Yu-Wei Lin

Pharmacokinetics/Pharmacodynamics of Pulmonary Delivery of Colistin against Pseudomonas aeruginosa in a Mouse Lung Infection Model.

ABSTRACT Colistin is often administered by inhalation and/or the parenteral route for the treatment of respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. However, limited pharmacokinetic (PK) and pharmacodynamic (PD) data are available to guide the optimization of dosage regimens of inhaled colistin. In the present study, PK of colistin in epithelial lining fluid (ELF) and plasma was determined following intratracheal delivery of a single dose of colistin solution in neutropenic lung-infected mice. The antimicrobial efficacy of intratracheal delivery of colistin against three P. aeruginosa strains (ATCC 27853, PAO1, and FADDI-PA022; MIC of 1 mg/liter for all strains) was examined in a neutropenic mouse lung infection model. Dose fractionation studies were conducted over 2.64 to 23.8 mg/kg of body weight/day. The inhibitory sigmoid model was employed to determine the PK/PD index that best described the antimicrobial efficacy of pulmonary delivery of colistin. In both ELF and plasma, the ratio of the area under the unbound concentration-time profile to MIC (fAUC/MIC) was the PK/PD index that best described the antimicrobial effect in mouse lung infection (R2 = 0.60 to 0.84 for ELF and 0.64 to 0.83 for plasma). The fAUC/MIC targets required to achieve stasis against the three strains were 684 to 1,050 in ELF and 2.15 to 3.29 in plasma. The histopathological data showed that pulmonary delivery of colistin reduced infection-caused pulmonary inflammation and preserved the integrity of the lung epithelium, although colistin introduced mild pulmonary inflammation in healthy mice. This study showed pulmonary delivery of colistin provides antimicrobial effects against MDR P. aeruginosa lung infections superior to those of parenteral administrations. For the first time, our results provide important preclinical PK/PD information for optimization of inhaled colistin therapy.

Measuring Bipolar Charge and Mass Distributions of Powder Aerosols by a Novel Tool (BOLAR).

The Bipolar Charge Analyzer (BOLAR) was evaluated for measuring bipolar electrostatic charge and mass distributions of powder aerosols generated from a dry powder inhaler. Mannitol powder (5, 10, and 20 mg) was dispersed using an Osmohaler inhaler into the BOLAR at air flow rates of 30 or 60 L/min. As the aerosol sample was drawn through the BOLAR, the air flow was divided into six equal fractions. Five of them entered individual detection tubes with a defined cutoff diameter in the range of 0.95 to 16.36 μm (depending on the flow rate) and the remaining (i.e., the sixth) fraction passed through a reference chamber. The aerosols that entered the detection tubes were separated according to the particle charge polarity (positive, negative, or neutral) and charge was measured by separate electrometers. The deposited powder of a single actuation from the inhaler was chemically assayed using high performance liquid chromatography. Additionally, the aerosol measurements were conducted on a modified Classic Electrical Low Pressure Impactor (ELPI) for comparison of the net specific charge per size fraction. Spray-dried mannitol carried significantly different positively and negatively charged particles in each of the five defined particle size fractions. The charge-to-mass ratio (q/m) of positively charged particles ranged from +1.11 to +32.57 pC/μg and negatively charged particles ranged from -1.39 to -9.25 pC/μg, resulting in a net q/m of -3.08 to +13.34 pC/μg. The net q/m values obtained on the modified ELPI ranged from -5.18 to +4.81 pC/μg, which were comparable to the BOLAR measurements. This is the first full report to utilize the BOLAR to measure bipolar charge and mass distributions of a powder aerosol. Positively and negatively charged particles were observed within each size fraction, and their corresponding q/m profiles were successfully characterized. Despite some potential drawbacks, the BOLAR has provided a new platform for investigating bipolar charge in powder aerosols for inhalation.

Aerosolized Polymyxin B for Treatment of Respiratory Tract Infections: Determination of Pharmacokinetic/Pharmacodynamic Indices for Aerosolized Polymyxin B against Pseudomonas aeruginosa in a Mouse Lung Infection Model

ABSTRACT Pulmonary administration of polymyxins is increasingly used for the treatment of respiratory tract infections caused by multidrug-resistant Gram-negative bacteria, such as those in patients with cystic fibrosis. However, there is a lack of pharmacokinetics (PK), pharmacodynamics (PD), and toxicity data of aerosolized polymyxin B to inform rational dosage selection. The PK and PD of polymyxin B following pulmonary and intravenous dosing were examined in neutropenic infected mice, and the data were analyzed by a population PK model. Dose fractionation study was performed for total daily doses between 2.06 and 24.8 mg base/kg of weight against Pseudomonas aeruginosa ATCC 27853, PAO1, and FADDI-PA022 (MIC of 1 mg/liter for all three strains). Histopathological examination of the lung was undertaken at 24 h posttreatment in both healthy and neutropenic infected mice. A two-compartment PK model was required for both epithelial lining fluid (ELF) and plasma drug exposure. The model consisted of central and peripheral compartments and was described by bidirectional first-order distribution clearance. The ratio of the area under the curve to the MIC (AUC/MIC) was the most predictive PK/PD index to describe the antimicrobial efficacy of aerosolized polymyxin B in treating lung infections in mice (R2 of 0.70 to 0.88 for ELF and 0.70 to 0.87 for plasma). The AUC/MIC targets associated with bacteriostasis against the three P. aeruginosa strains were 1,326 to 1,506 in ELF and 3.14 to 4.03 in plasma. Histopathological results showed that polymyxin B aerosols significantly reduced lung inflammation and preserved lung epithelial integrity. This study highlights the advantageous PK/PD characteristics of pulmonary delivery of polymyxin B over intravenous administration in achieving high drug exposure in ELF.

Potential Toxicity of Polymyxins in Human Lung Epithelial Cells

ABSTRACT Inhaled polymyxins are of considerable utility in achieving optimal exposure in the respiratory tract for the treatment of lung infections caused by multidrug-resistant Gram-negative pathogens. Current inhaled polymyxin therapy is empirical, and often large doses are used that may lead to potential pulmonary adverse effects. This study aimed to investigate the effect of polymyxins on human lung epithelial (A549) cells. The viability of A549 cells was examined after treatment with polymyxins by flow cytometry. Activation of caspases 3, 8, and 9, expression of Fas ligand (FasL), loss of mitochondrial membrane potential, and mitochondrial oxidative stress induced by polymyxin B were evaluated. The concentration of polymyxin B required to induce 50% of maximal cell death was 1.74 mM (95% confidence interval, 1.60 to 1.90 mM). Colistin was at least 2-fold less toxic than polymyxin B, while colistimethate was nontoxic. With 2.0 mM polymyxin B, 30.6% ± 11.5% (mean ± standard deviation) of the cells were apoptotic at 8 h and this increased to 71.3% ± 3.72% at 24 h. Concentration- and time-dependent activation of caspases 3, 8, and 9 was evident, while the activation of caspase 9 was more dramatic. Furthermore, polymyxin B caused concentration- and time-dependent FasL expression, production of mitochondrial reactive oxygen species, and changes in mitochondrial membrane potential. This is the first study to demonstrate that both extrinsic death receptor and intrinsic mitochondrial pathways are involved in polymyxin-induced toxicity in A549 cells. This knowledge base is critical for the development of novel strategies for the safe and effective inhalation therapy of polymyxins against Gram-negative “superbugs.”

Elucidating the pharmacokinetics/pharmacodynamics of aerosolized colistin against multidrug-resistant acinetobacter baumannii and klebsiella pneumoniae in a mouse lung infection model

ABSTRACT The pharmacokinetics/pharmacodynamics (PK/PD) of aerosolized colistin was investigated against Acinetobacter baumannii and Klebsiella pneumoniae over 24 h in a neutropenic mouse lung infection model. Dose fractionation studies were performed over 2.64 to 23.8 mg/kg/day, and the data were fitted to a sigmoid inhibitory model. The area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) in the epithelial lining fluid was the most predictive PK/PD index for aerosolized colistin against both pathogens. Our study provides important pharmacological information for optimizing aerosolized colistin.

Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling of Aerosolized Colistin in a Mouse Lung Infection Model

ABSTRACT Optimized dosage regimens of aerosolized colistin (as colistin methanesulfonate [CMS]) are urgently required to maximize bacterial killing against multidrug-resistant Gram-negative bacteria while minimizing toxicity. This study aimed to develop a mechanism-based pharmacokinetic (PK)/pharmacodynamic (PD) model (MBM) for aerosolized colistin based upon PK/PD data in neutropenic infected mice and to perform a deterministic simulation with the PK of aerosolized colistin (as CMS) in critically ill patients. In vivo time-kill experiments were carried out with three different strains of Pseudomonas aeruginosa. An MBM was developed in S-ADAPT and evaluated by assessing its ability to predict the PK/PD index associated with efficacy in mice. A deterministic simulation with human PK data was undertaken to predict the efficacy of current dosage regimens of aerosolized colistin in critically ill patients. In the final MBM, the total bacterial population for each isolate consisted of colistin-susceptible and -resistant subpopulations. The antimicrobial efficacy of aerosolized colistin was best described by a sigmoidal Emax model whereby colistin enhanced the rate of bacterial death. Deterministic simulation with human PK data predicted that an inhalational dosage regimen of 60 mg colistin base activity (CBA) every 12 h is needed to achieve a ≥2-log10 bacterial reduction (as the number of CFU per lung) in critically ill patients at 24 h after commencement of inhaled therapy. In conclusion, the developed MBM is a useful tool for optimizing inhalational dosage regimens of colistin. Clinical studies are warranted to validate and refine our MBM for aerosolized colistin.

Composite particle formulations of colistin and meropenem with improved in-vitro bacterial killing and aerosolization for inhalation

ABSTRACT Antibiotic combination therapy is promising for the treatment of lower respiratory tract infections caused by multi‐drug resistant Gram‐negative pathogens. Inhaled antibiotic therapy offers the advantage of direct delivery of the drugs to the site of infection, as compared to the parenteral administrations. In this study, we developed composite particle formulations of colistin and meropenem. The formulations were characterized for particle size, morphology, specific surface area, surface chemical composition, in‐vitro aerosolization performance and in‐vitro antibacterial activity. The combinations demonstrated enhanced antibacterial activity against clinical isolates of Acinetobacter baumannii N16870 and Pseudomonas aeruginosa 19147, when compared with antibiotic monotherapy. Spray‐dried meropenem alone showed a poor aerosolization performance as indicated by a low fine particle fraction (FPF) of 32.5±3.3%. Co‐spraying with colistin improved the aerosolization of meropenem with up to a two‐fold increase in the FPF. Such improvements in aerosolization can be attributed to the enrichment of colistin on the surface of composite particles as indicated by X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), and the increases in particle porosity. Intermolecular interactions between colistin and meropenem were observed for the combination formulations as measured by FT‐IR. In conclusion, our results show that co‐spray drying with colistin improves the antibacterial activity and aerosol performance of meropenem and produces a formulation with synergistic bacterial killing.

Lipidomic Analysis of the Outer Membrane Vesicles from Paired Polymyxin-Susceptible and -Resistant Klebsiella pneumoniae Clinical Isolates

Gram-negative bacteria produce outer membrane vesicles (OMVs) as delivery vehicles for nefarious bacterial cargo such as virulence factors, which are antibiotic resistance determinants. This study aimed to investigate the impact of polymyxin B treatment on the OMV lipidome from paired polymyxin-susceptible and -resistant Klebsiella pneumoniae isolates. K. pneumoniae ATCC 700721 was employed as a reference strain in addition to two clinical strains, K. pneumoniae FADDI-KP069 and K. pneumoniae BM3. Polymyxin B treatment of the polymyxin-susceptible strains resulted in a marked reduction in the glycerophospholipid, fatty acid, lysoglycerophosphate and sphingolipid content of their OMVs. Conversely, the polymyxin-resistant strains expressed OMVs richer in all of these lipid species, both intrinsically and increasingly under polymyxin treatment. The average diameter of the OMVs derived from the K. pneumoniae ATCC 700721 polymyxin-susceptible isolate, measured by dynamic light scattering measurements, was ~90.6 nm, whereas the average diameter of the OMVs isolated from the paired polymyxin-resistant isolate was ~141 nm. Polymyxin B treatment (2 mg/L) of the K. pneumoniae ATCC 700721 cells resulted in the production of OMVs with a larger average particle size in both the susceptible (average diameter ~124 nm) and resistant (average diameter ~154 nm) strains. In light of the above, we hypothesize that outer membrane remodelling associated with polymyxin resistance in K. pneumoniae may involve fortifying the membrane structure with increased glycerophospholipids, fatty acids, lysoglycerophosphates and sphingolipids. Putatively, these changes serve to make the outer membrane and OMVs more impervious to polymyxin attack.

Efficacy of systemically administered polymyxins in mouse burn wound infection caused by multidrug-resistant Gram-negative pathogens: A proof-of-concept study

ABSTRACT The efficacy of subcutaneously administered polymyxins against burn wound infections caused by Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae was examined in a murine infection model. Subcutaneously administered colistin and polymyxin B (30 mg/kg thrice daily) achieved a ≥2-log10 reduction in the bacterial load for P. aeruginosa and A. baumannii infections, whereas wound infections by K. pneumoniae were less responsive (<1-log10 reduction). This study highlights the potential therapeutic benefits of parenteral polymyxins for treating burn wound infections.

Polymyxin B in Combination with Enrofloxacin Exerts Synergistic Killing against Extensive Drug-resistant Pseudomonas aeruginosa

ABSTRACT Polymyxins are increasingly used as a last-resort class of antibiotics against extensively drug-resistant (XDR) Gram-negative bacteria. However, resistance to polymyxins can emerge with monotherapy. As nephrotoxicity is the major dose-limiting factor for polymyxin monotherapy, dose escalation to suppress the emergence of polymyxin resistance is not a viable option. Therefore, novel approaches are needed to preserve this last-line class of antibiotics. This study aimed to investigate the antimicrobial synergy of polymyxin B combined with enrofloxacin against Pseudomonas aeruginosa. Static time-kill studies were conducted over 24 h with polymyxin B (1 to 4 mg/liter) and enrofloxacin (1 to 4 mg/liter) alone or in combination. Additionally, in vitro one-compartment model (IVM) and hollow-fiber infection model (HFIM) experiments were performed against P. aeruginosa 12196. Polymyxin B and enrofloxacin in monotherapy were ineffective against all of the P. aeruginosa isolates examined, whereas polymyxin B-enrofloxacin in combination was synergistic against P. aeruginosa, with ≥2 to 4 log10 kill at 24 h in the static time-kill studies. In both IVM and HFIM, the combination was synergistic, and the bacterial counting values were below the limit of quantification on day 5 in the HFIM. A population analysis profile indicated that the combination inhibited the emergence of polymyxin resistance in P. aeruginosa 12196. The mechanism-based modeling suggests that the synergistic killing is a result of the combination of mechanistic and subpopulation synergy. Overall, this is the first preclinical study to demonstrate that the polymyxin-enrofloxacin combination is of considerable utility for the treatment of XDR P. aeruginosa infections and warrants future clinical evaluations.