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1.  ‘Old’ antibiotics for emerging multidrug-resistant bacteria 
Purpose of review
Increased emergence of bacterial resistance and the decline in newly developed antibiotics have necessitated the reintroduction of previously abandoned antimicrobial agents active against multidrug-resistant bacteria. Having never been subjected to contemporary drug development procedures, these ‘old’ antibiotics require redevelopment in order to optimize therapy. This review focuses on colistin as an exemplar of a successful redevelopment process and briefly discusses two other old antibiotics, fusidic acid and fosfomycin.
Recent findings
Redevelopment of colistin led to an improved understanding of its chemistry, pharmacokinetics and pharmacodynamics, enabling important steps towards optimizing its clinical use in different patient populations. A scientifically based dosing algorithm was developed for critically ill patients, including those with renal impairment. As nephrotoxicity is a dose-limiting adverse event of colistin, rational combination therapy with other antibiotics needs to be investigated.
Summary
The example of colistin demonstrated that state-of-the-art analytical, microbiological and pharmacokinetic/pharmacodynamic methods can facilitate optimized use of ‘old’ antibiotics in the clinic. Similar methods are now being applied to fosfomycin and fusidic acid in order to optimize therapy. To improve and preserve the usefulness of these antibiotics rational approaches for redevelopment need to be followed.
doi:10.1097/QCO.0b013e328358afe5
PMCID: PMC4242550  PMID: 23041772
colistin; fosfomycin; fusidic acid; pharmacodynamics; pharmacokinetics
2.  Pulmonary and Systemic Pharmacokinetics of Inhaled and Intravenous Colistin Methanesulfonate in Cystic Fibrosis Patients: Targeting Advantage of Inhalational Administration 
The purpose of this study was to define the pulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and formed colistin following intravenous (i.v.) and inhaled administration in cystic fibrosis (CF) patients. Six CF subjects were administered nebulized CMS doses of 2 and 4 million IU and an i.v. CMS infusion of 150 mg of colistin base activity. Blood plasma, sputum, and urine samples were collected for 12 to 24 h postdose. To assess the tolerability of the drug, lung function tests, blood serum creatinine concentrations, and adverse effect reports were recorded. All doses were well tolerated in the subjects. The pharmacokinetic parameters for CMS following i.v. delivery were consistent with previously reported values. Sputum concentrations of formed colistin were maintained at <1.0 mg/liter for 12 h postdose. Nebulization of CMS resulted in relatively high sputum concentrations of CMS and formed colistin compared to those resulting from i.v. administration. The systemic availability of CMS was low following nebulization of 2 and 4 million IU (7.93% ± 4.26% and 5.37% ± 1.36%, respectively), and the plasma colistin concentrations were below the limit of quantification. Less than 2 to 3% of the nebulized CMS dose was recovered in the urine samples in 24 h. The therapeutic availability and drug targeting index for CMS and colistin following inhalation compared to i.v. delivery were significantly greater than 1. Inhalation of CMS is an effective means of targeting CMS and formed colistin for delivery to the lungs, as high lung exposure and minimal systemic exposure were achieved in CF subjects.
doi:10.1128/AAC.01705-13
PMCID: PMC3993267  PMID: 24550334
3.  Clinical Population Pharmacokinetics and Toxicodynamics of Linezolid 
Thrombocytopenia is a common side effect of linezolid, an oxazolidinone antibiotic often used to treat multidrug-resistant Gram-positive bacterial infections. Various risk factors have been suggested, including linezolid dose and duration of therapy, baseline platelet counts, and renal dysfunction; still, the mechanisms behind this potentially treatment-limiting toxicity are largely unknown. A clinical study was conducted to investigate the relationship between linezolid pharmacokinetics and toxicodynamics and inform strategies to prevent and manage linezolid-associated toxicity. Forty-one patients received 42 separate treatment courses of linezolid (600 mg every 12 h). A new mechanism-based, population pharmacokinetic/toxicodynamic model was developed to describe the time course of plasma linezolid concentrations and platelets. A linezolid concentration of 8.06 mg/liter (101% between-patient variability) inhibited the synthesis of platelet precursor cells by 50%. Simulations predicted treatment durations of 5 and 7 days to carry a substantially lower risk than 10- to 28-day therapy for platelet nadirs of <100 ×109/liter. The risk for toxicity did not differ noticeably between 14 and 28 days of therapy and was significantly higher for patients with lower baseline platelet counts. Due to the increased risk of toxicity after longer durations of linezolid therapy and large between-patient variability, close monitoring of patients for development of toxicity is important. Dose individualization based on plasma linezolid concentration profiles and platelet counts should be considered to minimize linezolid-associated thrombocytopenia. Overall, oxazolidinone therapy over 5 to 7 days even at relatively high doses was predicted to be as safe as 10-day therapy of 600 mg linezolid every 12 h.
doi:10.1128/AAC.01885-13
PMCID: PMC4023770  PMID: 24514086
4.  Pharmacokinetics of Colistin Methanesulfonate and Formed Colistin in End-Stage Renal Disease Patients Receiving Continuous Ambulatory Peritoneal Dialysis 
Colistin, administered intravenously as its inactive prodrug colistin methanesulfonate (CMS), is increasingly used as last-line therapy to combat multidrug-resistant Gram-negative bacteria. CMS dosing needs to be adjusted for renal function. The impact of continuous ambulatory peritoneal dialysis (CAPD) on the pharmacokinetics of both CMS and colistin has not been studied. No CMS dosing recommendations are available for patients receiving CAPD. Eight CAPD patients received a single intravenous CMS dose (150 mg colistin base activity [CBA]) over 30 min. Serial blood and dialysate samples, and cumulative urine where applicable, were collected over 25 h. CMS and colistin concentrations were determined by high-performance liquid chromatography. Population pharmacokinetic modeling and Monte Carlo simulations were conducted. The total body clearance of CMS (excluding CAPD clearance) was 1.77 liters/h (44%) [population mean (between-subject variability)], while CAPD clearance was 0.088 liter/h (64%). The population mean terminal half-life of CMS was 8.4 h. For colistin, the total clearance/fraction of CMS metabolized to colistin (fm) (excluding CAPD clearance) was 2.74 liters/h (50%), the CAPD clearance was 0.101 liter/h (34%), and the mean terminal half-life was 13.2 h. Monte Carlo simulations suggested a loading dose of 300 mg CBA on day 1 and a maintenance dose of either 150 mg or 200 mg CBA daily to achieve a target average steady-state plasma colistin concentration of 2.5 mg/liter. Clearance by CAPD was low for both CMS and formed colistin. Therefore, CMS doses should not be increased during CAPD. Modeling and simulation enabled us to propose the first evidence-based CMS dosage regimen for CAPD patients.
doi:10.1128/AAC.01741-13
PMCID: PMC3910712  PMID: 24189256
5.  Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A 
Innate immunity  2012;19(3):265-277.
The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by 1H NMR, using a paired wild type (WT) and the Δ6lpxM mutant strains B5055 and B5055 ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055 ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055 ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains’ phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055 Δ6lpxM may be attributable to the favorable insertion of polymyxins into the more fluid OM compared with B5055. Molecular models of the polymyxin B–lipid A complex illuminate the key role of the lipid A acyl chains for complexation of polymyxin. The data provide important insight into the molecular basis for the increased polymyxin susceptibility of K. pneumoniae strains with under-acylated lipid A. Under-acylation appears to facilitate the integration of the N-terminal fatty-acyl chain of polymyxin into the OM resulting in an increased susceptibility to its antimicrobial activity/activities.
doi:10.1177/1753425912459092
PMCID: PMC4242410  PMID: 23008349
Polymyxin; Klebsiella pneumoniae; lipopolysaccharide
6.  Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae 
Innate immunity  2013;20(4):350-363.
This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by 1H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.
doi:10.1177/1753425913493337
PMCID: PMC4242413  PMID: 23887184
Polymyxin; colistin; Klebsiella pneumoniae; lipopolysaccharide; surface
7.  Synergistic Activity of Colistin and Rifampin Combination against Multidrug-Resistant Acinetobacter baumannii in an In Vitro Pharmacokinetic/Pharmacodynamic Model 
Combination therapy may be required for multidrug-resistant (MDR) Acinetobacter baumannii. This study systematically investigated bacterial killing and emergence of colistin resistance with colistin and rifampin combinations against MDR A. baumannii. Studies were conducted over 72 h in an in vitro pharmacokinetic (PK)/pharmacodynamic (PD) model at inocula of ∼106 and ∼108 CFU/ml using two MDR clinical isolates of A. baumannii, FADDI-AB030 (colistin susceptible) and FADDI-AB156 (colistin resistant). Three combination regimens achieving clinically relevant concentrations (constant colistin concentration of 0.5, 2, or 5 mg/liter and a rifampin maximum concentration [Cmax] of 5 mg/liter every 24 hours; half-life, 3 h) were investigated. Microbiological response was measured by serial bacterial counts. Population analysis profiles assessed emergence of colistin resistance. Against both isolates, combinations resulted in substantially greater killing at the low inoculum; combinations containing 2 and 5 mg/liter colistin increased killing at the high inoculum. Combinations were additive or synergistic at 6, 24, 48, and 72 h with all colistin concentrations against FADDI-AB030 and FADDI-AB156 in, respectively, 8 and 11 of 12 cases (i.e., all 3 combinations) at the 106-CFU/ml inoculum and 8 and 7 of 8 cases with the 2- and 5-mg/liter colistin regimens at the 108-CFU/ml inoculum. For FADDI-AB156, killing by the combination was ∼2.5 to 7.5 and ∼2.5 to 5 log10 CFU/ml greater at the low inoculum (all colistin concentrations) and high inoculum (2 and 5 mg/liter colistin), respectively. Emergence of colistin-resistant subpopulations was completely suppressed in the colistin-susceptible isolate with all combinations at both inocula. Our study provides important information for optimizing colistin-rifampin combinations against colistin-susceptible and -resistant MDR A. baumannii.
doi:10.1128/AAC.00703-13
PMCID: PMC3719722  PMID: 23716052
8.  Polymyxins and analogs bind to ribosomal RNA and interfere with eukaryotic translation in vitro 
doi:10.1002/cbic.201300496
PMCID: PMC3947458  PMID: 24105917
Antibiotics; Polymyxins; Colistin; RNA; Translation
9.  Pharmacokinetics of four different brands of colistimethate and formed colistin in rats 
Journal of Antimicrobial Chemotherapy  2013;68(10):2311-2317.
Objectives
Very different labelling conventions are employed by different products of colistimethate (CMS), an inactive prodrug of colistin that is used as a last-line defence against Gram-negative ‘superbugs’. This study examined the chemical composition and pharmacokinetics in rats of four commercial parenteral products of CMS.
Methods
Contents per vial of four brands of CMS from three different continents were weighed (n = 3). Elemental analysis and HPLC examination were conducted. The pharmacokinetics of CMS and formed colistin were investigated for each product after intravenous administration in rats (28.1 mg/kg CMS; n = 4). Blood was collected over 180 min, and concentrations of CMS and colistin were measured followed by pharmacokinetic analysis.
Results
X-GEN, Paddock and Atlantic products, labelled with 150 mg ‘colistin base activity’, contained 366.8 ± 0.80, 340.6 ± 0.08 and 380.0 ± 5.97 mg CMS (sodium) per vial, respectively; while the Forest product (labelled with 2 000 000 IU) contained 159.3 ± 1.75 mg CMS (sodium). The elemental compositions of the four products were similar; however, the HPLC profile of the Atlantic CMS was different from those of the other three products. The pharmacokinetics of CMS were generally comparable across brands; however, the molar ratios (%) of the AUC0–180min of colistin to CMS (1.68% ± 0.35% to 3.29% ± 0.43%) were significantly different (P = 0.0157).
Conclusion
This is the first study to demonstrate that although different brands of CMS from various parts of the world have similar elemental compositions, they lead to different exposures to the microbiologically active formed colistin. The study has significant implications for the interpretation of pharmacological studies of CMS conducted in different parts of the world.
doi:10.1093/jac/dkt207
PMCID: PMC3772743  PMID: 23749953
elemental analysis; HPLC; intravenous administration; colistin base activity
10.  New Dosing Strategies for an Old Antibiotic: Pharmacodynamics of Front-Loaded Regimens of Colistin at Simulated Pharmacokinetics in Patients with Kidney or Liver Disease 
Increasing evidence suggests that colistin monotherapy is suboptimal at currently recommended doses. We hypothesized that front-loading provides an improved dosing strategy for polymyxin antibiotics to maximize killing and minimize total exposure. Here, we utilized an in vitro pharmacodynamic model to examine the impact of front-loaded colistin regimens against a high bacterial density (108 CFU/ml) of Pseudomonas aeruginosa. The pharmacokinetics were simulated for patients with hepatic (half-life [t1/2] of 3.2 h) or renal (t1/2 of 14.8 h) disease. Front-loaded regimens (n = 5) demonstrated improvement in bacterial killing, with reduced overall free drug areas under the concentration-time curve (fAUC) compared to those with traditional dosing regimens (n = 14) with various dosing frequencies (every 12 h [q12h] and q24h). In the renal failure simulations, front-loaded regimens at lower exposures (fAUC of 143 mg · h/liter) obtained killing activity similar to that of traditional regimens (fAUC of 268 mg · h/liter), with an ∼97% reduction in the area under the viable count curve over 48 h. In hepatic failure simulations, front-loaded regimens yielded rapid initial killing by up to 7 log10 within 2 h, but considerable regrowth occurred for both front-loaded and traditional regimens. No regimen eradicated the high bacterial inoculum of P. aeruginosa. The current study, which utilizes an in vitro pharmacodynamic infection model, demonstrates the potential benefits of front-loading strategies for polymyxins simulating differential pharmacokinetics in patients with hepatic and renal failure at a range of doses. Our findings may have important clinical implications, as front-loading polymyxins as a part of a combination regimen may be a viable strategy for aggressive treatment of high-bacterial-burden infections.
doi:10.1128/AAC.00327-13
PMCID: PMC3957851  PMID: 24342636
11.  A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity 
The Journal of antibiotics  2013;67(2):147-151.
Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with E. coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.
doi:10.1038/ja.2013.111
PMCID: PMC3943757  PMID: 24169795
type II NADH-quinone oxidoreductase; polymyxin B; colistin; Escherichia coli; Klebsiella pneumoniae; Acinetobacter baumannii
12.  Case-case-control study on factors associated with vanB vancomycin-resistant and vancomycin-susceptible enterococcal bacteraemia 
BMC Infectious Diseases  2014;14:353.
Background
Enterococci are a major cause of healthcare-associated infection. In Australia, vanB vancomycin-resistant enterococci (VRE) is the predominant genotype. There are limited data on the factors linked to vanB VRE bacteraemia. This study aimed to identify factors associated with vanB VRE bacteraemia, and compare them with those for vancomycin-susceptible enterococci (VSE) bacteraemia.
Methods
A case-case-control study was performed in two tertiary public hospitals in Victoria, Australia. VRE and VSE bacteraemia cases were compared with controls without evidence of enterococcal bacteraemia, but may have had infections due to other pathogens.
Results
All VRE isolates had vanB genotype. Factors associated with vanB VRE bacteraemia were urinary catheter use within the last 30 days (OR 2.86, 95% CI 1.09-7.53), an increase in duration of metronidazole therapy (OR 1.65, 95% CI 1.17-2.33), and a higher Chronic Disease Score specific for VRE (OR 1.70, 95% CI 1.05-2.77). Factors linked to VSE bacteraemia were a history of gastrointestinal disease (OR 2.29, 95% CI 1.05-4.99) and an increase in duration of metronidazole therapy (OR 1.23, 95% CI 1.02-1.48). Admission into the haematology/oncology unit was associated with lower odds of VSE bacteraemia (OR 0.08, 95% CI 0.01-0.74).
Conclusions
This is the largest case-case-control study involving vanB VRE bacteraemia. Factors associated with the development of vanB VRE bacteraemia were different to those of VSE bacteraemia.
doi:10.1186/1471-2334-14-353
PMCID: PMC4091649  PMID: 24973797
Enterococci; Vancomycin-resistant; Vancomycin-susceptible; Bacteraemia
13.  Population Pharmacokinetics of Colistin Methanesulfonate in Rats: Achieving Sustained Lung Concentrations of Colistin for Targeting Respiratory Infections 
Antimicrobial Agents and Chemotherapy  2013;57(10):5087-5095.
Colistin methanesulfonate (CMS), the inactive prodrug of colistin, is administered by inhalation for the management of respiratory infections. However, limited pharmacokinetic data are available for CMS and colistin following pulmonary delivery. This study investigates the pharmacokinetics of CMS and colistin following intravenous (i.v.) and intratracheal (i.t.) administration in rats and determines the targeting advantage after direct delivery into the lungs. In addition to plasma, bronchoalveolar lavage (BAL) fluid was collected to quantify drug concentrations in lung epithelial lining fluid (ELF). The resulting data were analyzed using a population modeling approach in S-ADAPT. A three-compartment model described the disposition of both compounds in plasma following i.v. administration. The estimated mean clearance from the central compartment was 0.122 liters/h for CMS and 0.0657 liters/h for colistin. Conversion of CMS to colistin from all three compartments was required to fit the plasma data. The fraction of the i.v. dose converted to colistin in the systemic circulation was 0.0255. Two BAL fluid compartments were required to reflect drug kinetics in the ELF after i.t. dosing. A slow conversion of CMS (mean conversion time [MCTCMS] = 3.48 h) in the lungs contributed to high and sustained concentrations of colistin in ELF. The fraction of the CMS dose converted to colistin in ELF (fm,ELF = 0.226) was higher than the corresponding fractional conversion in plasma after i.v. administration. In conclusion, pulmonary administration of CMS achieves high and sustained exposures of colistin in lungs for targeting respiratory infections.
doi:10.1128/AAC.01127-13
PMCID: PMC3811457  PMID: 23917323
14.  Pharmacology of polymyxins: new insights into an ‘old’ class of antibiotics 
Future microbiology  2013;8(6):10.2217/fmb.13.39.
Increasing antibiotic resistance in Gram-negative bacteria, particularly in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, presents a global medical challenge. No new antibiotics will be available for these ‘superbugs’ in the near future due to the dry antibiotic discovery pipeline. Colistin and polymyxin B are increasingly used as the last-line therapeutic options for treatment of infections caused by multidrug-resistant Gram-negative bacteria. This article surveys the significant progress over the last decade in understanding polymyxin chemistry, mechanisms of antibacterial activity and resistance, structure–activity relationships and pharmacokinetics/pharmacodynamics. In the ‘Bad Bugs, No Drugs’ era, we must pursue structure–activity relationship-based approaches to develop novel polymyxin-like lipopeptides targeting polymyxin-resistant Gram-negative ‘superbugs’. Before new antibiotics become available, we must optimize the clinical use of polymyxins through the application of pharmacokinetic/pharmacodynamic principles, thereby minimizing the development of resistance.
doi:10.2217/fmb.13.39
PMCID: PMC3852176  PMID: 23701329
colistin; lipid A; lipopolysaccharide; pharmacokinetic/pharmacodynamic; polymyxin; resistance; structure–activity relationship
15.  Polymyxin B Induces Apoptosis in Kidney Proximal Tubular Cells 
The nephrotoxicity of polymyxins is a major dose-limiting factor for treatment of infections caused by multidrug-resistant Gram-negative pathogens. The mechanism(s) of polymyxin-induced nephrotoxicity is not clear. This study aimed to investigate polymyxin B-induced apoptosis in kidney proximal tubular cells. Polymyxin B-induced apoptosis in NRK-52E cells was examined by caspase activation, DNA breakage, and translocation of membrane phosphatidylserine using Red-VAD-FMK [Val-Ala-Asp(O-Me) fluoromethyl ketone] staining, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and double staining with annexin V-propidium iodide (PI). The concentration dependence (50% effective concentration [EC50]) and time course for polymyxin B-induced apoptosis were measured in NRK-52E and HK-2 cells by fluorescence-activated cell sorting (FACS) with annexin V and PI. Polymyxin B-induced apoptosis in NRK-52E cells was confirmed by positive labeling from Red-VAD-FMK staining, TUNEL assay, and annexin V-PI double staining. The EC50 (95% confidence interval [CI]) of polymyxin B for the NRK-52E cells was 1.05 (0.91 to 1.22) mM and was 0.35 (0.29 to 0.42) mM for HK-2 cells. At lower concentrations of polymyxin B, minimal apoptosis was observed, followed by a sharp rise in the apoptotic index at higher concentrations in both cell lines. After treatment of NRK-52E cells with 2.0 mM polymyxin B, the percentage of apoptotic cells (mean ± standard deviation [SD]) was 10.9% ± 4.69% at 6 h and reached plateau (>80%) at 24 h, whereas treatment with 0.5 mM polymyxin B for 24 h led to 93.6% ± 5.57% of HK-2 cells in apoptosis. Understanding the mechanism of polymyxin B-induced apoptosis will provide important information for discovering less nephrotoxic polymyxin-like lipopeptides.
doi:10.1128/AAC.02587-12
PMCID: PMC3754291  PMID: 23796937
16.  Species-Dependent Blood-Brain Barrier Disruption of Lipopolysaccharide: Amelioration by Colistin In Vitro and In Vivo 
The aim of this study was to use in vitro and in vivo models to assess the impact of lipopolysaccharide (LPS) from two different bacterial species on blood-brain barrier (BBB) integrity and brain uptake of colistin. Following repeated administration of LPS from Pseudomonas aeruginosa, the brain-to-plasma ratio of [14C]sucrose in Swiss outbred mice was not significantly increased. Furthermore, while the brain uptake of colistin in mice increased 3-fold following administration of LPS from Salmonella enterica, LPS from P. aeruginosa had no significant effect on colistin brain uptake. This apparent species-dependent effect did not appear to correlate with differences in plasma cytokine levels, as the concentrations of tumor necrosis factor alpha and interleukin-6 following administration of each LPS were not different (P > 0.05). To clarify whether this species-specific effect of LPS was due to direct effects on the BBB, human brain capillary endothelial (hCMEC/D3) cells were treated with LPS from P. aeruginosa or S. enterica and claudin-5 expression was measured by Western blotting. S. enterica LPS significantly (P < 0.05) reduced claudin-5 expression at a concentration of 7.5 μg/ml. In contrast, P. aeruginosa LPS decreased (P < 0.05) claudin-5 expression only at the highest concentration tested (i.e., 30 μg/ml). Coadministration of therapeutic concentrations of colistin ameliorated the S. enterica LPS-induced reduction in claudin-5 expression in hCMEC/D3 cells and the perturbation in BBB function in mice. This study demonstrates that BBB disruption induced by LPS is species dependent, at least between P. aeruginosa and S. enterica, and can be ameliorated by colistin.
doi:10.1128/AAC.00765-13
PMCID: PMC3754325  PMID: 23796941
17.  Relationship between Trough Plasma and Epithelial Lining Fluid Concentrations of Voriconazole in Lung Transplant Recipients 
Trough (predose) voriconazole concentrations in plasma and pulmonary epithelial lining fluid (ELF) of lung transplant recipients receiving oral voriconazole preemptive treatment were determined. The mean (± standard deviation [SD]) ELF/plasma ratio was 12.5 ± 6.3. A strong positive linear relationship was noted between trough plasma and ELF voriconazole concentrations (r2 = 0.87), suggesting the feasibility of using trough plasma voriconazole concentration as a surrogate to estimate the corresponding concentration in ELF of lung transplant recipients.
doi:10.1128/AAC.00942-13
PMCID: PMC3754345  PMID: 23817382
18.  Drug-binding energetics of human α-1-acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations 
This study utilizes sensitive, modern isothermal titration calorimetric (ITC) methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics were characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (ΔH°) and favorable (positive) entropic (ΔS°) contributions to the Gibbs free energy (ΔG°). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug-serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity.
doi:10.1002/jmr.2221
PMCID: PMC3513776  PMID: 23192962
human α-1-acid glycoprotein; thermodynamics; drug binding
19.  Colistin in the 21st Century 
Purpose of review
Colistin is a 50 year-old antibiotic that is being used increasingly as a ‘last-line’ therapy to treat infections caused by MDR Gram-negative bacteria, when essentially no other options are available. Despite its age, or because of its age, there has been a dearth of knowledge on its pharmacological and microbiological properties. This review focuses on recent studies aimed at optimizing the clinical use of this old antibiotic.
Recent findings
A number of factors, including the diversity in the pharmaceutical products available, have hindered the optimal use of colistin. Recent advances in understanding of the pharmacokinetics and pharmacodynamics of colistin, and the emerging knowledge on the relationship between the pharmacokinetics and pharmacodynamics, providing a solid base for optimization of dosage regimens. The potential for nephrotoxicity has been a lingering concern, but recent studies provide useful new information on the incidence, severity and reversibility of this adverse effect. Recent approaches to the use of other antibiotics in combination with colistin hold promise for increased antibacterial efficacy with less potential for emergence of resistance.
Summary
Because few, if any, new antibiotics with activity against MDR Gram-negative bacteria will be available within the next several years, it is essential that colistin is used in ways that maximize its antibacterial efficacy and minimize toxicity and development of resistance. Recent developments have improved use of colistin in the 21st century.
doi:10.1097/QCO.0b013e328332e672
PMCID: PMC2869076  PMID: 19797945
colistin; approaches to optimizing therapy; Gram-negative infections
20.  Pharmacokinetics and pharmacodynamics of ‘old’ polymyxins: what is new? 
‘Old’ colistin and polymyxin B are increasingly used as last-line therapy against multidrug-resistant Gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. For intravenous administration, colistin is dosed as its inactive prodrug colistin methanesulfonate (sodium), while polymyxin B is used as its sulfate (active antibacterial). Over the last decade significant progress has been made in understanding their chemistry, pharmacokinetics (PK) and pharmacodynamics (PD). The first scientifically based dosing suggestions are now available for colistin methanesulfonate to generate a desired target steady-state plasma concentration of formed colistin in various categories of critically-ill patients. As simply increasing polymyxin dosage regimens is not an option for optimizing their PK/PD due to nephrotoxicity, combination therapy with other antibiotics has great potential to maximize the efficacy of polymyxins while minimizing emergence of resistance. We must pursue rational approaches to the use of polymyxins and other existing antibiotics through the application of PK/PD principles.
doi:10.1016/j.diagmicrobio.2012.07.010
PMCID: PMC3477253  PMID: 22959816
Colistin; polymyxin B; pharmacokinetics; pharmacodynamics
21.  Cell surface hydrophobicity of colistin-susceptible versus -resistant Acinetobacter baumannii determined by contact angles: methodological considerations and implications 
Journal of applied microbiology  2012;113(4):940-951.
AIMS
Contact angle analysis of cell surface hydrophobicity (CSH) describes the tendency of a water droplet to spread across a lawn of filtered bacterial cells. Colistin-induced disruption of the Gram-negative outer membrane necessitates hydrophobic contacts with lipopolysaccharide (LPS). We aimed to characterize the CSH of Acinetobacter baumannii using contact angles, to provide insight into the mechanism of colistin resistance.
METHODS AND RESULTS
Contact angles were analysed for five paired colistin-susceptible and -resistant A. baumannii strains. Drainage of the water droplet through bacterial layers was demonstrated to influence results. Consequently, measurements were performed 0.66-sec after droplet deposition. Colistin-resistant cells exhibited lower contact angles (38.8±2.8° to 46.8±1.3°) compared to their paired-susceptible strains (40.7±3.0° to 48.0±1.4°; ANOVA; p<0.05). Contact angles increased at stationary phase (50.3±2.9° to 61.5±2.5° and 47.4±2.0° to 50.8±3.2°, susceptible and resistant, respectively, ANOVA; p<0.05), and in response to colistin 32-mgL−1 exposure (44.5±1.5° to 50.6±2.8° and 43.5±2.2° to 48.0±2.2°, susceptible and resistant, respectively; ANOVA; p<0.05). Analysis of complemented strains constructed with an intact lpxA gene, or empty vector, highlighted the contribution of LPS to CSH.
CONCLUSIONS
Compositional outer-membrane variations likely account for CSH differences between A. baumannii phenotypes, which influence the hydrophobic colistin-bacterium interaction.
SIGNIFICANCE AND IMPACT OF STUDY
Important insight into the mechanism of colistin resistance has been provided. Greater consideration of contact angle mehodology is nescessary to ensure accurate analyses are performed.
doi:10.1111/j.1365-2672.2012.05337.x
PMCID: PMC3434258  PMID: 22574702
Antimicrobials; Lipopolysaccharide; Mechanism of Action
22.  Interaction of Colistin and Colistin Methanesulfonate with Liposomes: Colloidal Aspects and Implications for Formulation 
Journal of pharmaceutical sciences  2012;101(9):3347-3359.
Interaction of colistin and colistin methanesulfonate (CMS) with liposomes has been studied with the view to understanding the limitations to the use of liposomes as a more effective delivery system for pulmonary inhalation of this important class of antibiotic. Thus, in this study, liposomes containing colistin or CMS were prepared and characterized with respect to colloidal behavior and drug encapsulation and release. Association of anionic CMS with liposomes induced negative charge on the particles. However, degradation of the CMS to form cationic colistin over time was directly correlated with charge reversal and particle aggregation. The rate of degradation of CMS was significantly more rapid when associated with the liposome bilayer than when compared with the same concentration in aqueous solution. Colistin liposomes carried positive charge and were stable. Encapsulation efficiency for colistin was approximately 50%, decreasing with increasing concentration of colistin. Colistin was rapidly released from liposomes on dilution. Although the studies indicate limited utility of colistin or CMS liposomes for long duration controlled-release applications, colistin liposomes were highly stable and may present a potential opportunity for coformulation of colistin with a second antibiotic to colocalize the two drugs after pulmonary delivery.
doi:10.1002/jps.23203
PMCID: PMC3437939  PMID: 22623044
colistin; polymyxin E; micelle; stability; self-assembly; liposome; controlled release/delivery; colloid
23.  Lipopolysaccharide-Deficient Acinetobacter baumannii Shows Altered Signaling through Host Toll-Like Receptors and Increased Susceptibility to the Host Antimicrobial Peptide LL-37 
Infection and Immunity  2013;81(3):684-689.
Infections caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious global health problem. We have shown previously that A. baumannii can become resistant to the last-line antibiotic colistin via the loss of lipopolysaccharide (LPS), including the lipid A anchor, from the outer membrane (J. H. Moffatt, M. Harper, P. Harrison, J. D. Hale, E. Vinogradov, T. Seemann, R. Henry, B. Crane, F. St. Michael, A. D. Cox, B. Adler, R. L. Nation, J. Li, and J. D. Boyce, Antimicrob. Agents Chemother. 54:4971–4977, 2010). Here, we show how these LPS-deficient bacteria interact with components of the host innate immune system. LPS-deficient A. baumannii stimulated 2- to 4-fold lower levels of NF-κB activation and tumor necrosis factor alpha (TNF-α) secretion from immortalized murine macrophages, but it still elicited low levels of TNF-α secretion via a Toll-like receptor 2-dependent mechanism. Furthermore, we show that while LPS-deficient A. baumannii was not altered in its resistance to human serum, it showed increased susceptibility to the human antimicrobial peptide LL-37. Thus, LPS-deficient, colistin-resistant A. baumannii shows significantly altered activation of the host innate immune inflammatory response.
doi:10.1128/IAI.01362-12
PMCID: PMC3584870  PMID: 23250952
24.  Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology 
The characterization of encapsulation efficiency and in vitro drug release from nanoparticle-based formulations often requires the separation of nanoparticles from unencapsulated drug. Inefficient separation of nanoparticles from the medium in which they are dispersed can lead to inaccurate estimates of encapsulation efficiency and drug release. This study establishes dynamic light scattering as a simple method for substantiation of the effectiveness of the separation process. Colistin-loaded liposomes, as an exemplar nano-sized delivery particle, were diluted to construct a calibration curve relating the amount of light scattering to liposome concentration. Dynamic light scattering revealed that, in the case of ultracentrifugation and centrifugal ultrafiltration, approximately 2.9% of the total liposomes remained in supernatants or filtrates, respectively. In comparison, filtrates obtained using pressure ultrafiltration contained less than 0.002% of the total liposomes from the formulation. Subsequent release studies using dialysis misleadingly implied a slow release of colistin over >48 h. In contrast, pressure ultrafiltration revealed immediate equilibration to the equilibrium distribution of colistin between the liposome and aqueous phases upon dilution. Pressure ultrafiltration is therefore recommended as the optimal method of choice for studying release kinetics of drug from nanomedicine carriers.
doi:10.1007/s13346-012-0064-4
PMCID: PMC3482165  PMID: 23110256
Nanoparticle; dynamic light scattering; encapsulation efficiency; in vitro release; centrifuge ultrafiltration; ultracentrifugation; pressure ultrafiltration
25.  The Combination of Colistin and Doripenem Is Synergistic against Klebsiella pneumoniae at Multiple Inocula and Suppresses Colistin Resistance in an In Vitro Pharmacokinetic/Pharmacodynamic Model 
Antimicrobial Agents and Chemotherapy  2012;56(10):5103-5112.
Multidrug-resistant (MDR) Klebsiella pneumoniae may require combination therapy. We systematically investigated bacterial killing with colistin and doripenem mono- and combination therapy against MDR K. pneumoniae and emergence of colistin resistance. A one-compartment in vitro pharmacokinetic/pharmacodynamic model was employed over a 72-h period with two inocula (∼106 and ∼108 CFU/ml); a colistin-heteroresistant reference strain (ATCC 13883) and three clinical isolates (colistin-susceptible FADDI-KP032 [doripenem resistant], colistin-heteroresistant FADDI-KP033, and colistin-resistant FADDI-KP035) were included. Four combinations utilizing clinically achievable concentrations were investigated. Microbiological responses were examined by determining log changes and population analysis profiles (for emergence of colistin resistance) over 72 h. Against colistin-susceptible and -heteroresistant isolates, combinations of colistin (constant concentration regimens of 0.5 or 2 mg/liter) plus doripenem (steady-state peak concentration [Cmax] of 2.5 or 25 mg/liter over 8 h; half-life, 1.5 h) generally resulted in substantial improvements in bacterial killing at both inocula. Combinations were additive or synergistic against ATCC 13883, FADDI-KP032, and FADDI-KP033 in 9, 9, and 14 of 16 cases (4 combinations at 6, 24, 48, and 72 h) at the 106-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 108-CFU/ml inoculum, respectively. Combinations at the highest dosage regimens resulted in undetectable bacterial counts at 72 h in 5 of 8 cases (4 isolates at 2 inocula). Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was virtually eliminated with combination therapy. Against the colistin-resistant isolate, colistin at 2 mg/liter plus doripenem (Cmax, 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.
doi:10.1128/AAC.01064-12
PMCID: PMC3457376  PMID: 22802247

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