Intracellular Pharmacodynamics: a Journey from Cell biology to the Design of New Chemotherapeutic Agents Paul M. Tulkens, MD, PhD Cellular and Molecular Pharmacology Unit Catholic University of Louvain, Brussels, Belgium & International Society of Antiinfective Pharmacology Advances in the Pharmacology of Antiinfective Therapy, Bangalore, India, January 20-22, 2000

What is Intracellular Infection ? The Cell (black box) antibioticbacteria

These they are… è OBLIGATORY OR MAINLY INTRACELLULAR: Ørespiratory infections (pneumopathies): Chlamydia pneumoniae: 10% in children Legionella pneumophila: frequent if immunosuppression Mycobacterium spp.:frequent if immunosuppression Øsexually transmitted diseases Chlamydia trachomatis: most common pathogen Ø CNS infections + other sites: Listeria monocytogenes: pregnant women; immunosuppression è FACULTATIVE OR MAINLY EXTRACELLULAR: Ø digestive tract infections Salmonella spp., Shigella spp. Ø respiratory, cutaneous, etc…tract infections Streptococcus spp., Staphylococcus spp.

1. where are the bacteria ? 2. which antibiotics accumulate in cells ? 3. where are antibiotics located ? 4. what is the intracellular expression of activity ? 5. what is the bacterial responsiveness ? 6. cooperation with the cell own defenses and cytokines? You need to consider (at least) the following questions:

1st question (and answer) : where do intracellular bacteria sojourn and thrive ? Legionella Chlamydia,... S. aureus Salmonella, M. leprae,... Listeria hly+, Shigella phagosomes phagolysosomes cytosol endosomes

Antibiotics and Intracellular Bacteria : a Bit of Theory Eur. J. Microbiol Infect. Dis. 10: , 1991

2d question (and answer): which antibiotics do accumulate in cells ? beta-lactams:  1x aminoglycosides: <1 to 2 x ansamycins: 2-3 x tetracyclines: 2-4 x fluoroquinolones: x macrolides: 4 to > 100 x

endosomes ? phagosomes ? phagolysososomes macrolides aminoglycosides cytosol fluoroquinolones beta-lactams ansamycins 3d question (and answer) : where are intracellular antibiotics located ?

endosomes phagosomes macrolides: reduced aminoglycosides: very reduced fluoroquinolones: yes ansamycins: excellent beta-lactams: yes 4th question: what are the antibiotic intracellular expressions of activity pH = 7 pH < 6

in endosomes: ? in phagosomes likely to excellent 5th question: what is the bacterial responsiveness to the antibiotics ? pH = 7 pH < 6 in cytosol: excellent in phagolysosomes: very variable

in endosomes: likely in phagosomes likely 6th question [1 of 2] : cooperation with the cell own defenses ? pH = 7 pH < 6 in cytosol: unlikely in phagolysosomes: very variable O 2. - NO. myeloperoxydase acidity, cationic proteins...

6th question [2 of 2] : cooperation or antagonism with the actions of cytokines ? The very black box antibiotic interferons ILx growth fact. TNF...

antibiotics: ampicillin sparfloxacin azithromycin interferon gamma Listeria monocytogenes hly+ and hly-

INTRACELLULAR INFECTION CYCLE OF Listeria monocytogenes hly + from Portnoy et al.

Bacteria are stained with fluorescein (FITC) [green] and cell actin with phalloidin rhodamin [red]; hly + : virulent hly - : non virulent Listeria and Interferon  : confocal microscopy

A: phagocytosis B: escape from vacuole C: surrounded by actin D,E: cells pre-treated with interferon-gamma, L. m. hly+ remains in vacuoles. F,G, H: L.m. hly- remains constantly in vacuoles A = 1 h post infection; B, C, D, F, G = 3 h post infection E, H = 5 h post infection Following the intracellular fate of Listeria m. in EM

control IFN  100 U/ml control IFN  100 U/ml IFN  contains L. monocytogenes hly + in phagosomes and impairs its growth. A: dose-dependency; B: specifity IFN  50 U/ml IFN  100 U/m + anti IFN  IgG

The growth containment of L. monocytogenes caused by IFN  is due to NO and H production...

IFN  and antibiotics... control + IFN + IFN  and LMMA / catalase Intracellular activity: 0 h 5 h log 10 Bacterial growth Bacterial kill

Listeria, ampicillin and Interferon gamma 1. Ampicillin is poorly active against intracellular Listeria; 2. Ampicillin activity is completely defeated when IFN  is present (IFN makes the job !) Why do you keep ampicillin ?  extracellular bacteria  get activity with very large doses

Listeria, fluoroquinolones and Interferon SP + INF  CT SP + INF  + LMMA + catalase sparfloxacin (Ouadrhiri et al, 1999) SP SP + INF  + LMMA + catalase T = 5h AccumulationActivity SP + INF 

Listeria, fluoroquinolones and Interferon 1. Sparfloxacin appears highly active against intracellular Listeria; 2. Sparfloxacin activity is enhanced when IFN  is present (IFN and Sp cooperate !) Why don’t you use fluoroquinolones ?  too low intrinsic activity...

Where published ? Scorneaux et al., Antimicrob. Agents Chemother. 40: , 1996 Ouadrhiri et al., Antimicrob. Agents Chemother. 43: , 1999 Ouadrhiri et al., J. Infect. Dis. 180: , 1999

CN O COOH R C H N O H 2 N CH 3 3 D-alanine beta-lactam

O COOH R ALL beta-lactam have (and must have) a free carboxyl group (or an equivalent acidic group [electron donor]) — at appropriate distance from the C — N bound NC

phagolysososomes pH  5-6 exatracellular pH  7.4 Why do beta-lactams not accumulate in cells (1 of 2) ? cytosol pH  7.2

Why beta-lactams do not accumulate in cells (2 of 2) ? AH A - AH A - AH A - H+H+ H+H+ H+H+

What if you are basic ? B BH + B BH + B BH + H+H+ H+H+ H+H+

O CO- R R To get accumulation, make a basic beta-lactam... NC - NH 2

O CO - NH-CH 2 -CH 2 -CH 2 -N (CH 3 ) 2 R N-(3-dimethylaminopropyl)benzylpenicillamide... NC S CH 3 H

N-(3-dimethylaminopropyl) benzylpenicillamide…  get accumulated in cells (5 to 10-fold  concentrates in lysosomes but…  is inactive because the amide linkage is not splitted by lysosomal enzymes...

O CO - O - CH 2 - X - NH 2 R You need to make a chemically labile ester... NC S CH 3 H

What you can do with old pivampicillin... N S COO CH 3 O 3 H N C O NH 2 O O H 2 C O CC O CH C N S 3 O 3 H N C O NH 2 N S COOH CH 3 O 3 H N C O NH 3 O O H 2 C O CC O CH C N S 3 O 3 H N C O NH 3 ampicillin pivampicillin pH 7 pH <

Old pivampicillin does bring ampicillin in cells... free AMPI PIVA AMPI FROM PIVA CTAMPIPIVA T = 24h (Fan et al, 1997)

Where published ? Renard et al., J. Med. Chem. 29: , 1986 Renard et al., Antimicrob. Agents Chemother. 31: , 1987 Fan et al., Bioorg. Med. Chem. Letters 24: , 1997

Thanks to the crew... Zubida Bumkhalla (beta-lactams [chemistry]) Marie-Béatrice Carlier (macrolides, fluoroquinolones) Huajuan Fan (betlactams [chemistry] ) Christine Renard (beta-lactams) Yousef Ouadrhiri (Listeria, interferon, IL6, fluoroquinolones) Isabelle Paternotte (beta-lactams [chemistry], efflux mech.) Bernard Scorneaux (Listeria, interferon, fluoroquinolones) Etienne Sonveaux (beta-lactams [chemistry] ) Andrée Zenebergh (lincosaminides, macrolides)

But where do they work ? Catholic University of Louvain Faculty of Medicine Brussels, Belgium

But, where is this university? Cellular and Molecular Pharmacology Unit Catholic University of Louvain, Belgium

intracellular pharmacodynamics AUIC postantibiotic effect... population pharmacokinetics tissue concentrations efficacy/toxicity ratios But don’t forget ISAP...

intracellular pharmacodynamics AUIC postantibiotic effect... population pharmacokinetics tissue concentrations efficacy/toxicity ratios And thank you to Bangalore...