Computational Fluid Dynamics (CFD) Guided Spray Drying Recommendations for Improved Aerosol Performance of a Small-Particle Antibiotic Formulation.

PURPOSE: The objective of this study was to implement computational fluid dynamics (CFD) simulations and aerosol characterization experiments to determine best-case spray drying conditions of a tobramycin excipient enhanced growth (Tobi-EEG) formulation for use in a pediatric air-jet dry powder inhaler (DPI). METHODS: An iterative approach was implemented in which sets of spray drying conditions were explored using CFD simulations followed by lead candidate selection, powder production and in vitro aerosol testing. CFD simulations of a small-particle spray dryer were performed to capture droplet drying parameters and surface-averaged temperature and relative humidity (RH) conditions in the powder collection region. In vitro aerosol testing was performed for the selected powders using the pediatric air-jet DPI, cascade impaction, and aerosol transport through a pediatric mouth-throat (MT) model to a tracheal filter. RESULTS: Based on comparisons of CFD simulations and in vitro powder performance, recommended drying conditions for small-particle powders with electrostatic collection include: (i) reducing the CFD-predicted drying parameters of κavg and κmax to values below 3 µm2/ms and 114 µm2/ms, respectively; (ii) maintaining the Collector Surface RH within an elevated range, which for the Tobi-EEG formulation with l-leucine was 20-30 %RH; and (iii) ensuring that particles reaching the collector were fully dried, based on a mass fraction of solute CFD parameter. CONCLUSIONS: Based on the newly recommended spray dryer conditions for small particle aerosols, delivery performance of the lead Tobi-EEG formulation was improved resulting in >60% of the DPI loaded dose passing through the pediatric MT model.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Amplified electrochemical antibiotic aptasensing based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework.

A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study.

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/wPLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 µm and its internal diameter was 2.61 ± 0.05 mm. The graft's antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.

Overcoming ß-Lactam resistance in Pseudomonas aeruginosa using non-canonical tobramycin-based antibiotic adjuvants.

ß-Lactam antibiotics have for long been a mainstay in antimicrobial chemotherapy. However, due to its ubiquitous usage, bacteria have evolved multiple concerted pathways to evade its actions, underscoring the complexity of resistance to this class of drug. Current strategies to mitigate this problem are geared towards developing inhibitors that can shield the ß-lactam core from enzymatic hydrolysis. In reality, a combination of factors including porin loss, overexpressed efflux pumps, expression of ß-lactamases, reduced outer membrane permeability, and target modifications are characteristics of phenotypes that are microbiologically resistant to ß-lactam antibiotics, especially Pseudomonas aeruginosa. Herein, we describe a strategy that may simultaneously address multiple mechanisms of resistance to ß-lactams. A triple combination with ß-lactam/ß-lactamase inhibitors offers better microbiological response against carbapenem-resistant P. aeruginosa than the current standard of care. The observed interactions are also unaffected by efflux pumps. We conclude that a multicomponent combination therapy may be the way forward in addressing the myriads of emerging therapy failure associated with ß-lactam resistance.

Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms.

BACKGROUND: Novel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area. RESULTS: We report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of ~ 16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to ~ 230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of ~ 232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies. CONCLUSIONS: Positive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite-tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

Comparison of two therapeutic approaches for the management of ventilator-associated pneumonia due to multidrug-resistant Acinetobacter: a randomized clinical trial study.

Management of ventilator-associated pneumonia (VAP) is a puzzling issue for infectious disease specialist. The present clinical trial study was aimed to comparing the effects of injectable colistin plus nebulized colistin and injectable colistin plus nebulized tobramycin on management of patients with VAP due to multidrug-resistant Acinetobacter. VAP patients were randomly divided into two groups (n = 30/each): Group 1 - patients that received intravenous (IV) meropenem, injectable colistin plus nebulized colistin, as a routine treatment, and Group 2 - patients that received IV meropenem, injectable colistin plus nebulized tobramycin. A total of 14 days of therapeutic intervention are required for every case. Follow-up for subjects was performed at five time-points: days 1, 3, 5, 7, and 14 after intervention. Also, a mean of creatinine levels of patients was determined in five times. In the present study, the clinical pulmonary infection score (CPIS) was determined on the basis of points assigned for various clinically manifestations of VAP. Based on our statistical analysis, there was no significant difference between CPIS and creatinine level in both Groups 1 and 2 (p > .05). CPIS and other clinical investigation appeared effectiveness of the treatment with injected colistin plus nebulized tobramycin; on the other hand, the results of present clinical trial showed that aforementioned therapeutic approach can be used as an alternative treatment for the management of infection in VAP patients.

A colorimetric nanoprobe based on dynamic aggregation of SDS-capped silver nanoparticles for tobramycin determination in exhaled breath condensate.

A colorimetric nanoprobe was developed for the quantification of tobramycin in exhaled breath condensate (EBC). The nanoprobe consists of silver nanoparticles (Ag NPs) modified with sodium dodecyl sulfate (SDS), which is applied in the presence of sodium metaborate. Characterization of the synthesized SDS-capped Ag NPs by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX) showed that the nanoparticles were well synthesized with nearly uniform size and an average diameter of < 30 nm. Interaction of sodium metaborate with the SDS-capped Ag NPs and tobramycin results in aggregation of the nanoparticles and consequently decreases the absorbance intensity, leading to the production of a new absorbance peak and a color change from yellow to purple. The absorbance intensity was recorded at λmax = 400 nm and 522 nm and λ522/λ400 was used as the analytical signal. The experimental parameters were investigated and optimized using a multivariate optimization method (central composite design). The current nanoprobe gives a linear response for tobramycin from 1.0 to 50.0 ng mL-1 with a detection limit of 0.5 ng mL-1. The intra- and inter-day relative standard deviations for five replicated analyses of 10.0 ng mL-1 tobramycin are 2.8% and 4.2%, respectively. Graphical abstractSchematic representation of SDS-capped silver nanoparticles's response to tobramycin in the presence of sodium metaborate.

Potentiation of ß-lactam antibiotics and ß-lactam/ß-lactamase inhibitor combinations against MDR and XDR Pseudomonas aeruginosa using non-ribosomal tobramycin-cyclam conjugates.

OBJECTIVES: To develop a multifunctional adjuvant molecule that can rescue ß-lactam antibiotics and ß-lactam/ß-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates. METHODS: Preparation of adjuvant was guided by structure-activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model. RESULTS: Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time-kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of ß-lactam/ß-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations. CONCLUSIONS: Non-ribosomal tobramycin-cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates ß-lactam/ß-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to ß-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of ß-lactam antibiotics.

Enhanced of antibacterial activity of antibiotic-functionalized silver nanocomposites with good biocompatibility.

Antimicrobial resistance to traditional antibiotics leads to a serious concern for medical care owing to ineffective antibiotic therapies. This study focused on the preparation of silver nanocomposites (AgNPs@Tob&PAGA) by modifying AgNPs with tobramycin (Tob) and carbohydrate polymer of poly(2-(acrylamido) glucopyranose) (PAGA). The enhanced antibacterial activities of nanocomposites against common pathogens of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were explored. The introduction of PAGA onto silver nanocomposites improved both citocompatibility and antibacterial activity. Compared with nude Tob, AgNPs@Tob&PAGA showed more fascinating antimicrobial effect against E. coli and S. aureus with about 20-fold increase in the antibacterial activity, simultaneously no detectable resistance was observed. Consequently, the silver nanocomposite as an antimicrobial agent presents promising prospects in the treatment of bacterial infections caused by antimicrobial resistant bacteria.

PEGylation of Tobramycin Improves Mucus Penetration and Antimicrobial Activity against Pseudomonas aeruginosa Biofilms in Vitro.

Pseudomonas aeruginosa is the predominant pathogen in the persistent lung infections of cystic fibrosis (CF) patients among other diseases. One of the mechanisms of resistance of P. aeruginosa infections is the formation and presence of biofilms. Previously, we demonstrated that PEGylated-tobramycin (Tob-PEG) had superior antimicrobial activity against P. aeruginosa biofilms compared to tobramycin (Tob). The goal of this study was to optimize the method of PEGylation of Tob and assess its activity in an in vitro CF-like mucus barrier biofilm model. Tob was PEGylated using three separate chemical conjugation methods and analyzed by 1H NMR. A comparison of the Tob-PEG products from the different conjugation methods showed significant differences in the reduction of biofilm proliferation after 24 h of treatment. In the CF-like mucus barrier model, Tob-PEG was significantly better than Tob in reducing P. aeruginosa proliferation after only 5 h of treatment ( p < 0.01). Finally, Tob-PEG caused a reduction in the number of surviving P. aeruginosa biofilm colonies higher than that of Tob ( p < 0.0001). We demonstrate the significantly improved antimicrobial activity of Tob-PEG against P. aeruginosa biofilms compared to Tob using two PEGylation methods. Tob-PEG had better in vitro activity compared to that of Tob against P. aeruginosa biofilms growing in a CF-like mucus barrier model.

Characterisation of 40 mg/ml and 100 mg/ml tobramycin formulations for aerosol therapy with adult mechanical ventilation.

BACKGROUND: Preservative-free tobramycin is commonly used as aerosolized therapy for ventilator associated pneumonia. The comparative delivery profile of the formulations of two different concentrations (100 mg/ml and 40 mg/ml) is unknown. This study aims to evaluate the aerosol characteristics of these tobramycin formulations in a simulated adult mechanical ventilation model. METHODS: Simulated adult mechanical ventilation set up and optimal settings were used in the study. Inhaled mass study was performed using bacterial/viral filters at the tip of the tracheal tube and in the expiratory limb of circuit. Laser diffractometer was used for characterising particle size distribution. The physicochemical characteristics of the formulations were described and nebulization characteristics compared using two airways, an endotracheal tube (ET) and a tracheostomy tube (TT). For each type of tube, three internal tube diameters were studied, 7 mm, 8 mm and 9 mm. RESULTS: The lung dose was significantly higher for 100 mg/ml solution (mean 121.3 mg vs 41.3 mg). Viscosity was different (2.11cp vs 1.58cp) for 100 mg/ml vs 40 mg/ml respectively but surface tension was similar. For tobramycin 100 mg/ml vs 40 mg/ml, the volume median diameter (2.02 vs 1.9 µm) was comparable. The fine particle fraction (98.5 vs 85.4%) was higher and geometric standard deviation (1.36 vs 1.62 µm) was significantly lower for 100 mg/ml concentration. Nebulization duration was longer for 100 mg/ml solution (16.9 vs 10.1 min). The inhaled dose percent was similar (30%) but the exhaled dose was higher for 100 mg/ml solution (18.9 vs 10.4%). The differences in results were non-significant for type of tube or size except for a small but statistically significant reduction in inhaled mass with TT compared to ET (0.06%). CONCLUSION: Aerosolized tobramycin 100 mg/ml solution delivered higher lung dose compared to tobramycin 40 mg/ml solution. Tracheal tube type or size did not influence the aerosol characteristics and delivery parameters.

Influence of Manufacturing Process Variables on the Properties of Ophthalmic Ointments of Tobramycin.

PURPOSE: The main purpose of this study was to evaluate qualitative (Q1) and quantitative (Q2) equivalent oleaginous ophthalmic ointments of tobramycin (TOB) with different physicochemical properties and identify critical process/quality attributes using various in vitro methods of characterization. METHODS: Various sources of petrolatum and TOB, and two mixing methods were employed to generate Q1/Q2 equivalent ointments. Characterization studies included content uniformity, microscopy, modulated temperature differential scanning calorimetry (MTDSC), gas chromatography-mass spectrometry (GC/MS), thermogravimetric analysis (TGA) and rheology. RESULTS: The particle size distribution of TOB influenced the content uniformity of ointments. Differences in the MTDSC endothermic and exothermic peaks of TOB suggested the presence of different polymorphic forms. GC/MS revealed variations in the composition and distribution of linear and branched hydrocarbons of petrolatums. Differences were also observed in the TGA derivative weight loss peaks demonstrating differences in the composition of petrolatum that may be the source of the observed variations in the rheological parameters of the ointments. CONCLUSIONS: Source and composition of the petrolatum played a more critical role in determining the rheological properties compared to the method of preparation. Results demonstrated the impact of the source of TOB, excipients and manufacturing processes on the quality attributes of TOB ophthalmic ointments.

Influence of Excipients on the Antimicrobial Activity of Tobramycin Against Pseudomonas aeruginosa Biofilms.

PURPOSE: It is unknown if inactive pharmaceutical ingredients influence the activity of antibiotics they are co-formulated with. Recently it was found that materials acting as carbon nutrient sources for bacteria can promote bacterial dispersion from a biofilm and/or reverse the persister state of a subpopulation of bacteria within the biofilms. Both can make bacteria more susceptible to antibiotics. Thus, the aim was to identify potential excipients to improve antibiotic activity in Pseudomonas aeruginosa biofilms. METHODS: We screened 190 potential excipients alone, and in combination with tobramycin sulfate against P. aeruginosa (strain PAO1) grown planktonically or as biofilms. After the excipient screening stage, we investigated the effect of 10 selected excipients against a more virulent strain (luminescent strain UCBPP-PA14). Temporal changes in luminescence, as an indicator of bacterial proliferation, and surviving colony forming units (CFUs) from the treated PA14 biofilms were quantified. RESULTS: Forty-eight materials tested caused a reduction of PAO1 proliferation either alone or combined with tobramycin. L-alanine (p < 0.05), D-alanine (p > 0.05), and N-acetyl-D-glucosaminitol (p > 0.05) improved the activity of tobramycin measured by PA14 luminometry. Additionally, L-alanine and succinic acid significantly reduced the survival of PA14 biofilms (p < 0.05). CONCLUSIONS: L-alanine, succinic acid, and N-acetyl-D-glucosaminitol may be useful as antibiotic adjuvants in future tobramycin anti-biofilm formulations.

In vivo efficacy of tobramycin-loaded synthetic calcium phosphate beads in a rabbit model of staphylococcal osteomyelitis.

BACKGROUND: Osteomyelitis is an acute or chronic inflammatory process of the bone following infection with pyogenic organisms like Staphylococcus aureus. Tobramycin (TOB) is a promising aminoglycoside antibiotic used to treat various bacterial infections, including S. aureus. The aim of this study was to investigate the efficacy of tobramycin-loaded calcium phosphate beads (CPB) in a rabbit osteomyelitis model. METHODS: Tobramycin (30 mg/mL) was incorporated into CPB by dipping method and the efficacy of TOB-loaded CPB was studied in a rabbit osteomyelitis model. For juxtaposition, CPB with and without TOB were prepared. Twenty-five New Zealand white rabbits were grouped (n = 5) as sham (group 1), TOB-loaded CPB without S. aureus (group 2), S. aureus only (group 3), S. aureus + CPB (group 4), and S. aureus + TOB-loaded CPB (group 5). Groups infected with S. aureus followed by CPB implantation were immediately subjected to surgery at the mid-shaft of the tibia. After 28 days post-surgery, all rabbits were euthanized and the presence or absence of chronic osteomyelitis and the extent of architectural destruction of the bone were assessed by radiology, bacteriology and histological studies. RESULTS: Tobramycin-loaded CPB group potentially inhibited the growth of S. aureus causing 3.2 to 3.4 log10 reductions in CFU/g of bone tissue compared to the controls. Untreated groups infected with S. aureus showed signs of chronic osteomyelitis with abundant bacterial growth and alterations in bone architecture. The sham group and TOB-loaded CPB group showed no evidence of bacterial growth. CONCLUSIONS: TOB-incorporated into CPB for local bone administration was proven to be more successful in increasing the efficacy of TOB in this rabbit osteomyelitis model and hence could represent a good alternative to other formulations used in the treatment of osteomyelitis.

Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms.

Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.

Altering polymerization temperature of antibiotic-laden cement can increase porosity and subsequent antibiotic elution.

PURPOSE: To examine the role of polymerization temperature on the cement porosity and antibiotic elution to optimize antibiotic release from antibiotic-laden cement (ABLC). METHODS: Elution profiles of vancomycin and tobramycin from ABLC discs prepared with low- and high-dose antibiotic dosages, cured at 8, 21, and 37 °C, and placed in phosphate buffered saline (PBS) at 37 °C were examined. Samples were collected at one, four, eight, 24, 72, 168, 336, and 1008 hours to calculate the quantity of antibiotic eluted. Porosity was determined by MicroCT analysis. RESULTS: ABLC porosity and antibiotic elution were increased up to five times the amount eluted from room temperature discs (p < 0.05). Low-dose ABLC group saw decreased but similar porosity at 8 °C and 21 °C compared to cement cured at 37 °C (p < 0.001). High-dose ABLC group porosities were all significantly different (p < 0.02). CONCLUSIONS: Altering the polymerization temperature of ABLC led to more porous constructs yielding increased antibiotic elution.

Differential Effects of Linkers on the Activity of Amphiphilic Tobramycin Antifungals.

As the threat associated with fungal infections continues to rise and the availability of antifungal drugs remains a concern, it becomes obvious that the need to bolster the antifungal armamentarium is urgent. Building from our previous findings of tobramycin (TOB) derivatives with antifungal activity, we further investigate the effects of various linkers on the biological activity of these aminoglycosides. Herein, we analyze how thioether, sulfone, triazole, amide, and ether functionalities affect the antifungal activity of alkylated TOB derivatives against 22 Candida, Cryptococcus, and Aspergillus species. We also evaluate their impact on the hemolysis of murine erythrocytes and the cytotoxicity against mammalian cell lines. While the triazole linker appears to confer optimal activity overall, all of the linkers incorporated into the TOB derivatives resulted in compounds that are very effective against the Cryptococcus neoformans species, with MIC values ranging from 0.48 to 3.9 µg/mL.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2 O and D2 O for the binary enzyme-sisomicin complex but remained the same in both solvents for the ternary enzyme-CoASH-sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Physical Characterization of Tobramycin Inhalation Powder: II. State Diagram of an Amorphous Engineered Particle Formulation.

Tobramycin Inhalation Powder (TIP) is a spray-dried engineered particle formulation used in TOBI Podhaler, a drug-device combination for treatment of cystic fibrosis (CF). A TIP particle consists of two phases: amorphous, glassy tobramycin sulfate and a gel-phase phospholipid (DSPC). The objective of this work was to characterize both the amorphous and gel phases following exposure of TIP to a broad range of RH and temperature. Because, in principle, changes in either particle morphology or the solid-state form of the drug could affect drug delivery or biopharmaceutical properties, understanding physical stability was critical to development and registration of this product. Studies included morphological assessments of particles, thermal analysis to measure the gel-to-liquid crystalline phase transition (Tm) of the phospholipid and the glass transition temperature (Tg) of tobramycin sulfate, enthalpy relaxation measurements to estimate structural relaxation times, and gravimetric vapor sorption to measure moisture sorption isotherms of TIP and its components. Collectively, these data enabled development of a state diagram for TIP-a map of the environmental conditions under which physical stability can be expected. This diagram shows that, at long-term storage conditions, TIP is at least 50 °C below the Tg of the amorphous phase and at least 40 °C below the Tm of the gel phase. Enthalpy relaxation measurements demonstrate that the characteristic structural relaxation times under these storage conditions are many orders of magnitude greater than that at Tg. These data, along with long-term physicochemical stability studies conducted during product development, demonstrate that TIP is physically stable, remaining as a mechanical solid over time scales and conditions relevant to a pharmaceutical product. This met a key design goal in the development of TIP: a room-temperature-stable formulation (3-year shelf life) that obviates the need for refrigeration for long-term storage. This has enabled development of TOBI Podhaler-an approved inhaled drug product that meaningfully reduces the treatment burden of CF patients worldwide.