Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria

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1 Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Unité de Pharmacologie cellulaire et moléculaire F. Van Bambeke

2 Magic bullets need to reach their target

3 Magic bullets need to reach their target glycopeptides β-lactams quinolones macrolides aminoglycosides

4 Target accessibility is critical for intracellular activity

5 Active efflux reduces antibiotic cellular concentration

6 Why efflux transporters?

7 Why efflux transporters? polar drug lipophilic drug physico-chemical properties are inadequate for reaching an intracellular target! Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

8 Why efflux transporters? amphipathic drug most drugs are amphipathic by design, to be able to cross membrane barriers! Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

9 Why efflux transporters? But a diffusible compound may have potentially harmful effects! Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

10 Why efflux transporters? Van Bambeke et al. (2000) Biochem. Pharmacol. 60: Extrusion by efflux pumps

11 Why efflux transporters? Extrusion by efflux pumps general mean of protection against cell invasion by diffusible molecules Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

12 Typical toxic diffusible substances as substrates for efflux pumps antibiotics antifungals anticancer agents

13 Mechanisms of active efflux Van Bambeke et al. (2000) Biochem. Pharmacol. 60: flip-flop flippase vacuum cleaner ATP ADP+Pi membrane insertion / release

14 Most antibiotics are amphiphilic! Van Bambeke et al. Biochem. Pharmacol. (2000) 60: cationic amphiphiles anionic amphiphiles + + macrolide tetracycline fluoroquinolone rifampicin - β-lactam fluoroquinolone + + lincosamide - fusidic acid sulfamide

15 Van Bambeke et al. (2000) Biochem. Pharmacol. 60: Antibiotics as substrates of efflux pumps Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes β-lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones

16 Consequences of antibiotic efflux from eucaryotic cells Van Bambeke et al, JAC (2003) 51: alteration of pharmacokinetics single cell: accumulation, localization whole organism: absorption, distribution, elimination alteration of pharmacodynamics cellular level: activity against intracellular bacteria body level: drug concentration in the infected compartment

17 Van Bambeke et al. (2000) Biochem. Pharmacol. 60: Antibiotics as substrates of efflux pumps Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes β-lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones

18 Antibiotics as substrates of efflux pumps azithromycin CH 3 F O O OH H 3 C OH N CH 3 OH HN N N H 3 C H 3 CH 2 C O OH H 3 C O CH 3 O O CH 3 (H 3 C) 2 N OHO O H 3 CO CH 3 CH 3 ciprofloxacin F N HN OCH 3 O N O C OH H 3 C OH moxifloxacin

19 Macrolides and quinolones as cell-associated antibiotics

20 Characterization of antibiotic efflux pumps in macrophages

21 Efflux pumps expressed in J774 macrophages

22 ABC multidrug transporters cationic amphiphiles MDR-1 (P-glycoprotein) anionic amphiphiles MRP1-10 ATP ADP

23 How to inhibit ABC transporters? cationic amphiphiles MDR-1 (P-glycoprotein) anionic amphiphiles MRP1-10 deoxyglucose NaN 3 ATP ADP

24 How to inhibit ABC transporters? H 3 CO OCH 3 verapamil OCH 3 OCH 3 cationic amphiphiles H 3 CO H 3 CO CN CH(CH 3 ) 2 N CH 3 H 3 CO MDR-1 (P-glycoprotein) N N H O C H N O GF ATP ADP

25 How to inhibit ABC transporters? CH 3 C 3 H 7 O N S C 3 H 7 O probenecid COOH H 3 C gemfibrozil S CH 3 (CH 2 ) 3 N(CH 3 ) 2 C CH 3 COOH anionic amphiphiles Cl N S O MRP1-10 MK571 HOOC ATP ADP

equilibrium azithromycin & P-glycoprotein ciprofloxacin &

26 Differential recognition by MDR pumps Influence of ATP-depletion and pump inhibitors on accumulation at equilibrium azithromycin & P-glycoprotein ciprofloxacin & MRP extracell. conc. 5 mg/l; AZM 3 h; CIP 2 h Michot et al. AAC (2004) 48:

27 Efflux of macrolides: azithromycin azithromycin CH 3 H 3 C N CH 3 OH OH H 3 C H 3 CH 2 C OH H 3 C O CH 3 (H 3 C) 2 N OHO O CH 3 O CH 3 O O H 3 CO CH 3 H 3 C OH

28 extracell. conc. 5 mg/l Seral et al (2003) AAC 47: Kinetics of accumulation Azithromycin accumulates to high levels in eucaryotic cells

29 Macrolide subcellular distribution Carlier et al, JAC (1987) 20 Suppl B:47-56 Macrolides accumulate in the lysosomal compartment lysosomal enzymes

30 Mechanism of accumulation for macrolides De Duve et al, Biochem Pharmacol. (1974) 23: Macrolide accumulation proceeds by diffusion / segregation B B B H + BH + BH + BH +

31 Kinetics of accumulation and efflux for azithromycin extracell. conc. 5 mg/l Seral et al (2003) AAC 47: Azithromycin concentration is high but still suboptimal

32 Kinetics of accumulation and efflux for azithromycin extracell. conc. 5 mg/l; verapamil 20 µm Seral et al (2003) AAC 47: Inhibition of P-gp by verapamil increases accumulation

33 Kinetics of accumulation and efflux for azithromycin extracell. conc. 5 mg/l; verapamil 20 µm Seral et al (2003) AAC 47: Accumulation markedly increased; efflux marginally affected

34 Azithromycin, kick-back model Gaj et al. (1998) Biochem. Pharmacol. 55:

35 Efflux of quinones (ciprofloxacin, moxifloxacin) O O F OH HN N N ciprofloxacin O O F C OH HN N N OCH 3 moxifloxacin

36 Carlier et al JAC (1990) 26 Suppl B:27-39 Kinetics of accumulation Quinolones accumulate to moderate levels in eucaryotic cells

37 Quinolone subcellular distribution Quinolones are found in the soluble fraction LDH (cytosol) NABgase (lysosomes) proteins Carlier et al JAC (1990) 26 Suppl B:27-39

38 Kinetics of accumulation and efflux for ciprofloxacin extracell. conc. 17 mg/l; probenecid 5 mm Michot et al. AAC (2004) 48: both accumulation and efflux markedly affected

39 Ciprofloxacin, classical model Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42

40 Kinetics of accumulation and efflux for moxifloxacin extracell. conc. 17 mg/l; probenecid 5 mm Michot et al. AAC (2005) 49: neither accumulation nor efflux affected

41 Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin Michot et al. AAC (2005) 49:

42 Quinolones as inhibitors of ciprofloxacin efflux Michot et al. AAC (2005) 49: ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin not affected

43 Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin Michot et al. AAC (2005) 49: CIP CIP MXF

44 Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin Michot et al. AAC (2005) 49: CIP CIP MXF moxifloxacin also able to interact with the transporter!

45 Moxifloxacin, futile-cycle model Eytan et al. (1996) JBC 271:

46 Influence of efflux pumps on antibiotic activity against intracellular infections

47 Efflux from eucaryotic cells and intracellular activity Carryn et al, Infect Dis Clin North Am. (2003) 17:615-34

48 Models of intracellular infection L. monocytogenes S. aureus cytosol phagolysosomes

49 verapamil 20 µm; 24 h Seral et al (2003) JAC 51: Influence of pump inhibitors on intracellular activity azithromycin and L. monocytogenes L. monocytogenes AZM AZM log CFU (5 h 0 h)

50 verapamil 20 µm; 24 h Seral et al (2003) JAC 51: Influence of pump inhibitors on intracellular activity azithromycin and S. aureus S. aureus AZM AZM

51 gemfibrozil 250 µm; 24 h Seral et al (2003) JAC 51: Influence of pump inhibitors on intracellular activity ciprofloxacin and L. monocytogenes L. monocytogenes CIP log CFU (5 h 0 h)

52 gemfibrozil 250 µm; 24 h Seral et al (2003) JAC 51: Influence of pump inhibitors on intracellular activity ciprofloxacin and S. aureus S. aureus CIP

Seral et al (2003) JAC 51:1167-73 Influence of pump inhibitors on antibiotic distribution

53 Seral et al (2003) JAC 51: Influence of pump inhibitors on antibiotic distribution verapamil enhances azithromycin concentration in cytosol and vacuoles L. monocytogenes AZM AZM S. aureus

54 Seral et al (2003) JAC 51: Influence of pump inhibitors on antibiotic distribution gemfibrozil enhances ciprofloxacin cytosolic content L. monocytogenes CIP S. aureus

55 Conclusion constitutive efflux of antibiotics in macrophages pharmacokinetics: suboptimal cellular accumulation pharmacodynamics: suboptimal intracell. activity pharmacology: differences in affinity within a AB class pharmacokinetics: wide spectrum transporters resistance? drug interactions?

56 Perspectives for the future of chemotherapy

57 Perspectives for the future of chemotherapy use of poor substrates of efflux pumps (moxi vs cipro) development of specific inhibitors of efflux pumps caution for «cross resistance» with other substrates (over expression of efflux pumps)

58 Quinolones differ by the susceptibility to efflux 20 cellular accumulation control ATP-depletion probenecid gemfibrozil MK571 0 CIP LVX GAR MXF Michot et al. AAC (2005) 49:

59 Quinolones differ by their activity against intracellular Listeria Seral et al. JAC (2005) 5:511-7

60 Perspectives for the future of chemotherapy use of poor substrates of efflux pumps (moxi vs cipro) development of specific inhibitors of efflux pumps caution for «cross resistance» with other substrates (over expression of efflux pumps)

61 Specific inhibitors GF : a specific MDR inhibitor currently in clinical evaluation in cancer chemotherapy

62 Specific inhibitors GF increases efficacy of doxorubicin in mice with resistant tumors GF 20mg/kg GF 2.5mg/kg GF 0 mg/kg Hyafil et al, Cancer Res. (1993) 53:

63 Specific inhibitors GF is more potent than verapamil to increase azithromycin cellular accumulation Seral et al, AAC (2003) 47:

64 Perspectives for the future of chemotherapy use of poor substrates of efflux pumps (moxi vs cipro) development of specific inhibitors of efflux pumps caution for «cross resistance» with other substrates (over expression of efflux pumps)

65 Over-expression of efflux pumps as mechanism of resistance anticancer agent antibiotic antibiotic????? anticancer agent????? anticancer agent antibiotic?????

66 Over-expression of efflux pumps as mechanism of resistance How to get resistant cells? Gottesman et al, Methods Enzymol. (1998) 292: step-wise increase in CIP concentration several passages at each step multifactorial multidrug resistance time and drug consumming!

67 Over-expression of efflux pumps as mechanism of resistance Heremans et al. ICAAC 2004 Ciprofloxacin accumulation is reduced in resistant cells cellular accumulation 25 WT RS extracellular concentration (mg/l)

68 Over-expression of efflux pumps as mechanism of resistance influence of probenecid on quinolone accumulation in wild-type cells Heremans et al. ICAAC ciprofloxacin moxifloxacin cellular accumulation wild-type wild-type probenecid concentration (mm)

69 Over-expression of efflux pumps as mechanism of resistance influence of probenecid on quinolone accumulation in wild-type and CIP-resistant cells 25 ciprofloxacin moxifloxacin cellular accumulation wild-type CIP-resistant wild-type CIP-resistant probenecid concentration (mm) Heremans et al. ICAAC 2004

70 Over-expression of efflux pumps as mechanism of resistance CIP is ineffective in CIP-resistant cells infected by L. monocytogenes Heremans et al. ECCMID 2005 control log CFU from time 0 h RS WT Log CIP concentration (X MIC)

71 Over-expression of efflux pumps as mechanism of resistance Probenecid restores CIP activity in CIP-resistant cells infected by L. monocytogenes Heremans et al. ECCMID 2005 control + probenecid log CFU from time 0 h RS WT RS WT log CFU from time 0 h Log CIP concentration (X MIC)

72 Over-expression of efflux pumps as mechanism of resistance Heremans et al. ICAAC 2004 the CIP-resistant phenotype is not easily reversible

73 Over-expression of efflux pumps as mechanism of resistance Heremans et al. ICAAC 2004 the CIP-resistant phenotype is not easily reversible

74 Take home message constitutive efflux is part of the game accumulation efflux Take it into account in the choice of your «magic bullets» for their optimal targeting

75 Thanks to F Van Bambeke JM Michot C Seral M Heremans MP Mingeot- Leclercq PM Tulkens come and see us at <

Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria

Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Françoise Van Bambeke on behalf of J.M. Michot, C. Seral, M. Heremans, M.P. Mingeot-Leclercq, P.M.