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Paul M. Tulkens, MD, PhD Cellular and Molecular Pharmacology Unit

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.

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Paul M. Tulkens, MD, PhD Cellular and Molecular Pharmacology Unit

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  1. Intracellular Pharmacodynamics: a Journey from Cell biology to the Design of New Chemotherapeutic Agents Paul M. Tulkens, MD, PhDCellular 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) antibiotic bacteria

  3. These they are… • OBLIGATORY OR MAINLY INTRACELLULAR: • respiratory infections (pneumopathies): Chlamydia pneumoniae: 10% in children Legionella pneumophila: frequent ifimmunosuppression 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.

  4. You need to consider (at least)the following questions: 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?

  5. 1st question (and answer) : where do intracellular bacteria sojourn and thrive ? cytosol endosomes Listeria hly+, Shigella phagolysosomes phagosomes S. aureus Salmonella, M. leprae, ... Legionella Chlamydia, ...

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

  9. 4th question: what are the antibiotic intracellular expressions of activity • fluoroquinolones: yes • ansamycins: excellent • beta-lactams: yes endosomes pH = 7 pH < 6 phagosomes • macrolides: reduced • aminoglycosides: very reduced

  10. 5th question: what is the bacterial responsiveness to the antibiotics ? in cytosol: excellent in endosomes: ? pH = 7 pH < 6 in phagolysosomes: very variable in phagosomes likely to excellent

  11. 6th question [1 of 2]: cooperation with the cell own defenses ? in cytosol: unlikely in endosomes: likely pH = 7 pH < 6 O2. - NO. in phagolysosomes: very variable in phagosomes likely myeloperoxydase acidity, cationic proteins...

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

  13. A first story : Listeria in phagosomes • antibiotics: • ampicillin • sparfloxacin • azithromycin interferon gamma Listeria monocytogenes hly+ and hly-

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

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

  16. Following the intracellular fateof Listeria m. in EM 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

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

  18. The growth containment of L. monocytogenes caused by IFNis due to NO and H202 production...

  19. Intracellular activity: CFU @ 0 h CFU @ 5 h log10 control + IFN + IFN  and LMMA / catalase IFN  and antibiotics... Bacterial kill Bacterial growth

  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 Activity sparfloxacin Accumulation T = 5h SP + INF + LMMA + catalase SP + INF SP + INF + LMMA + catalase CT SP SP SP + INF (Ouadrhiri et al, 1999)

  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. A story with beta-lactams R H N CH 2 3 CH H 3 C N C N O O COOH COOH D-alanine beta-lactam

  25. R C N O COOH 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

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

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

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

  29. R C N O - NH2 CO- R To get accumulation, make a basic beta-lactam...

  30. S R CH3 CH3 C N O H CO- NH-CH2-CH2-CH2-N (CH3)2 N-(3-dimethylaminopropyl)benzylpenicillamide...

  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. S R CH3 CH3 C N O H CO - O - CH2 - X - NH2 You need to make a chemically labile ester...

  33. What you can do with old pivampicillin... pH 7 pH < 6 NH 2 + H NH 3 N C S CH H 3 N C O S CH N CH 3 3 - O O COO N CH 3 ampicillin O COOH + NH 3 NH 2 H N C S H CH 3 N C S CH 3 O N CH 3 O CH O 3 O N CH 3 H O CH C 2 O 3 C C CH C 3 H C O O 2 O C C CH C 3 CH O O 3 O CH 3 pivampicillin

  34. Old pivampicillin does bring ampicillin in cells... free AMPI PIVA T = 24h AMPI FROM PIVA CT AMPI PIVA (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. But don’t forget ISAP... tissue concentrations efficacy/toxicity ratios population pharmacokinetics postantibiotic effect... AUIC intracellular pharmacodynamics http://www.isap.org

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

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