Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.

Rescuing the Last-Line Polymyxins: Achievements and Challenges.

Antibiotic resistance is a major global health challenge and, worryingly, several key Gram negative pathogens can become resistant to most currently available antibiotics. Polymyxins have been revived as a last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram negative bacteria, in particular Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterales. Polymyxins were first discovered in the late 1940s but were abandoned soon after their approval in the late 1950s as a result of toxicities (e.g., nephrotoxicity) and the availability of "safer" antibiotics approved at that time. Therefore, knowledge on polymyxins had been scarce until recently, when enormous efforts have been made by several research teams around the world to elucidate the chemical, microbiological, pharmacokinetic/pharmacodynamic, and toxicological properties of polymyxins. One of the major achievements is the development of the first scientifically based dosage regimens for colistin that are crucial to ensure its safe and effective use in patients. Although the guideline has not been developed for polymyxin B, a large clinical trial is currently being conducted to optimize its clinical use. Importantly, several novel, safer polymyxin-like lipopeptides are developed to overcome the nephrotoxicity, poor efficacy against pulmonary infections, and narrow therapeutic windows of the currently used polymyxin B and colistin. This review discusses the latest achievements on polymyxins and highlights the major challenges ahead in optimizing their clinical use and discovering new-generation polymyxins. To save lives from the deadly infections caused by Gram negative "superbugs," every effort must be made to improve the clinical utility of the last-line polymyxins. SIGNIFICANCE STATEMENT: Antimicrobial resistance poses a significant threat to global health. The increasing prevalence of multidrug-resistant (MDR) bacterial infections has been highlighted by leading global health organizations and authorities. Polymyxins are a last-line defense against difficult-to-treat MDR Gram negative pathogens. Unfortunately, the pharmacological information on polymyxins was very limited until recently. This review provides a comprehensive overview on the major achievements and challenges in polymyxin pharmacology and clinical use and how the recent findings have been employed to improve clinical practice worldwide.

Liposomal Formulation Reduces Transport and Cell Uptake of Colistin in Human Lung Epithelial Calu-3 Cell and 3D Human Lung Primary Tissue Models.

Respiratory tract infections caused by multi-drug-resistant (MDR) bacteria have been a severe risk to human health. Colistin is often used to treat the MDR Gram-negative bacterial infections as a last-line therapy. Inhaled colistin can achieve a high concentration in the lung but none of aerosolized colistin products has been approved in the USA. Liposome has been reported as an advantageous formulation strategy for antibiotics due to its controlled release profile and biocompatibility. We have developed colistin liposomal formulations in our previous study. In the present study, the cellular uptake and transport of colistin in colistin liposomes were examined in two human lung epithelium in vitro models, Calu-3 monolayer and EpiAirway 3D tissue models. In both models, cellular uptake (p < 0.05) and cellular transport (p < 0.01) of colistin were significantly reduced by the colistin liposome compared to the colistin solution. Our findings indicate that inhaled colistin liposomes could be a promising treatment for extracellular bacterial lung infections caused by MDR Pseudomonas aeruginosa (P. aeruginosa).

Synchrotron-based X-ray fluorescence microscopy reveals accumulation of polymyxins in single human alveolar epithelial cells.

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity; while inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic to the infection site directly. However, polymyxin inhalation therapy has not been optimized and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with an iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 µM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± SEM, n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4 and 24 h of treatment at 10 µM, respectively. The corresponding intracellular concentrations following the treatment at 5 µM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). FADDI-096 was mainly localized throughout the cytoplasm and nuclear region over 24 h. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells and provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation safer polymyxins.

Recent Advances in Inhaled Nanoformulations of Vaccines and Therapeutics Targeting Respiratory Viral Infections.

With the rapid outbreak of respiratory viral infections, various biological (e.g. vaccines, peptides, recombinant proteins, antibodies and genes) and antiviral agents (e.g. ribavirin, palivizumab and valaciclovir) have been successfully developed for the treatment of respiratory virus infections such as influenza, respiratory syncytial virus and SARS-CoV-2 infections. These therapeutics are conventionally delivered via oral, intramuscular or injection route and are associated with several adverse events due to systemic toxicity. The inherent in vivo instability of biological therapeutics may hinder them from being administered without proper formulations. Therefore, we have witnessed a boom in nanotechnology coupled with a needle-free administration approach such as the inhalation route for the delivery of complex therapeutics to treat respiratory infections. This review discussed the recent advances in the inhalation strategies of nanoformulations that target virus respiratory infections.

Effect of Buffer Salts on Physical Stability of Lyophilized and Spray-Dried Protein Formulations Containing Bovine Serum Albumin and Trehalose.

This study examined the effect of buffer salts on the physical stability of spray-dried and lyophilized formulations of a model protein, bovine serum albumin (BSA). BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as Brunauer-Emmett-Teller (BET) surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40°C. mDSC and PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. Moreover, our results suggested that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.

Improved Aerosolization Stability of Inhalable Tobramycin Powder Formulation by Co-Spray Drying with Colistin.

PURPOSE: Tobramycin shows synergistic antibacterial activity with colistin and can reduce the toxic effects of colistin. The purpose of this study is to prepare pulmonary powder formulations containing both colistin and tobramycin and to assess their in vitro aerosol performance and storage stability. METHODS: The dry powder formulations were manufactured using a lab-scale spray dryer. In vitro aerosol performance was measured using a Next Generation Impactor. The storage stability of the dry powder formulations was measured at 22°C and two relative humidity levels - 20 and 55%. Colistin composition on the particle surface was measured using X-ray photoelectron spectroscopy. RESULTS: Two combination formulations, with 1:1 and 1:5 molar ratios of colistin and tobramycin, showed fine particle fractions (FPF) of 85%, which was significantly higher than that of the spray dried tobramycin (45%). FPF of the tobramycin formulation increased significantly when stored for four weeks at both 20% and 55% RH. In contrast, FPF values of both combination formulations and spray dried colistin remained stable at both humidity levels. Particle surface of each combination was significantly enriched in colistin molecules; 1:5 combination showed 77% by wt. colistin. CONCLUSIONS: The superior aerosol performance and aerosolization stability of 1:1 and 1:5 combination formulations of colistin and tobramycin could be attributed to enrichment of colistin on the co-spray dried particle surface. The observed powder properties may be the result of a surfactant-like assembly of these colistin molecules during spray drying, thus forming a hydrophobic particle surface.

Polymyxin-Induced Metabolic Perturbations in Human Lung Epithelial Cells.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log2 fold change [log2FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log2FC = -1.34 ± 0.64) and hypotaurine (log2FC = -1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log2FC = -1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log2FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log2FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log2FC ≤ -1); several phosphatidylserines were also increased (log2FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial ß-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Understanding the Impact of Protein-Excipient Interactions on Physical Stability of Spray-Dried Protein Solids.

Mannitol, leucine, and trehalose have been widely used in spray-dried formulations, especially for inhalation formulations. The individual contribution of these excipients on protein physical stability in spray-dried solids was studied here using bovine serum albumin (BSA) as a model protein. The spray-dried solids were characterized with scanning electron microscopy, powder X-ray diffraction, and solid-state Fourier-transform infrared spectroscopy to analyze particle morphology, crystallinity, and secondary structure change, respectively. Advanced solid-state characterizations were conducted with solid-state hydrogen-deuterium exchange (ssHDX) and solid-state nuclear magnetic resonance (ssNMR) to explore protein conformation and molecular interactions in the context of the system physical stability. Trehalose remained amorphous after spray-drying and was miscible with BSA, forming hydrogen bonds to maintain protein conformation, whereby this system showed the least monomer loss in the stability study. As indicated by ssNMR, both crystalline and amorphous forms of mannitol existed in the spray-dried BSA-mannitol solids, which explained its partial stabilizing effect on BSA. Leucine showed the strongest crystallization tendency after spray-drying and did not provide a stabilizing effect due to substantial immiscibility and phase separation with BSA as a result of crystal formation. This work showed novel applications of ssNMR in examining protein conformation and protein-excipient interaction in dry formulations. Overall, our results demonstrate the pivotal role of advanced solid-state characterization techniques in understanding the physical stability of spray-dried protein solids.

Polymyxin-Induced Cell Death of Human Macrophage-Like THP-1 and Neutrophil-Like HL-60 Cells Associated with the Activation of Apoptotic Pathways.

Innate immunity is crucial for the host to defend against infections, and understanding the effect of polymyxins on innate immunity is important for optimizing their clinical use. In this study, we investigated the potential toxicity of polymyxins on human macrophage-like THP-1 and neutrophil-like HL-60 cells. Differentiated THP-1 human macrophages (THP-1-dMs) and HL-60 human neutrophils (HL-60-dNs) were employed. Flow cytometry was used to measure the concentration-dependent effects (100 to 2,500 µM for THP-1-dMs and 5 to 2,500 µM for HL-60-dNs) and time-dependent effects (1,000 µM for THP-1-dMs and 300 µM for HL-60-dNs) of polymyxin B over 24 h. Effects of polymyxin B on mitochondrial activity, activation of caspase-3, caspase-8, and caspase-9, and Fas ligand (FasL) expression in both cell lines were examined using fluorescence imaging, colorimetric, and fluorometric assays. In both cell lines, polymyxin B induced concentration- and time-dependent loss of viability at 24 h with 50% effective concentration (EC50) values of 751.8 µM (95% confidence interval [CI], 692.1 to 816.6 µM; Hill slope, 3.09 to 5.64) for THP-1-dM cells and 175.4 µM (95% CI, 154.8 to 198.7 µM; Hill slope, 1.42 to 2.21) for HL-60-dN cells. A concentration-dependent loss of mitochondrial membrane potential and generation of mitochondrial superoxide was also observed. Polymyxin B-induced apoptosis was associated with concentration-dependent activation of all three tested caspases. The death receptor apoptotic pathway activation was demonstrated by a concentration-dependent increase of FasL expression. For the first time, our results reveal that polymyxin B induced concentration- and time-dependent cell death in human macrophage-like THP-1 and neutrophil-like HL-60 cells associated with mitochondrial and death receptor apoptotic pathways.

Understanding Dynamics of Polymorphic Conversion during the Tableting Process Using In Situ Mechanical Raman Spectroscopy.

The objective of this study is to achieve a fundamental understanding of polymorphic interconversion during the tableting process, including during compaction, dwell, decompression/unloading, and ejection using an in situ mechanical Raman spectroscopy. The fit-for-purpose in situ mechanical Raman spectroscopy developed herein can provide simultaneous measurement of Raman spectra and densification for the powder compacts. Chlorpropamide (CPA), an antidiabetic drug, was selected as a model pharmaceutical compound because of its mechanical shear-induced polymorphic conversions. The results confirm that CPA polymorph A (CPA-A) was transformed to CPA polymorph C (CPA-C) under different compaction stresses. We also observed that the converted polymorph CPA-C could be reverted to the CPA-A due to the elastic recovery of powder compacts as detected during dwelling and unloading. This study is the first depiction of the dynamics of CPA polymorphic interconversion during compression, dwell, unloading, and ejection. Mechanistically, this study illustrates a correlation between the change in the powder compact's relative density and polymorphic interconversion of the drug substance in different solid-state forms. The present research suggests that the process-induced polymorph conversion is a complicated dynamic process, which could be affected by the compaction pressure, the elasticity/plasticity of the material, the level of elastic recovery, and the dissipation of residual stress. In summary, this study demonstrates that the in situ mechanical Raman spectroscopy approach enables the simultaneous detection of mechanical and chemical information of the powder compact throughout the tableting process.

Inhalable Nanocomposite Microparticles with Enhanced Dissolution and Superior Aerosol Performance.

Previous studies have shown that combining colistin (Col), a cationic polypeptide antibiotic, with ivacaftor (Iva), a cystic fibrosis (CF) drug, could achieve synergistic antibacterial effects against Pseudomonas aeruginosa. The purpose of this study was to develop dry powder inhaler formulations for co-delivery of Col and Iva, aiming to treat CF and lung infection simultaneously. In order to improve solubility and dissolution for the water-insoluble Iva, Iva was encapsulated into bovine serum albumin (BSA) nanoparticles (Iva-BSA-NPs). Inhalable composite microparticles of Iva-BSA-NPs were produced by spray-freeze-drying using water-soluble Col as the matrix material and l-leucine as an aerosol enhancer. The optimal formulation showed an irregularly shaped morphology with fine particle fraction (FPF) values of 73.8 ± 5.2% for Col and 80.9 ± 4.1% for Iva. Correlations between "D×ρtapped" and FPF were established for both Iva and Col. The amorphous solubility of Iva is 66 times higher than the crystalline solubility in the buffer. Iva-BSA-NPs were amorphous and remained in the amorphous state after spray-freeze-drying, as examined by powder X-ray diffraction. In vitro dissolution profiles of the selected DPI formulation indicated that Col and Iva were almost completely released within 3 h, which was substantially faster regarding Iva release than the jet-milled physical mixture of the two drugs. In summary, this study developed a novel inhalable nanocomposite microparticle using a synergistic water-soluble drug as the matrix material, which achieved reduced use of excipients for high-dose medications, improved dissolution rate for the water-insoluble drug, and superior aerosol performance.

Surface Composition and Aerosolization Stability of an Inhalable Combinational Powder Formulation Spray Dried Using a Three-Fluid Nozzle.

PURPOSE: This study aims to understand the impact of spray drying nozzles on particle surface composition and aerosol stability. METHODS: The combination formulations of colistin and azithromycin were formulated by 2-fluid nozzle (2 N) or 3-fluid (3 N) spray drying in a molar ratio of 1:1. A 3-factor, 2-level (23) factorial design was selected to investigate effects of flow rate, inlet temperature and feed concentration on yield of spray drying and the performance of the spray dried formulations for the 3 N. RESULTS: FPF values for the 2 N formulation (72.9 ± 1.9% for azithromycin & 73.4 ± 0.8% for colistin) were higher than those for the 3 N formulation (56.5 ± 3.8% for azithromycin & 55.1 ± 1.6% for colistin) when stored at 20% RH for 1 day, which could be attributed to smaller physical size for the 2 N. There was no change in FPF for both drugs in the 2 N formulation after storage at 75% RH for 90 days; however, there was a slight increase in FPF for colistin in the 3 N formulation at the same storage conditions. Surface enrichment of hydrophobic azithromycin was measured by X-ray photoelectron spectroscopy for both 2 N and 3 N formulations and interactions were studied using FTIR. CONCLUSIONS: The 3-fluid nozzle provides flexibility in choosing different solvents and has the capability to spray dry at higher feed solid concentrations. This study highlights the impact of hydrophobic azithromycin enrichment on particle surface irrespective of the nozzle type, on the prevention of moisture-induced deterioration of FPF for hygroscopic colistin.

Dry powder aerosol containing muco-inert particles for excipient enhanced growth pulmonary drug delivery.

Tenacious sputum poses a critical diffusion barrier for aerosol antibiotics used to treat cystic fibrosis (CF) lung infection. We conducted a proof-of-concept study using dense poly(ethylene glycol) coated polystyrene nanoparticles (PS-PEG NPs) as model muco-inert particles (MIPs) formulated as a powder using an excipient enhanced growth (EEG) strategy, aiming to minimize extrathoracic airway loss, maximize deposition in the airway and further overcome the sputum barrier in the CF lungs. The EEG aerosol formulation containing PS-PEG MIPs was prepared by spray drying and produced discrete spherical particles with geometric diameter of approximately 2 µm; and >80% of the powder dose was delivered from a new small-animal dry powder inhaler (DPI). The MIPs released from the EEG aerosol had human airway mucus and CF sputum diffusion properties comparable to the suspension formulation. These properties make this formulation a promising pulmonary drug delivery system for CF lung infections.

Novel Polymyxin Combination with the Antiretroviral Zidovudine Exerts Synergistic Killing against NDM-Producing Multidrug-Resistant Klebsiella pneumoniae.

Polymyxins are used as a last-line therapy against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae However, polymyxin resistance can emerge with monotherapy; therefore, novel strategies are urgently needed to minimize the resistance and maintain their clinical utility. This study aimed to investigate the pharmacodynamics of polymyxin B in combination with the antiretroviral drug zidovudine against K. pneumoniae Three isolates were evaluated in static time-kill studies (0 to 64 mg/liter) over 48 h. An in vitro one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model (IVM) was used to simulate humanized dosage regimens of polymyxin B (4 mg/liter as continuous infusion) and zidovudine (as bolus dose thrice daily to achieve maximum concentration of drug in broth [Cmax] of 6 mg/liter) against K. pneumoniae BM1 over 72 h. The antimicrobial synergy of the combination was further evaluated in a murine thigh infection model against K. pneumoniae 02. In the static time-kill studies, polymyxin B monotherapy produced rapid and extensive killing against all three isolates followed by extensive regrowth, whereas zidovudine produced modest killing followed by significant regrowth at 24 h. Polymyxin B in combination with zidovudine significantly enhanced the antimicrobial activity (≥4 log10 CFU/ml) and minimized bacterial regrowth. In the IVM, the combination was synergistic and the total bacterial loads were below the limit of detection for up to 72 h. In the murine thigh infection model, the bacterial burden at 24 h in the combination group was ≥3 log10 CFU/thigh lower than each monotherapy against K. pneumoniae 02. Overall, the polymyxin B-zidovudine combination demonstrates superior antimicrobial efficacy and minimized emergence of resistance to polymyxins.

Intracellular localization of polymyxins in human alveolar epithelial cells.

Background: Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. Objectives: To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. Methods: A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Results: Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells. Conclusions: Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.

Surface Composition and Formulation Heterogeneity of Protein Solids Produced by Spray Drying.

PURPOSE: The aim of this study is to determine the effects of saccharide-containing excipients on the surface composition of spray-dried protein formulations and their matrix heterogeneity. METHODS: Spray-dried formulations of myoglobin or bovine serum albumin (BSA) were prepared without excipient or with sucrose, trehalose, or dextrans. Samples were characterized by solid-state Fourier-transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while conformational differences were determined by solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). RESULTS: Structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextran-containing formulations. Using ssHDX-MS, the amount of deuterium incorporation and populations present were the largest in the samples processed with dextrans. Linear correlation was found between protein surface coverage and ssHDX-MS peak area (R2 = 0.853) for all formulations with saccharide-containing excipients. CONCLUSIONS: Lower molecular weight species of saccharides tend to enrich the particle surface and reduce protein concentration at the air-liquid interface, resulting in reduced population heterogeneity and improved physical stability, as identified by ssHDX-MS.

Pharmacokinetics of salvianolic acid B, rosmarinic acid and Danshensu in rat after pulmonary administration of Salvia miltiorrhiza polyphenolic acid solution.

A sensitive and accurate LC-MS/MS method was established for quantifying salvianolic acid B (Sal B), rosmarinic acid (Ros A) and Danshensu (DA) in rat plasma. Salvia miltiorrhiza polyphenolic acid (SMPA), active water-soluble ingredients isolated and purified from Salvia miltiorrhiza Bge included Sal B, Ros A and DA. The pharmacokinetic analysis of Sal B, Ros A and DA after pulmonary administration of SMPA solution to rat was performed by LC-MS/MS. Results from the pharmacokinetic studies showed that the peak concentration of DA was 21.85 ± 6.43 and 65.39 ± 3.83 ng/mL after pulmonary and intravenous administration, respectively. DA was not detected at 2 h after administration. The absolute bioavailabilities of Sal B and Ros A were respectively 50.37 ± 27.04 and 89.63 ± 12.16% after pulmonary administration of 10 mg/kg SMPA solution in rats. The absolute bioavailability of Sal B increased at least 10-fold after pulmonary administration, compared with oral administration. It was concluded that the newly established LC-MS/MS method was suitable for describing the pharmacokinetic characteristics of Sal B, Ros A and DA in rat after pulmonary administration of SMPA solution. The data from this study will provide a preclinical insight into the feasibility of pulmonary administration of SMPA.

A Proof-of-Concept Study of the Efficacy of Systemically Administered Polymyxins in Mouse Burn Wound Infection Caused by Multidrug-Resistant Gram-Negative Pathogens.

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.