1. 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

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

3. 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.

4. 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:

5. 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

6. Antibiotics and Intracellular Bacteria : a Bit of Theory Eur. J. Microbiol Infect. Dis. 10: 100-106, 1991

7. 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: 10-20 x macrolides: 4 to > 100 x

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

9. 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

10. 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

11. 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...

12. 6th question [2 of 2] : cooperation or antagonism with the actions of cytokines ? The very black box antibiotic interferons ILx growth fact. TNF...

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

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

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

16. 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

17. 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

18. The growth containment of L. monocytogenes caused by IFN  is due to NO and H 2 0 2 production...

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

20. 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

21. 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 

22. 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...

23. Where published ? Scorneaux et al., Antimicrob. Agents Chemother. 40:1225-30, 1996 Ouadrhiri et al., Antimicrob. Agents Chemother. 43:1242-51, 1999 Ouadrhiri et al., J. Infect. Dis. 180:1195-204, 1999

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

25. 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

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

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

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

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

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

31. 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...

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

33. 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 3 3 3 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 3 3 3 C N S 3 O 3 H N C O NH 3 ampicillin pivampicillin pH 7 pH < 6 + + -

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

35. Where published ? Renard et al., J. Med. Chem. 29:1291-1293, 1986 Renard et al., Antimicrob. Agents Chemother. 31:410-416, 1987 Fan et al., Bioorg. Med. Chem. Letters 24:3107-3112, 1997

36. 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)

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

38. But, where is this university? Cellular and Molecular Pharmacology Unit Catholic University of Louvain, Belgium http://www.md.ucl.ac.be/facm

39. intracellular pharmacodynamics AUIC postantibiotic effect... population pharmacokinetics tissue concentrations efficacy/toxicity ratios http://www.isap.org But don’t forget ISAP...

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