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Due to the lack of new antibiotics facing the increasing emergence of resistances, it is important to understand the mechanism and dynamics of these phenomenon. Lipopolysaccharide (LPS) is the polymyxin main target and the most contributing component to polymyxin resistance. Plasmid-mediated colistin resistance MCR-1 (Mobile Colistin Resistance) carrying by Escherichia coli and Klebsiella pneumoniae encodes a phosphoethanolamine transferase leads to the addition of phosphoethanolamine to lipid A of LPS. However, chromosomally-encoded LPS-modifying phosphoethanolamine transferase and loss of LPS are the two primary mechanisms that have been described in colistin-resistant Acinetobacter baumannii to date. Polymyxin-resistance occurred during polymyxin treatment, but the mechanism and dynamics how these strains acquired resistance is poorly understood. Sequential time-kill (TK) were developed as an alternative approach to discriminate heterogenous subpopulations (S/R) versus adaptive resistance (AR) during colistin and polymyxin B exposure.
In this thesis we:
1. Confirm that sequential TK could discriminate between a single homogenous population of bacteria without or with adaptation (AR) and two independent subpopulations with different antibiotic susceptibilities (S/R) demonstrated by pharmacokinetics/pharmacodynamics (PK/PD) modelling.
2. Determine the molecular impact of MCR-1 in the progressive adaptation of E. coli and K. pneumoniae over chromosomal genes involved to LPS modification by using the sequential TK approach.
3. Determine the mechanisms involved in polymyxin resistance in two clinical strains of A. baumannii, isolated from a patient before and after treated with colistin.
These studies not only provide a simple approach to discriminate between two PK/PD models, but also an indication that the MCR-1 presence favor another resistance mechanism leading to high-level resistance to polymyxin. By the similar approach, we determine how colistin-susceptible and resistant A. baumannii isolates respond under polymyxin pressure, including the genes involved. Furthermore, polymyxin B showed a lower capacity to induce high-level of resistance than colistin for all bacterial species.
Keywords: colistin, polymyxin B, E. coli, K. pneumoniae, A. baumannii, sequential time-kill
Mycobacterium abscessus is rapidly growing non-tuberculous mycobacteria responsible for difficult-to-treat pulmonary infections in humans. Current recommended treatment is associated with high treatment failure and emergence of resistance to most of the antibiotics. Also, with only a few new antibiotic drugs active against multidrug-resistant bacteria approved every year, it is important to optimize the use of already existing antibiotics using biopharmaceutical approach like Pharmacokinetic/pharmacodynamic (PK/PD). In pulmonary infections, direct administration of low permeability drugs such as cefoxitin (FOX) and amikacin (AMK) into lungs as therapeutic aerosols should increase their efficiency and minimize whole body exposure responsible for adverse effects, particularly in the case of prolonged treatments. Moreover, the use of antibiotics in combination may reduce the risk of resistance. Several points have been addressed in this thesis:
Biopharmaceutical studies of AMK and FOX: It was shown that after nebulization of AMK and FOX, pulmonary concentrations were almost 1000-fold higher than after intravenous administration for both antibiotics, making them a good candidate for nebulization.
Pharmacokinetic/pharmacodynamic (PK/PD) study of cefoxitin: a semi-mechanistic PK/PD model was developed from in vitro time kill-kinetics assay data, enabling identification of concentration-effect relationships for two bacterial sub-populations while taking into account the unstability degradation of cefoxitin.
PK/PD study of bi-combination: Using a mechanism-based mathematical model and data obtained from time kill-kinetics study, it was shown that the combined effect of AMK and FOX was additive to synergistic at different concentration.
Bi-or tri-combinations: several tri-combinations including AMK, FOX and a 3rd antibiotic (including clarithromycin, linezolid, clofazimine, ciprofloxacin, moxifloxacin, rifampicin and rifabutin) were tested against reference strain, clarithromycin resistance-clinical isolate (Ma1611) and multidrug-resistance-clinical isolate (T28). All tri-combinations were active against reference strain. Similar observation was made with Ma1611 except combination with clofazimine and clarithromycin. Any combination was active against T28. Bi-combinations with highest concentrations of FOX and rifamycins were effective against T28. The synergy between FOX and fluoroquinolones or rifamycins suggests a potent role of these combinations that may warrant further optimization of treatment regimen for the treatment of M. abscessus pulmonary infections.
Tri-combination including AMK, FOX and moxifloxacin (MXF) up to 21 days against clarithromycin-resistance clinical isolate has shown no importance of using MXF as tri-combination was not more effective than the bi-combination of AMK and FOX.
In the face of the shortage of new anti-infectives and the ever-increasing emergence of multi-resistant infectious agents, it is essential to make better use of the therapeutic arsenal at our disposal. This may involve the use of new routes of administration in order to act more effectively locally. To treat lung infections, inhalation or nebulisation appear to be a good option. It has been shown previously that rather hydrophilic molecules were good candidates for this route of administration because the pulmonary epithelium was not very permeable to them and that the active drug was sequestered in the lung. On the other hand, for lipophilic compounds that diffuse rapidly through the epithelium, pulmonary administration is of little therapeutic interest. The situation becomes more complex in the case of prodrug nebulization and/or when efflux pumps are involved in the pulmonary distribution of anti-infectives. In order to better characterize the role of efflux pumps in the pulmonary distribution of anti-infectives in vitro and their impact in vivo, the pulmonary distribution of several anti-infectives was evaluated according to a standardized protocol in rats, allowing measurement and comparison of free plasma and pulmonary epithelial fluid (ELF) concentrations after systemic and intratracheal administration. The first study focuses on the pulmonary distribution of oseltamivir, an anti-viral active against influenza, which is administered as a prodrug (oseltamivir phosphate (OP)). It has been shown in vitro that OP (non-active) is a substrate for efflux pumps and that this active transport is characterized in vivo by higher local concentrations of OP in ELF than in plasma regardless of the route of administration (intravenous or nebulization). However, these high pulmonary prodrug concentrations have little effect on pulmonary concentrations of the active molecule (oseltamivir carboxylate (OC)), due to low local conversion to the active compound and pulmonary permeability of the OC. The second study presents the case of oxazolidinones (linezolide and tedizolide) used to treat Gram-positive infections for which in vivo studies in humans had previously shown local concentrations (ELF) higher than plasma concentrations after oral administration. These data were found in rats according to our standardized protocol and supplemented with post-nebulization data, suggesting a major role for transporters in the pulmonary diffusion of tedizolide. However, membrane permeability and in vitro inhibition studies conducted in a cellular model (NCI-H441) were unable to demonstrate the role of these efflux transporters on high pulmonary concentrations. Other explanations have been considered such as protein binding in ELF or intracellular penetration of active compound. In conclusion, these in vivo and in vitro studies on 4 active compounds have allowed us to improve our knowledge of the parameters that govern the pulmonary diffusion of anti-infectives such as permeability and affinity for efflux transporters and to show the complexity of extrapolation in vitro/in vivo.
Fighting against multidrug-resistant bacteria is a major priority set by World Health Organisation, since it is forecasted that multi-drug-resistant bacteria will be responsible for more deaths than cancer by 2050. In the current context, with only a few new antibiotic drugs active against multidrug-resistant bacteria approved every year, it is of importance to optimize the use of already available antibiotics. It is with this goal in mind, that semi-mechanistic models used to analyse results from PK/PD studies of antibiotics, can be developed. These mathematical tools enable quantification of concentration-effect relationships of drugs, used alone or in combination, in order to optimize their efficacy, prevent bacterial resistance, thus lengthening the period of usability of antibiotics. In this work, after a presentation of the methods used to study PK/PD of antibiotics alone and in combination, results from two projects are presented:
A study of cefoxitin PK/PD against a Mycobacterium abscessus strain. Firstly, it was shown that after nebulisation of cefoxitin, pulmonary concentrations were 1000-fold higher than after intravenous administration, making cefoxitin a good candidate for nebulisation. In a second part, a semi-mechanistic PK/PD model was developed from in vitro data, enabling identification of concentration-effect relationships for two bacterial sub-populations while taking into account degradation of cefoxitin.
A study of the PK/PD of polymyxin B and minocycline association against a polymyxin B resistant Acinetobacter baumannii strain. This in vitro study incorporates data from time-kill experiments with quantification of a bacterial sub-population resistant to polymyxin B, enriched by complementary experiments providing information on the characteristics of this resistant sub-population. This data was analysed by semi-mechanistic PK/PD modelling, which made possible quantification of the strength of interaction between the two drugs and to form hypotheses about the mechanisms of the observed interaction.
The rapid increase in antibiotic resistance during the last decades and the few numbers of recently approved new antibiotics lead to a significant interest to drug combinations. Among these combinations, the β-lactam-β-lactamase inhibitor combination, such as aztreonam-avibactam (ATM-AVI), is one strategy that aims to overcome the resistance due to β-lactamases production, one of the most relevant mechanisms of resistance in Gram-negative bacteria. However, drug interactions can be complex. To better understand the PK/PD of ATM-AVI, two issues have been addressed in this thesis: i. ATM-AVI PK at the infection site. A microdialysis study performed in rats with or without peritonitis showed that ATM and AVI distribution in intraperitoneal fluid was rapid and that concentrations at the target site could be predicted from blood concentrations.ii. PD interaction between ATM and AVI. Checkerboard experiments analyzed with an Emax model have been used to characterize AVI effect on ATM MIC in terms of efficacy and potency in the presence of various multi-drug resistant strains. A PK/PD model was developed based on in vitro data to describe the time-course of ATM-AVI combined effect and to investigate the individual contribution of each of the AVI effects to the combined activity. According to the modeling results, the combined bactericidal activity was mainly explained by AVI enhancing effect, even though AVI demonstrated high efficiency to prevent ATM hydrolysis.
Ciprofloxacin (CIP) is an antibiotic that has been clinically trialed for the treatment of P. aeruginosa (PA) lung infections by aerosolization. However, CIP is rapidly systemically absorbed after lung delivery, increasing the risk for subtherapeutic concentrations and resistant bacteria selection. The aim of this study was to develop an inhalable dry powder (DP) of CIP which would allow the concentration of CIP in the lung epithelial lining fluid (ELF) to be controlled to provide a more efficient effect against extracellular PA. CIP can form complexes with cations (Ca2+,…) reducing its intestinal permeability. While these interactions prove a limitation in terms of oral delivery, it was envisaged as beneficial to slowdown CIP absorption through the lung. This work includes two main parts. First, a proof of concept study that has shown the ability to precisely control the CIP apparent permeability across a pulmonary epithelium model thanks to a Ca−CIP interaction, while keeping antibacterial activity. In this study, CaCO3-based particles were developed to deliver CIP as (CIP-Ca)2+ complex to the lung by a DP inhaler. A second study allowed validating the concept in vivo in healthy rats. In this study, two types of inhalable microparticles loaded with the low-affinity CIP-calcium complex (CIP-Ca)2+ or with the high-affinity CIP-copper complex(CIP-Cu)2+ were developed. Then, ELF and plasma pharmacokinetics of CIP were studied after intratracheal delivery of these particles and of a CIP solution. The dry powder loaded with (CIP-Cu)2+ allowed a 100-times higher pulmonary ELF CIP exposure to be obtained compared to the intratracheal administration of a CIP solution.
Directors: Jean-Christophe Olivier and Julien Brillault.
The rapid emergence of resistant bacteria and the lack of new efficient treatments lead to re-use old forgotten, but still effective, antimicrobials. In particular, chloramphenicol (CHL) and thiamphenicol (THA) have been proposed to treat multidrug-resistant pulmonary bacterial infections. Their direct administration into the lungs as therapeutic aerosols should increase their efficiency and minimize whole body exposure responsible for adverse effects, particularly in the case of prolonged treatments. The purpose of these PhD. works was to perform biopharmaceutical studies and to develop an effective aerosol formulation for lung delivery. The membrane permeability of CHL and THA was evaluated in vitro in the Calu-3 bronchial epithelial cell model and pharmacokinetic (PK) studies were carried out in rats after intratracheal and intravenous administration. In vitro membrane permeability of CHL was high, but intermediate for THA. Both compounds were shown to be substrates of membrane efflux transporters. In agreement with these findings, the PK studies showed that the administration route had no impact in the case of CHL and a moderate one in the case of THA. Therefore, in order to prolong lung exposure to CHL and THA, nanoparticle-based formulations with sustained release properties were formulated using the palmitate ester prodrugs of CHL and THA. To ease administration, nanoparticles were included in microsphere-based dry powder for inhalation. These powders showed an optimal content, satisfactory aerodynamic properties and sustained drug release, which make them promising formulations for lung delivery of CHL and THA as aerosols.
Colistin is an old antibiotic used in human and veterinary medicine. However, as less and less antibiotics are discovered, colistin is considered as a last-line antibiotic to fight against multi-drug resistant bacteria in human. In order to preserve the efficacy of colistin, two issues were investigated in this thesis:(i) Risks of selection of bacteria resistant to colistin, in conjunction with the discovery by the end of 2015 of a plasmid-mediated resistance gene (mcr-1). Thus, the impact of oral use of colistin in pigs was assessed in vivo and no selection was observed in our experimental conditions. Similarly, the use of colistin in human medicine for selective digestive decontamination was studied thanks to human flora‐associated rats. Preliminary results were also neither in favour of a selective effect of colistin.(ii) development of a physiologically-based pharmacokinetic model (PBPK) in pigs for the systemic use of colistin and its prodrug, the colistimethate sodium (CMS). This model provided a further insight into CMS and colistin tissue distribution, especially in kidneys where toxic effects are frequent. As a model application, the withdrawal period after use of CMS in pigs was estimated. Then, we used the ability of PBPK models to carry out intra and inter-species extrapolations in order to adapt this model in adults and children and eventually predict the plasmatic concentrations of colistin during a treatment with CMS.
Ventilator-associated pneumonia (VAP) is associated with high mortality. Nebulization of antibiotics improves outcome of patient with VAP. However, pharmacokinetic data concerning colistin and gentamicin allowing for optimal dosing regimen recommendation are lacking.We compared systemic and pulmonary concentrations of colistin (administered as an inactive prodrug, colistin methanesulfonate or CMS) and gentamicin according to the route of administration (nebulization and intravenous infusion) in critically ill patients with VAP.Intra-pulmonary concentrations of colistin and gentamicin were 10 to 40-fold and 50 to 70-fold much higher after nebulization than after the same dose by intravenous route, respectively. Nebulization has also the theoretical potential advantage to improve patients’ safety in relation to the colistin biodisponibility lower than 10%.With high intra-pulmonary concentrations and very low systemic absorption, CMS and gentamicin nebulization may be good alternatives to intravenous infusion for VAP treatment.
Antibiotics are among the most commonly prescribed drugs, however optimal dosages are not yet well defined. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamics (PK/PD) models that characterize the course of antimicrobial drug concentrations and effects over time, with an emphasis on the development of resistance. These models were applied to optimize dosing regimens of antimicrobial therapies.<br/>A population PK model for colistin and its prodrug, colistin methanesulfonate (CMS) was developed in critically ill patients receiving colistin by nebulization and/or undergoing an intermittent hemodialysis (HD). Results predicted clear beneﬁts of using aerosol delivery of 2MIU CMS dose for the treatment of pulmonary infections. For patients with HD session dosing regimen of CMS should be 1.5 MIU twice daily with an additional dose of 1.5 MIU after each HD session.<br/>An assessment of the performances of different PK-PD models by using a simulation approach have shown the importance of longer study designs and of complementary microbiological data to predict accurately bacterial resistance development.<br/> A semi-mechanistic PK/PD model that incorporates mutation rate and adaptive resistance development of a bioluminescent strain of Pseudomonas aeruginosa against colistin was developed based on in-vitro data. A high, quick and partially reversible resistance was described. These results confirm that the first 24 h of treatment are critical in the management of infections, that colistin alone cannot eradicate completely the mutants of Pseudomonas aeruginosa that were selected during the experiments and that combination therapies seem necessary.
To exert their effect while avoiding adverse events, drugs must reach sufficient concentrations in their site of action. Antibiotics are used for treating infection that bacterial target is in the cerebrospinal fluid (CSF) or brain extracellular fluid (ECF) which is also a target for adverse events. Drug distribution in the brain and CSF may be limited by the presence of the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB). In addition, efflux mechanisms may decrease tissue concentrations of drugs.To optimize the use of antibiotics and reduce adverse events, it is important to obtain tissue pharmacokinetics. Collecting ECF samples via brain microdialysis and CSF samples via external ventricular drain in critically ill patients allow comparison of free drug concentrations. This work is a study of the distribution in plasma, CSF and ECF of two antibiotics, cefotaxime and metronidazole.Patients (after a head injury or stroke) were treated with cefotaxime and metronidazole for pneumonia. Four pharmacokinetic studies were performed at equilibrium by brain microdialysis (n = 11) for collecting ECF or CSF samples (n = 9) by an external ventricular drain. The results showed that metronidazole distributes extensively in both ECF and CSF while the diffusion of cefotaxime was limited, probably due to efflux transporters.
The aim of this study was to investigate the efficiency of intrapulmonary administration and the biopharmaceutical parameters regulating the pulmonary diffusion following nebulization. We examined whether certain efflux pumps were present in an in vitro model of rat lung cells and whether these efflux pumps could be beneficial by increasing lung concentrations in vivo. Fluoroquinolones and colistin were the molecules used as reference. These different molecules allowed an overview of the intrapulmonary diffusion characteristics of antibiotics. The in vivo study with fluoroquinolones showed that their lung concentrations are higher than in plasma, probably due to glycoprotein-P. The presence of this efflux pump was confirmed in the model with rat lung cells. The in vivo study with colistin showed that a slow diffusion may confer an advantage for nebulization over intravenous administration. In conclusion, the nebulization molecules passing slowly (colistin) across the tissues may be advantageous, whereas for others, with a fast passage across the barrier (fluoroquinolones), the pulmonary route may not provide an advantage over the intravenous administration. Moreover, the results showed that a slow permeability across the lung (colistin) may confer an advantage for the antibiotic nebulization, while affinity by transporters (fluoroquinolones) is beneficial for both nebulization and intravenous administration.