monocytogenes
hly+
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10/09/2004

11. Intracellular infection cycle of Listeria monocytogenes hly+
from Portnoy et al.
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10/09/2004

12. Following the intracellular fate of Listeria m. by EM
in cytosol
escape from vacuole
phagocytosis
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10/09/2004

13. 1st question: is there a simple relation between MIC, accumulation and intracellular activity (5 h model)
MIC
AMPI AZ SP
0.0
0.5
1.0
1.5
Accumulation
AMPI AZ SP 25 50 75
100
Activity *
AMPI AZ SP
0.0
0.5
1.0
1.5
*  log CFU 5h Ce = 10 x MIC
Ouadhriri et al., AAC,1999
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10/09/2004

14. Listeria m. and ampicillin
Ampicillin is poorly active against intracellular Listeria m. in spite of its favourable MIC;
lack of accumulation ...
Why do you keep ampicillin ?
extracellular bacteria
get intracellular activity with very large doses ?? (but -lactams are NOT dose-dependent…)
but may be you just have to wait ...
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10/09/2004

15. -lactams become bactericidal intracellularly after 24h and if you give a dose high enough-2 -1 1 2 3
ampicilline
méropénème
Multiples of MIC (log10)
Change from original inoculum (log10)
(Lemaire et al, 2004, unpublished
(Carryn et al., 2003, JAC 51:
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10/09/2004

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 ?
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10/09/2004

17. Listeria m. and fluoroquinolones
In a pharmacological model*, fluoroquinolones appear most active in spite of relatively unfavourable MICs and modest cellular accumulation compared to macrolides …
Fluoroquinolones have a large subcellular bioavailability are highly bactericidal
* all Ce = 10 X the MIC
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10/09/2004

18. Comparative intracellular activities at multiples of Cmax
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5 25 50 75
100
ampicillin
moxifloxacin
Change from original inoculum ( D
log)
(Carryn et al., 2002, AAC 43: )
Moxifloxacin is profoundly bactericidal at clinically achievable concentrations
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10/09/2004

19. broth
cells
However, intracellular moxifloxacin is NOT more active intracellularly than extracellularly when using the extracellular concentration as comparison basis ...
Carryn et al., AAC, 2002
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10/09/2004

20. And this is obvious if you compare intracellular and extracellular activities at the same apparent concentration * / MIC ratio …. 1
extracellular
levofloxacine
moxifloxacine
log CFU (5 h - 0h)
intracellular
levofloxacine -1
moxifloxacine 
Seral et al., unpublished -2
0.1 1 10
100
extracellular = actual
intracellular = calculated
concentration / MIC
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10/09/2004

21. A basic prodrug of a -lactam ?
pH 7.4
pH 7.0
pH 5.4
Proton pump
ProD
ProD
ProDH+ H+ D
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10/09/2004

22. regenerate ampicillin
Basic compounds that
H2N
Phthalimidomethylampicillin (PIMA)
regenerate ampicillin
accumulate in J774 macrophages
Fan et al, Bioorg. Med. Chem. Let.1997
Pivaloyloxymethylampicillin (PIVA)
Paternotte et al, Biorg. Med. Chem. 2001
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10/09/2004

23. Intracellular activity for extracellular concentrations of ...
PIVA
PIMA
10X MIC
CFU/mg prot
Time (h)
Same activity for PIVA, PIMA and AMPI
0.5X MIC
Only PIVA is active
CTRL
AMPI
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10/09/2004
Chanteux et al, JAC, 2003

24. But maintains it if the medium is renewed every 5 h
At low extracellular concentration, PIVA loses its activity after 5 h if the medium is not renewed 5 10 15 20 4 6 8
CTRL
Extracellular concentration
PIVA
0.5X MIC
CFU/mg prot
But maintains it if the medium is renewed every 5 h
Time (h)
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10/09/2004
Chanteux et al, JAC, 2003

25. PIVA releases large amount of intracellular ampicillin5 10 15 20 25 30
AMPI from PIVA
AMPI from AMPI
AMPI from PIVA with changes of medium)
Time (h)
Cc/Ce
Chanteux et al, JAC, 2003
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10/09/2004

26. The seven pillars of intracellular / intratissular activity ?D* 4
4. Subcell. bioavailability 7
7. Cooper. with host def. D 1
1. Penetration D
3. Accumulation 3 5
5. Expression of activity 2
2. No efflux
6. Bacterial responsiveness
and pharmacodynamics 6
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10/09/2004

27. The 6 pillars of intracellular / intratissular accumulation and activity of antibiotics...
Hugues Chanteux 4
Christina Seral D D 2 D
Françoise Van Bambeke
Stéphane Carryn 5
Sandrine Lemaire
Françoise Van Bambeke, Stéphane Carryn, Cristina Seral, Hugues Chanteux, Sandrine Lemaire ...
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10/09/2004