Intracellular antibiotics D 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 www.facm.ucl.ac.be Magic Bullets ... 10/09/2004

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

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... Magic Bullets ... 10/09/2004

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

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

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

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

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

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

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

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

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

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 Magic Bullets ... 10/09/2004

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 ... Magic Bullets ... 10/09/2004

-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 51:1051-52 Magic Bullets ... 10/09/2004

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 ? Magic Bullets ... 10/09/2004

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 Magic Bullets ... 10/09/2004

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 43:2095-103) Moxifloxacin is profoundly bactericidal at clinically achievable concentrations Magic Bullets ... 10/09/2004

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 Magic Bullets ... 10/09/2004

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 Magic Bullets ... 10/09/2004

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

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 Magic Bullets ... 10/09/2004

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 Magic Bullets ... 10/09/2004 Chanteux et al, JAC, 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) Magic Bullets ... 10/09/2004 Chanteux et al, JAC, 2003

PIVA releases large amount of intracellular ampicillin 5 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 Magic Bullets ... 10/09/2004

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 Magic Bullets ... 10/09/2004

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 ... Magic Bullets ... 10/09/2004