Intracellular - UCLouvain

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*


Why intracellular antibiotics ?

The Cellantibiotic bacteria

Black Box...


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...


Subcellular localization of antibiotics ?

cytosol

• fluoroquinolones• beta-lactams• ansamycins• macrolides (1/3)

lysososomes• macrolides (2/3)• aminoglycosides ?

?endosomes

phagosomes

phago-lysososomes

?


Mechanisms of localisation and accumulation ...

cytosol

• fluoroquinolones

• Mechanism unknown(loose binding to lipids / lipoproteins ? …)

• Efflux demonstrated (MRP ?)


Mechanisms of localisation and accumulation ...

lysososomes• macrolides • aminoglycosides

� proton trapping� binding to phospholipids� for aminoglycosides:

inability to cross membranes


Subcellular bioavailability of antibiotics ?

High Fair Nil

FQ / Ansamyc. / cytosol. ML lysosom. ML / AG



FQ

Fluoroquinolonesmove easilyacrossmembranes



aminoglycosidesand lysosomal macrolidesreamain largely if not totally sequesteredin an acidic environment ...


Listeria monocytogeneshly+

antibiotics:

• ampicillin/meropenem• azithromycin• sparfloxacin/moxifloxcin• pivampicillin


Intracellular infection cycle of Listeria monocytogenes hly+

from Portnoy et al.


phagocytosis

Following the intracellular fate of Listeria m. by EM

escape from vacuole

in cytosol


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


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 ...


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


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 ?


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


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


However, intracellularmoxifloxacin is NOT moreactiveintracellularlythan extracellularly when using the extracellular concentration as comparison basis ...

broth

cells

Carryn et al., AAC, 2002


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


A basic prodrug of a β-lactam ?

ProD

DDD

ProDH+H+

ProDH+

H+ProD ProD

pH 7.4 pH 7.0 pH 5.4


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


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


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



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



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 ?


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

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Intracellular - UCLouvain