Understanding the impact of mannitol on physical stability and aerosolization of spray-dried protein powders for inhalation.

Pulmonary delivery of protein-based therapeutics, including antibodies, is a promising option for treating respiratory diseases. Spray drying is a widely used method for producing dry powder formulations with mannitol being a commonly used excipient for these inhalation formulations. There is limited research available concerning the utilization of mannitol as an excipient in the spray drying of proteins and its impact on aerosol performance. This study highlights the importance to understand mannitol's potential role and impact in this context. To investigate the impact of mannitol on physical stability and aerosolization of spray-dried protein formulations, bovine serum albumin (BSA) was employed as a model protein and formulated with different concentrations of mannitol via spray drying. The spray-dried solids were characterized for their particle size using Malvern mastersizer and aerodynamic particle size using next generation impactor (NGI). Additionally, the solids were characterized with solid-state Fourier-transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) and solid-state nuclear magnetic resonance spectroscopy (ssNMR) to analyze the change in their secondary structure, crystallinity, particle morphology, and protein-excipient interaction, respectively. Size exclusion chromatography (SEC) was used to investigate changes in monomer content resulting from storage under stressed condition of 40 °C. Protein formulations containing more than 33 % mannitol by weight showed crystallization tendencies, causing an increase in monomer loss over time. ssNMR data also showed mixing heterogeneity of BSA and mannitol in the formulations with high mannitol contents. Futhermore, fine particle fraction (FPF) was found to decrease over time for the formulations containing BSA: Mannitol in the ratios of 2:1, 1:2, and 1:5, due to particle agglomeration induced by crystallization of mannitol. This study underscores the significant influence of excipients such as mannitol on the aerosol performance and storage stability of spray-dried protein formulations.

Spray dried inhalable ivacaftor co-amorphous microparticle formulations with leucine achieved enhanced in vitro dissolution and superior aerosol performance.

The present study aimed to develop inhalable powder formulations with both dissolution enhancement and superior aerodynamic properties for potential pulmonary delivery of a poorly water-soluble drug, ivacaftor (IVA). The IVA-leucine (LEU) microparticle formulations were produced by spray drying and the physicochemical, aerosolization and cytotoxicity properties were characterized. Co-amorphous microparticle formulation was formed at the IVA: LEU 3:1 M ratio with hydrogen bond interactions as indicated by Fourier transform infrared spectroscopy (FTIR) results. Dissolution rate of the co-spray dried formulations was significantly improved as compared with the IVA alone or physical mixtures. The co-spray dried formulations exhibited > 80% fine particle fraction (FPF) and > 95% emitted dose percentage (ED) values respectively, with superior physical and aerosolization stability under 40℃ at 75% RH for 30 days. The laser scanning confocal microscopy results demonstrated that more IVA was uptake by Calu-3 cell lines for the co-spray dried formulation. In summary, our results demonstrated that co-spray drying IVA with LEU could achieve enhanced in vitro release and superior aerodynamic properties for pulmonary delivery of IVA.

Pharmacokinetics and pharmacodynamics of peptide antibiotics.

Antimicrobial resistance is a major global health challenge. As few new efficacious antibiotics will become available in the near future, peptide antibiotics continue to be major therapeutic options for treating infections caused by multidrug-resistant pathogens. Rational use of antibiotics requires optimisation of the pharmacokinetics and pharmacodynamics for the treatment of different types of infections. Toxicodynamics must also be considered to improve the safety of antibiotic use and, where appropriate, to guide therapeutic drug monitoring. This review focuses on the pharmacokinetics/pharmacodynamics/toxicodynamics of peptide antibiotics against multidrug-resistant Gram-negative and Gram-positive pathogens. Optimising antibiotic exposure at the infection site is essential for improving their efficacy and minimising emergence of resistance.

In vitro evaluation of drug delivery behavior for inhalable amorphous nanoparticle formulations in a human lung epithelial cell model.

Respiratory tract infections caused by multidrug-resistant (MDR) Gram-negative bacteria such as Pseudomonas aeruginosa are serious burdens to public health, especially in cystic fibrosis patients. The combination of colistin, a cationic polypeptide antibiotic, and ivacaftor, a cystic fibrosis transmembrane regulator (CFTR) protein modulator, displays a synergistic antibacterial effect against P. aeruginosa. The primary aim of the present study is to investigate the transport, accumulation and toxicity of a novel nanoparticle formulation containing colistin and ivacaftor in lung epithelial Calu-3 cells. The cell viability results demonstrated that ivacaftor alone or in combination with colistin in the physical mixture showed significant toxicity at an ivacaftor concentration of 10 µg/mL or higher. However, the cellular toxicity was significantly reduced in the nanoparticle formulation. Ivacaftor transport into the cells reached a plateau rapidly as compared to colistin. Colistin transport across the Calu-3 cell monolayer was less than ivacaftor. A substantial amount (46-83%) of ivacaftor, independent of dose, was accumulated in the cell monolayer following transport from the apical into the basal chamber, whereas the intracellular accumulation of colistin was relatively low (2-15%). The nanoparticle formulation significantly reduced the toxicity of colistin and ivacaftor to Calu-3 cells by reducing the accumulation of both drugs in the cell and potential protective effects by bovine serum albumin (BSA), which could be a promising safer option for the treatment of respiratory infections caused by MDR P. aeruginosa.

Pharmaceutical amorphous solid dispersion: A review of manufacturing strategies.

Amorphous solid dispersions (ASDs) are popular for enhancing the solubility and bioavailability of poorly water-soluble drugs. Various approaches have been employed to produce ASDs and novel techniques are emerging. This review provides an updated overview of manufacturing techniques for preparing ASDs. As physical stability is a critical quality attribute for ASD, the impact of formulation, equipment, and process variables, together with the downstream processing on physical stability of ASDs have been discussed. Selection strategies are proposed to identify suitable manufacturing methods, which may aid in the development of ASDs with satisfactory physical stability.

Comparative metabolomics reveals key pathways associated with the synergistic activity of polymyxin B and rifampicin combination against multidrug-resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii presents a critical challenge to human health worldwide and polymyxins are increasingly used as a last-line therapy. Due to the rapid emergence of resistance during polymyxin monotherapy, synergistic combinations (e.g. with rifampicin) are recommended to treat A. baumannii infections. However, most combination therapies are empirical, owing to a dearth of understanding on the mechanism of synergistic antibacterial killing. In the present study, we employed metabolomics to investigate the synergy mechanism of polymyxin B-rifampicin against A. baumannii AB5075, an MDR clinical isolate. The metabolomes of A. baumannii AB5075 were compared at 1 and 4 h following treatments with polymyxin B alone (0.75 mg/L, i.e. 3 × MIC), rifampicin alone (1 mg/L, i.e. 0.25 × MIC) and their combination. Polymyxin B monotherapy significantly perturbed glycerophospholipid and fatty acid metabolism at 1 h, reflecting its activity on bacterial outer membrane. Rifampicin monotherapy significantly perturbed glycerophospholipid, nucleotide and amino acid metabolism, which are related to the inhibition of RNA synthesis. The combination treatment significantly perturbed the metabolism of nucleotides, amino acids, fatty acids and glycerophospholipids at 1 and 4 h. Notably, the intermediate metabolite pools from pentose phosphate pathway were exclusively enhanced by the combination, while most metabolites from the nucleotide and amino acid biosynthesis pathways were significantly decreased. Overall, the synergistic activity of the combination was initially driven by polymyxin B which impacted pathways associated with outer membrane biogenesis; and subsequent effects were mainly attributed to rifampicin via the inhibition of RNA synthesis. This study is the first to reveal the synergistic killing mechanism of polymyxin-rifampicin combination against polymyxin-susceptible MDR A. baumannii at the network level. Our findings provide new mechanistic insights for optimizing this synergistic combination in patients.

Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens.

The status quo for combating uprising antibacterial resistance is to employ synergistic combinations of antibiotics. Nevertheless, the currently available combination therapies are fast becoming untenable. Combining antibiotics with various FDA-approved non-antibiotic drugs has emerged as a novel strategy against otherwise untreatable multi-drug resistant (MDR) pathogens. The apex of this study was to investigate the mechanisms of antibacterial synergy of the combination of polymyxin B with the phenothiazines against the MDR Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. The synergistic antibacterial effects were tested using checkerboard and static time-kill assays. Electron microscopy (EM) and untargeted metabolomics were used to ascertain the mechanism(s) of the antibacterial synergy. The combination of polymyxin B and the phenothiazines showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. EM revealed that the polymyxin B-prochlorperazine combination resulted in greater damage to the bacterial cell compared to each drug monotherapy. In metabolomics, at 0.5 h, polymyxin B monotherapy and the combination (to a greatest extent) disorganised the bacterial cell envelope as manifested by a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan and lipopolysaccharide (LPS) biosynthesis. At the late time exposure (4 h), the aforementioned effects (except LPS biosynthesis) perpetuated mainly with the combination therapy, indicating the disorganising bacterial membrane biogenesis is potentially behind the mechanisms of antibacterial synergy. In conclusion, the study highlights the potential usefulness of the combination of polymyxin B with phenothiazines for the treatment of polymyxin-resistant Gram-negative infections (e.g. CNS infections).

An "Unlikely" Pair: The Antimicrobial Synergy of Polymyxin B in Combination with the Cystic Fibrosis Transmembrane Conductance Regulator Drugs KALYDECO and ORKAMBI.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type IIα topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.