1. 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
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2. 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
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3. 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
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4. 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
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5. 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.
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6. 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
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7. 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:
Darling et al, Cell Microbiol. 2004;6:521-33
Mittal et al, J. Immunol, 2009, 183:
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8. 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
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9. 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
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10. 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.
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11. 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: ….
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12. 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
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13. 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
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. ( ,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
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14. 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:
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15. 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)
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16. 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
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17. 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)
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18. 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
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19. 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
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20. 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)
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21. 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
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22. 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…
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23. 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
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