D D Intracellular antibiotics and Listeria monocytogenes D

D* D Intracellular antibiotics and Listeria monocytogenes D Paul M. Tulkens, MD, Ph. D Hugues Chanteux, Ph. D Stéphane Carryn, Ph. D Cellular and Molecular Pharmacology Catholic University of Louvain, Brussels, Belgium Presented at the 5 th European Congress of Chemotherapy, Rhodos, Greece, October 17 th-20 th, 2003 5 th ECC Rhodes, Greece 19/10/2003 1

Why intracellular / intratissular antibiotics ? antibiotic The Cell bacteria Black Box. . . 5 th ECC Rhodes, Greece 19/10/2003 2

Subcellular localization ? cytosol • fluoroquinolones • beta-lactams • ansamycins • macrolides (1/3) endosomes ? ? phagolysososomes • macrolides (2/3) • aminoglycosides lysososomes 5 th ECC Rhodes, Greece 19/10/2003 phagosomes ? 3

Mechanisms of localisation and accumulation. . . cytosol • fluoroquinolones Mechanism unknown (loose binding to lipid-containing proteins ? …) 5 th ECC Rhodes, Greece 19/10/2003 4

Mechanisms of localisation and accumulation. . . 4 proton trapping 4 binding to phospholipids 4 for aminoglycosides: inability to cross membranes • macrolides • aminoglycosides lysososomes 5 th ECC Rhodes, Greece 19/10/2003 5

Subcellular bioavailability of antibiotics ? High Fair FQ / Ansamyc. / cytosol. ML 5 th ECC Rhodes, Greece 19/10/2003 Nil lysosom. ML / AG 6

Subcellular bioavailability of antibiotics ? Fluoroquinolones move easily across FQ membranes 5 th ECC Rhodes, Greece 19/10/2003 7

Subcellular bioavailability of antibiotics ? aminoglycosides and lysosomal macrolides reamain largely if not totally sequestered in an acidic environment. . . 5 th ECC Rhodes, Greece 19/10/2003 8

antibiotics: • ampicillin/meropenem Listeria monocytogenes • azithromycin hly+ • sparfloxacin/moxifloxcin • pivampicillin 5 th ECC Rhodes, Greece 19/10/2003 9

Intracellular infection cycle of Listeria monocytogenes hly+ from Portnoy et al. 5 th ECC Rhodes, Greece 19/10/2003 10

Following the intracellular fate of Listeria m. by EM escape from vacuole phagocytosis 5 th ECC Rhodes, Greece 19/10/2003 in cytosol 11

1 st question: is there a simple relation between MIC, accumulation and intracellular activity (5 h model) MIC Accumulation 1. 5 100 Activity * 1. 5 75 1. 0 50 0. 5 25 0. 0 0 0. 0 AMPI AZ AMPI SP AZ SP Ouadhriri et al. , AAC, 1999 5 th ECC Rhodes, Greece 19/10/2003 AMPI AZ SP * log CFU 5 h Ce = 10 x MIC 12

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 ? è extracellular bacteria è get intracellular activity with very large doses ? ? (but -lactams are NOT dose-dependent…) è but may be you just have to wait. . . 5 th ECC Rhodes, Greece 19/10/2003 13

-lactams become bactericidal intracellularly after 24 h 10 9 10 8 control ampicillin 10 7 10 6 10 5 10 4 0 5 10 15 20 25 Changee from original inuculum CFU / mg protein 10 10 4 3 2 1 0 INTRA 5 h INTRA 24 h -1 EXTRA 24 h -2 -3 -4 -5 Control Time (h) Ampicillin Meropenm (Carryn et al. , 2002, JAC 51: 1051 -52 5 th ECC Rhodes, Greece 19/10/2003 14

Listeria m. and azithromycin Azitromycin is also 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 ? 5 th ECC Rhodes, Greece 19/10/2003 15

Listeria m. and fluoroquinolones In this pharmacological model *, sparfloxacin IS the most active in spite of a unfavourable MIC (1. 4 µg/ml) and modest cellular accumulation (12 x) Fluoroquinolones have a large subcellular bioavailability are highly bactericidal Why don’t you use fluoroquinolones today ? è too low intrinsic activity **. . . * all Ce = 10 X the MIC 5 th ECC Rhodes, Greece 19/10/2003 16

But look at moxifloxacin (5 h model). . . Carryn et al. , AAC, 2002 5 th ECC Rhodes, Greece 19/10/2003 17

Change from original inoculum. D(log) Comparative intracellular activities 1. 5 ampicillin moxifloxacin 1. 0 0. 5 0. 0 -0. 5 Moxifloxacin is profoundly bactericidal at clinically achievable concentrations -1. 0 -1. 5 -2. 0 -2. 5 25 50 % of Cmax 5 th ECC Rhodes, Greece 19/10/2003 75 100 (Carryn et al. , 2002, AAC 43: 2095 -103) 18

However, intracellular moxifloxacin is NOT more active intracellularly than extracellularly. . . broth cells Carryn et al. , AAC, 2002 5 th ECC Rhodes, Greece 19/10/2003 19

A basic prodrug of a -lactam ? p. H 7. 4 p. H 7. 0 Pro. D + H Pro. DH+ D 5 th ECC Rhodes, Greece D 19/10/2003 p. H 5. 4 + H Pro. D + Pro. DH D 20

H 2 N Phthalimidomethylampicillin (PIMA) Basic compounds that • regenerate ampicillin • accumulate in J 774 macrophages H 2 N Fan et al, Bioorg. Med. Chem. Let. 1997 Pivaloyloxymethylampicillin (PIVA) 5 th ECC Rhodes, Greece 19/10/2003 Paternotte et al, Biorg. Med. Chem. 2001 21

Intracellular activity for extracellular concentrations of. . . PIVA PIMA CTRL AMPI CFU/mg prot 10 X MIC 10 7 0. 5 X MIC 10 7 10 6 0 1 2 3 4 5 Time (h) Same activity for PIVA, PIMA and AMPI 5 th ECC Rhodes, Greece 19/10/2003 Only PIVA is active Chanteux et al, JAC, 2003 22

At low extracellular concentration, PIVA loses its activity after 5 h if the medium is not renewed 10 10 CFU/mg prot CTRL 10 8 10 6 10 4 Extracellular concentration PIVA 0 5 10 15 0. 5 X MIC 20 But maintains it if the medium is renewed every 5 h Time (h) 5 th ECC Rhodes, Greece 19/10/2003 Chanteux et al, JAC, 2003 23

PIVA releases large amount of intracellular ampicillin AMPI from PIVA 30 AMPI from PIVA with changes of medium) Cc/Ce 25 AMPI from PIMA 15 10 5 AMPI from AMPI 0 0 5 10 15 20 25 Time (h) 5 th ECC Rhodes, Greece 19/10/2003 Chanteux et al, JAC, 2003 24

The seven pillars of intracellular / intratissular activity ? D* 1 D D D 3 7 5 4 4 5 4 2 6 1. Penetration 2. No efflux 3. Accumulation 4. Subcell. bioavailability 5 th ECC Rhodes, Greece 19/10/2003 6 7 5. Expression of activity 6. Bacterial responsiveness and pharmacodynamics 7. Cooper. with host def. 25

The 6 pillars of intracellular / intratissular accumulation and activity of antibiotics. . . D* D D D 4 5 2 Françoise Van Bambeke, Stéphane Carryn, Cristina Seral, Hugues Chanteux, Donatienne Tyteca, Marie-Paule Mingeot-Leclercq. . . 5 th ECC Rhodes, Greece 19/10/2003 26