A novel in vitro metric predicts in vivo efficacy of inhaled silver-based antimicrobials in a murine Pseudomonas aeruginosa pneumonia model.

To address the escalating problem of antimicrobial resistance and the dwindling antimicrobial pipeline, we have developed a library of novel aerosolizable silver-based antimicrobials, particularly for the treatment of pulmonary infections. To rapidly screen this library and identify promising candidates, we have devised a novel in vitro metric, named the "drug efficacy metric" (DEM), which integrates both the antibacterial activity and the on-target, host cell cytotoxicity. DEMs calculated using an on-target human bronchial epithelial cell-line correlates well (R2 > 0.99) with in vivo efficacy, as measured by median survival hours in a Pseudomonas aeruginosa pneumonia mouse model following aerosolized antimicrobial treatment. In contrast, DEMs derived using off-target primary human dermal fibroblasts correlate poorly (R2 = 0.0595), which confirms our hypothesis. SCC1 and SCC22 have been identified as promising drug candidates through these studies, and SCC22 demonstrates a dose-dependent survival advantage compared to sham treatment. Finally, silver-bearing biodegradable nanoparticles were predicted to exhibit excellent in vivo efficacy based on its in vitro DEM value, which was confirmed in our mouse pneumonia model. Thus, the DEM successfully predicted the efficacy of various silver-based antimicrobials, and may serve as an excellent tool for the rapid screening of potential antimicrobial candidates without the need for extensive animal experimentation.

Antimicrobial Activity of Ibuprofen against Cystic Fibrosis-Associated Gram-Negative Pathogens.

Clinical trials have demonstrated the benefits of ibuprofen therapy in cystic fibrosis (CF) patients, an effect that is currently attributed to ibuprofen's anti-inflammatory properties. Yet, a few previous reports demonstrated an antimicrobial activity of ibuprofen as well, although none investigated its direct effects on the pathogens found in the CF lung, which is the focus of this work. Determination of ibuprofen's in vitro antimicrobial activity against Pseudomonas aeruginosa and Burkholderia species strains through measurements of the endpoint number of CFU and growth kinetics showed that ibuprofen reduced the growth rate and bacterial burden of the tested strains in a dose-dependent fashion. In an in vitroPseudomonas biofilm model, a reduction in the rate of biomass accumulation over 8 h of growth with ibuprofen treatment was observed. Next, an acute Pseudomonas pneumonia model was used to test this antimicrobial activity after the oral delivery of ibuprofen. Following intranasal inoculation, ibuprofen-treated mice exhibited lower CFU counts and improved survival compared with the control animals. Preliminary biodistribution studies performed after the delivery of ibuprofen to mice by aerosol demonstrated a rapid accumulation of ibuprofen in serum and minimum retention in lung tissue and bronchoalveolar lavage fluid. Therefore, ibuprofen-encapsulated polymeric nanoparticles (Ibu-NPs) were formulated to improve the pharmacokinetic profile. Ibu-NPs formulated for aerosol delivery inhibited the growth of P. aeruginosa in vitro and may provide a convenient dosing method. These results provide an additional explanation for the previously observed therapeutic effects of ibuprofen in CF patients and further strengthen the argument for its use by these patients.

Synthesis, characterization, and in vivo efficacy of shell cross-linked nanoparticle formulations carrying silver antimicrobials as aerosolized therapeutics.

The use of nebulizable, nanoparticle-based antimicrobial delivery systems can improve efficacy and reduce toxicity for treatment of multi-drug-resistant bacteria in the chronically infected lungs of cystic fibrosis patients. Nanoparticle vehicles are particularly useful for applying broad-spectrum silver-based antimicrobials, for instance, to improve the residence time of small-molecule silver carbene complexes (SCCs) within the lung. Therefore, we have synthesized multifunctional, shell cross-linked knedel-like polymeric nanoparticles (SCK NPs) and capitalized on the ability to independently load the shell and core with silver-based antimicrobial agents. We formulated three silver-loaded variants of SCK NPs: shell-loaded with silver cations, core-loaded with SCC10, and combined loading of shell silver cations and core SCC10. All three formulations provided a sustained delivery of silver over the course of at least 2-4 days. The two SCK NP formulations with SCC10 loaded in the core each exhibited excellent antimicrobial activity and efficacy in vivo in a mouse model of Pseudomonas aeruginosa pneumonia. SCK NPs with shell silver cation-load only, while efficacious in vitro, failed to demonstrate efficacy in vivo. However, a single dose of core SCC10-loaded SCK NPs (0.74 ± 0.16 mg Ag) provided a 28% survival advantage over sham treatment, and administration of two doses (0.88 mg Ag) improved survival to 60%. In contrast, a total of 14.5 mg of Ag(+) delivered over 5 doses at 12 h intervals was necessary to achieve a 60% survival advantage with a free-drug (SCC1) formulation. Thus, SCK NPs show promise for clinical impact by greatly reducing antimicrobial dosage and dosing frequency, which could minimize toxicity and improve patient adherence.

Aerosolized antimicrobial agents based on degradable dextran nanoparticles loaded with silver carbene complexes.

Degradable acetalated dextran (Ac-DEX) nanoparticles were prepared and loaded with a hydrophobic silver carbene complex (SCC) by a single-emulsion process. The resulting particles were characterized for morphology and size distribution using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The average particle size and particle size distribution were found to be a function of the ratio of the organic phase to the surfactant containing aqueous phase with a 1:5 volume ratio of Ac-DEX CH(2)Cl(2) (organic):PBS (aqueous) being optimal for the formulation of nanoparticles with an average size of 100 ± 40 nm and a low polydispersity. The SCC loading was found to increase with an increase in the SCC quantity in the initial feed used during particle formulation up to 30% (w/w); however, the encapsulation efficiency was observed to be the best at a feed ratio of 20% (w/w). In vitro efficacy testing of the SCC loaded Ac-DEX nanoparticles demonstrated their activity against both Gram-negative and Gram-positive bacteria; the nanoparticles inhibited the growth of every bacterial species tested. As expected, a higher concentration of drug was required to inhibit bacterial growth when the drug was encapsulated within the nanoparticle formulations compared with the free drug illustrating the desired depot release. Compared with free drug, the Ac-DEX nanoparticles were much more readily suspended in an aqueous phase and subsequently aerosolized, thus providing an effective method of pulmonary drug delivery.

Synthesis, characterization, and antimicrobial activity of silver carbene complexes derived from 4,5,6,7-tetrachlorobenzimidazole against antibiotic resistant bacteria.

Silver N-heterocyclic carbene complexes have been shown to have great potential as antimicrobial agents, affecting a wide spectrum of both Gram-positive and Gram-negative bacteria. A new series of three silver carbene complexes (SCCs) based on 4,5,6,7-tetrachlorobenzimidazole has been synthesized, characterized, and tested against a panel of clinical strains of bacteria. The imidazolium salts and their precursors were characterized by elemental analysis, mass spectrometry, (1)H and (13)C NMR spectroscopy, and single crystal X-ray diffraction. The silver carbene complexes, SCC32, SCC33, and SCC34 were characterized by elemental analysis, (1)H and (13)C NMR spectroscopy, and single crystal X-ray diffraction. These complexes proved highly efficacious with minimum inhibitory concentrations (MICs) ranging from 0.25 to 6 µg mL(-1). Overall, the complexes were effective against highly resistant bacteria strains, such as methicillin-resistant Staphylococcus aureus (MRSA), weaponizable bacteria, such as Yersinia pestis, and pathogens found within the lungs of cystic fibrosis patients, such as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, and Burkholderia gladioli. SCC33 and SCC34 also showed clinically relevant activity against a silver-resistant strain of Escherichia coli based on MIC testing.