Emilien Drouot, Paul M. Tulkens, Françoise Van Bambeke OS0469 Pharmacodynamic evaluation of the intracellular activity of neglected and disused antibiotics (NDAB) towards Pseudomonas aeruginosa after phagocytosis by human THP-1 monocytes Emilien Drouot, Paul M. Tulkens, Françoise Van Bambeke Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain, Brussels, Belgium <www.facm.ucl.ac.be> 23/04/2017 ECCMID 2017- OS0469

The battle against superbugs: who is going to win ? Become MDR … Nowadays, we are facing the challenge of treating infections caused by so-called superbugs that have acquired resistance to virtually all classes of available antibiotics https://loonylabs.org/2015/01/04/antibiotic-resistance-2/ 23/04/2017 ECCMID 2017- OS0469

Worrying Superbugs ESKAPE pathogens Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumanii Pseudomonas aeruginosa Enterobacter species These superbugs were classified as ESKAPE pathogens and recently ranked by WHO based on their priority. This is why the IDSA has highlighted the urgent need of discovering new antibiotics, with the objective of registering ten new drugs before 2020 23/04/2017 ECCMID 2017- OS0469

The battle against superbugs: who is going to win ? Become MDR … Old or New drug ? In the mean time, the question is: what can we do ? Maybe revive old antibiotics, which are active against these superbugs but have not been used because not needed at the time they were discovered, so that resistance did not develop https://loonylabs.org/2015/01/04/antibiotic-resistance-2/ 23/04/2017 ECCMID 2017- OS0469

Reviving old antibiotics: why and how ? The interest of this old, neglected drugs is highlighted in a series of recent papers, suggesting even, as a reply to IDSA, that we have in hands 10 old antibiotics of high interest. These old drugs do reply to a medical need in the fight against resistance. 23/04/2017 ECCMID 2017- OS0469

The battle against superbugs: who is going to win ? Adopt antibiotic “tolerant” lifestyles Now, beside resistance, bacteria are also difficult to eradicate because they adopt specific lifestyles poorly responsive to antibiotics https://loonylabs.org/2015/01/04/antibiotic-resistance-2/ 23/04/2017 ECCMID 2017- OS0469

Intracellular Superbugs ESKAPE pathogens Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumanii Pseudomonas aeruginosa Enterobacter species Sinha et al, Cell. Microbiol. 1999; 1:101-17 Korsley et al, PLoS One. 2013;8:e83637 Among these lifestyles: their capacity to survive within eukaryotic cells, which has been described for all ESKAPE pathogens Bist et al, Infect Immun. 2014; 82: 1112–22 Rosen et al, Infect. Immun. 2008; 76:3337-45 Darling et al, Cell Microbiol. 2004;6:521-33 Mittal et al, J. Immunol, 2009, 183:6588-99 23/04/2017 ECCMID 2017- OS0469

The battle against superbugs: who is going to win ? Adopt antibiotic “tolerant” lifestyles Optimize PK/PD ? To act upon these intracellular forms, we need to consider PK/PD in these specific environments https://loonylabs.org/2015/01/04/antibiotic-resistance-2/ 23/04/2017 ECCMID 2017- OS0469

PK/PD parameters and intracellular infections bacterial responsiveness physico-chemical conditions cooperation with host defenses pharmacodynamics accumulation and bioavailability metabolism binding influx efflux pharmacokinetics To act upon intracellular bacteria, antibiotics have to fulfill a series of characteristics. With respect to PK, they have to accumulate and be bioavailable in the infected compartment, which implies a low efflux, metabolism, or binding to cell constituents. With respect to PD, we have to take into account that the intracellular environment can modulate bacterial responsiveness or antibiotic expression of activity due to the specific physico-chemical properties found in the infected compartment. We have also to consider a possible cooperation with host cell defense mechanisms. Carryn et al, Infect Dis Clin North Am 2003; 17:615-34 23/04/2017 ECCMID 2017- OS0469

Reviving old antibiotics: why and how ? With respect to old antibiotics, we are lacking of PK-PD data, as there were discovered before the development of modern pharmacology We also need to determine which ones could offer a real interest. 23/04/2017 ECCMID 2017- OS0469

Objectives of the study Evaluation of antibiotic activity in a model of intracellular infection by P. aeruginosa To determine the activity of different neglected and disused antibiotics (NDAB) towards intracellular infection using a previously developed in vitro model To compare pertinent pharmacological descriptors of antibiotic activity (maximal efficacy, relative potency) between NDAB and old, conventional antipseudomonal drugs In this context, the aim of this study was: …. 23/04/2017 ECCMID 2017- OS0469

Reviving old antibiotics: Bug and Drugs used in this work ESKAPE pathogens Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumanii Pseudomonas aeruginosa Enterobacter species Old, conventional drugs Old, neglected and disused drugs drugs MIC* (mg/L) Cellular accumul. MIC* (mg/L) Colistin 2 ND Polymyxin B 4 Meropenem 1 1 x Chloramphenicol 16 2-5 x Amikacin 2-4 x (slow) Rifampicin 2-10 x Ciprofloxacin 0.06 5 x Minocycline 2-4 x We selected 4 old conventional antibiotics representative of the classes used today in the clinics and 4 neglected antibiotics with activity against Gram(-) bacteria. MICs are quite high, but at first glance, it was tempting to test them as they accumulate in eukaryotic cells to variable extents. * P. aeruginosa PAO1 23/04/2017 ECCMID 2017- OS0469

In vitro model of intracellular infection in THP-1 human monocytes Phagocytosis (2 h) 750,000 cells/mL 10 bact/cell Opsonization (60 min, 37°C) 9 mL RPMI-1640 + 1 mL fresh human serum [Reference strain PAO1] Extracellular Wash gentamicin 100 µg/mL (60 min – 37°C) Typical post-phagocytosis inoculum: ~ 1x106 CFU/mg prot. Incubation with antibiotics over a wide range of conc. (0.001-1,000 x MIC) (0  24h) This slide illustrates the model used . Cell washing, collection, and lysis Cell-associated CFUs counting Cell protein content determination Buyck et al, AAC 2013; 57:2310-8 23/04/2017 ECCMID 2017- OS0469

Definition of pharmacodynamic parameters for intracellular infection extracellular concentration (total drug) resulting in no apparent bacterial growth (vs. initial inoculum), as calculated from the Hill equation of the concentration-response curve. Cs: log CFU decrease (at 24 h) from the corresponding original inoculum, as extrapolated for an infinitely large antibiotic concentration. Emax: 23/04/2017 ECCMID 2017- OS0469

Intracellular activities of old, conventional antipseudomonal drugs Pharmacological comparison at equipotent concentrations Cs Emax Here, we compare conventional drugs at equipotent concentrations. Activity develops following a sigmoidal concentration-response curve. Cs is close to the MIC for all drugs Emax is low, ranging from -1 log to – 3 log decrease in CFU from original inoculum Cs: close to the MIC Emax low (no bactericidal effect) Emax: low (ciprofloxacin ~ bactericidal effect) 23/04/2017 ECCMID 2017- OS0469

Intracellular activities of old, conventional antipseudomonal drugs Clinically-oriented comparison at serum concentrations If we now compare these drugs in a clinically-oriented perspective, looking at the effect that can be reached at concentrations observed in the serum of treated patients, we see that cipro and to a lower extent mero show useful activity while colistin and amika are only bacteriostatic. Meropenem  1 log decrease in CFU Ciprofloxacin  ~ 3 log decrease in CFU at clinically-achievable concentrations 23/04/2017 ECCMID 2017- OS0469

Intracellular activities of neglected and disused antibiotics Pharmacological comparison at equipotent concentrations Cs Emax Move now to neglected antibiotics, we do the same type of observations: Cs close to the MIC and low Emax (ranging from bacteriostatic effect to 2 log decrease for minocycline Cs: close to the MIC Emax low (no bactericidal effect) Emax: low (no bactericidal effect) 23/04/2017 ECCMID 2017- OS0469

Intracellular activities of neglected and disused antibiotics Clinically-oriented comparison at serum concentrations At clinically relevant concentrations, we notice here that a static effect can be achieved for poly B while the other drugs fail to prevent intracellular growth. Polymyxin B  bacteriostatic Others  barely active at clinically-achievable concentrations 23/04/2017 ECCMID 2017- OS0469

Summary of antibiotic PD parameters against intracellular P. aeruginosa Pharmacological comparison - Relative potency: To sum up the data, we see here the Cs of all tested drugs, which is always close to the MIC, whatever the accumulation level of the drug inside the cells. Intracellular MIC ~ extracellular MIC (2 dilution difference max) whatever the cellular accumulation level of the drug 23/04/2017 ECCMID 2017- OS0469

Summary of antibiotic PD parameters against intracellular P. aeruginosa Pharmacological comparison – Maximal efficacy: Emax are low, reaching barely a 10 % decrease in intra inoculum for most of the drugs. This is unexpected as most of these drugs are well known as highly bactericidal in broth, as illustrated here for 4 of them. The Emax is at the limit of detection against extra bacteria but much lower intracellularly Intracellular efficacy low (<<< extracellular efficacy) 23/04/2017 ECCMID 2017- OS0469

Summary of antibiotic PD parameters against intracellular P. aeruginosa Clinically-oriented comparison – Efficacy at Cmax: At clinically relevant conc, we see that neglected drugs do not show a useful activity Neglected, disused antibiotics show no useful activity against intracellular P. aeruginosa at clinically-achievable concentrations 23/04/2017 ECCMID 2017- OS0469

Still room for better ideas… Conclusions Relative potency is close to the MIC (whatever the antibiotic cell accumulation) Activity as determined in broth can to some extent predict intracellular potency (~ MIC) but not intracellular efficacy. Both old conventional and neglected and disused antibiotics are poorly effective against intracellular P. aeruginosa, regardless their mode of action. Among the antibiotics tested, only ciprofloxacin reached a bactericidal effect against intracellular P. aeruginosa at clinically-achievable concentrations. Neglected and disused antibiotics cannot be used alone to act against intracellular forms of [MDR] pathogens. Still room for better ideas… 23/04/2017 ECCMID 2017- OS0469

The other teams of our consortium Acknowledgments The other teams of our consortium J.W. Mouton, Rotterdam, The Netherlands L. Friberg, Uppsala, Sweden E. Nielsens, Uppsala, Sweden T. Tangden, Uppsala, Sweden W. Couet, Poitiers, France A. Bousquet-Melou, Toulouse, France J. Meletiadis, Athens, Greece V. Yfantis and M.C. Cambier for expert technical assistance 23/04/2017 ECCMID 2017- OS0469