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

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

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

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

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

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

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

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

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

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

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

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 5th ECC Rhodes, Greece 19/10/2003

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 ... 5th ECC Rhodes, Greece 19/10/2003

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

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 ? 5th ECC Rhodes, Greece 19/10/2003

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 ? too low intrinsic activity ** ... * all Ce = 10 X the MIC 5th ECC Rhodes, Greece 19/10/2003

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

Comparative intracellular activities 1.5 log) D 1.0 ampicillin moxifloxacin 0.5 0.0 Change from original inoculum ( -0.5 Moxifloxacin is profoundly bactericidal at clinically achievable concentrations -1.0 -1.5 -2.0 -2.5 25 50 75 100 % of Cmax (Carryn et al., 2002, AAC 43:2095-103) 5th ECC Rhodes, Greece 19/10/2003

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

A basic prodrug of a -lactam ? pH 7.4 pH 7.0 pH 5.4 Proton pump ProD ProD ProDH+ H+ D 5th ECC Rhodes, Greece 19/10/2003

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 5th ECC Rhodes, Greece 19/10/2003

Intracellular activity for extracellular concentrations of ... PIVA PIMA 10X MIC 0.5X MIC CTRL AMPI 1 2 3 4 5 10 6 7 1 2 3 4 5 10 6 7 CFU/mg prot Time (h) Same activity for PIVA, PIMA and AMPI Only PIVA is active Chanteux et al, JAC, 2003 5th ECC Rhodes, Greece 19/10/2003

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) Chanteux et al, JAC, 2003 5th ECC Rhodes, Greece 19/10/2003

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

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 5th ECC Rhodes, Greece 19/10/2003

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