Alginates are a family of linear anionic polysaccharides produced by brown algae and some bacteria and consist of 1 → 4 linked β-d-mannuronic acid (M), and its C5-epimer α-l-guluronic acid (G). Research has shown that oligoguluronates possess biofilm disruption properties, which combined with their high water-solubility and low toxicity, represent ideal candidates for creating novel bifunctional antifungal compounds. The progressive increase in antifungal resistance is a major global human health threat. This intrinsic resistance illustrates the need to develop improved antifungal drugs with greater efficacy against antifungal growth, while also reducing the potential for drug toxicity, resistance and tolerance. Amphotericin B is a polyene macrolide antifungal with broad specificity but has poor water solubility and is limited by infusion-related reactions and nephrotoxicity. In this work, we developed two types of Alginate-Amphotericin B conjugates with low molecular weight oligoguluronates conjugated through the reducing end. Conjugation to amphotericin B was through the carboxylic acid group or the amine group. The Alginate-Amphotericin B conjugates have reduced toxicity towards liver cells (HepG2) and kidney cells (LLC-PK1) and highly improved solubility characteristics, while maintaining anti-fungal activity.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Airway mucus is a major barrier to the delivery of lipid-based nanoparticles in chronic airway diseases such as cystic fibrosis (CF). Receptor-Targeted Nanocomplexes (RTN), comprise mixtures of cationic lipids and bifunctional peptides with receptor-targeting and nucleic acid packaging properties. The aim of this study is to improve the mucus-penetrating properties of cationic siRNA and mRNA RTNs by combining them with low molecular weight alginate oligosaccharides, OligoG and OligoM. Cationic RTNs formulated with either alginate become strongly anionic, while PEGylated messenger RNA (mRNA) and short interfering RNA (siRNA) RTNs remain cationic. Both alginates enhance mucus diffusion rates of cationic siRNA and mRNA RTNs in a static mucus barrier diffusion model, with OligoG particularly effective. PEGylation also enhance mucus diffusion rates of siRNA RTNs but not mRNA RTNs. Electron microscopy shows that RTNs remained intact after mucosal transit. The transfection efficiency of OligoM-coated mRNA RTNs is better than those coated with OligoG or PEG, and similar to cationic RTNs. In siRNA RTN transfections, OligoM is better than OligoG although 1% PEG is slightly better than both. The combination of cationic RTNs and alginate oligosaccharides represents a promising alternative to PEGylation for epithelial delivery of genetic therapies across the mucus barrier while retaining transfection efficiency.

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)–DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Background The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

OligoG has previously shown potentiation of aztreonam against Burkholderia cepacia complex (Bcc) through biofilm disruption. A randomized, double-blind, placebo-controlled cross-over design was used to evaluate safety and efficacy of inhaled OligoG as a therapy for Bcc-infected CF patients taking aztreonam. Subjects received OligoG (1050 mg daily) or matching placebo for 28-days. Of 14 subjects completing the study, 8 showed a mean decrease in total bacterial CFU's (0.82 log10) after OligoG treatment. There was a reduction in mean Bcc CFU's (2.19 log10) after OligoG treatment but this was not statistically signifi-cant. Rheology analysis showed improvements in phase-angle after OligoG, but there was no statistically significant improvement in lung function parameters. Six out of 12 QoL summary scores showed relative improvement after OligoG treatment compared to placebo. There was a favourable safety profile for Oli-goG. Potential for reducing Bcc warrants further investigation of OligoG for the treatment of infection in CF.(c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing ( p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers . The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure.

Culture-independent microbiota analysis is widely used in research and being increasingly used in translational studies. However, methods for standardisation and application of these analyses in clinical trials are limited. Here we report the microbiota analysis that accompanied two phase 2b clinical trials of the novel, non-antibiotic therapy OligoG CF-5/20 for cystic fibrosis (CF) lung infection. Standardised protocols (DNA extraction, PCR, qPCR and 16S rRNA gene sequencing analysis) were developed for application to the Pseudomonas aeruginosa (NCT02157922) and Burkholderia cepacia complex (NCT02453789) clinical trials involving 45 and 13 adult trial participants, respectively. Microbiota analysis identified that paired sputum samples from an individual participant, taken within 2 h of each other, had reproducible bacterial diversity profiles. Although culture microbiology had identified patients as either colonised by P. aeruginosa or B. cepacia complex species at recruitment, microbiota analysis revealed patient lung infection communities were not always dominated by these key CF pathogens. Microbiota profiles were patient-specific and remained stable over the course of both clinical trials (6 sampling points over the course of 140 days). Within the Burkholderia trial, participants were infected with B. cenocepacia (n = 4), B. multivorans (n = 6), or an undetermined species (n = 3). Colonisation with either B. cenocepacia or B. multivorans influenced the overall bacterial community structure in sputum. Overall, we have shown that sputum microbiota in adults with CF is stable over a 2 h time-frame, suggesting collection of a single sample on a collection day is sufficient to capture the microbiota diversity. Despite the uniform pathogen culture-positivity status at recruitment, trial participants were highly heterogeneous in their lung microbiota. Understanding the microbiota profiles of individuals with CF ahead of future clinical trials would be beneficial in the context of patient stratification and trial design.

The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients.

Background: OligoG is a low molecular-weight alginate oligosaccharide that improves the viscoelastic properties of cystic fibrosis (CF) mucus and disrupts biofilms, thereby potentiating the activity of antimicrobial agents. The efficacy of inhaled OligoG was evaluated in adult patients with CF. Methods: A randomised, double-blind, placebo-controlled multicentre crossover study was used to demonstrate safety and efficacy of inhaled dry powder OligoG. Subjects were randomly allocated to receive OligoG 1050 mg per day (10 capsules three times daily) or matching placebo for 28 days, with 28-day washout periods following each treatment period. The primary end-point was absolute change in percentage predicted forced expiratory volume in 1 s (FEV1) at the end of 28-day treatment. The intention-to-treat (ITT) population (n=65) was defined as randomised to treatment with at least one administration of study medication and post-dosing evaluation. Results: In this study, 90 adult subjects were screened and 65 were randomised. Statistically significant improvement in FEV1 was not observed in the ITT population. Adverse events included nasopharyngitis, cough and pulmonary exacerbation. The number and proportions of patients with adverse events and serious adverse events were similar between OligoG and placebo group. Conclusions: Inhalation of OligoG-dry powder over 28 days was safe in adult CF subjects. Statistically significant improvement of FEV1 was not reached. The planned analyses did not indicate a significant treatment benefit with OligoG compared to placebo. Post hoc exploratory analyses showed subgroup results that indicate that further studies of OligoG in this patient population are justified.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide (“OligoG”)–polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG–polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200–12,800 g/mol and antibiotic loading of 6.1–12.9% w/w, were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2–9.3-fold) and polymyxin B (2.9–27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG–polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2–4-fold). Both OligoG–colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro “time-to-kill” (TTK) model using Acinetobacter baumannii, only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG–polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Cystic fibrosis (CF) is an autosomal recessive, life-limiting condition characterized by progressive lung disease, which is a major cause of morbidity and mortality for these patients. The inhalation therapy, OligoG CF-5/20, is a low molecular weight (mean Mn 3200 g/mol) alginate oligomer, with a high guluronic acid content (>85%). The ability of OligoG CF-5/20 to enhance the activity of antimicrobial/antibiotic therapies, modify the rheological properties of CF sputum and interact with mucin, has previously been shown. To further characterize the physicochemical interactions of OligoG CF-5/20 with CF sputum, Fourier-transform infrared (FTIR) spectroscopy was used to analyze ex vivo sputum samples from adolescent CF patients (n = 13) following treatment with 0.2% OligoG CF-5/20. FTIR analysis confirmed the interaction of OligoG CF-5/20 with mucin glycans in CF sputum, which showed a shift in wavenumber from 1078 cm(-1) to 1070 cm(-1) and subsequent loss of the 1053 cm(-1) peak in the OligoG CF-5/20 treated samples. OligoG CF-5/20 interaction with key terminal moieties in mucin were also evident, with a significant change in sulphation at wavenumber 1116 cm(-1), suggesting a link with sulphated Lewis x antigen. There were also significant shifts at wavenumber 1637 cm(-1) indicative of beta-sheet conformational changes in the mucin peptide caused by action of OligoG. The alterations in charge of glycan and mucin structures support previous observations wherein OligoG CF-5/20 modifies the viscoelastic properties of CF sputum. These findings suggest a possible mechanism of action for the rheological changes observed with this novel therapy.

Cellulose nanofibrils (CNFs) from wood pulp are a renewable material possessing advantages for biomedical applications because of their customizable porosity, mechanical strength, translucency, and environmental biodegradability. Here, we investigated the growth of multispecies wound biofilms on CNF formulated as aerogels and films incorporating the low-molecular-weight alginate oligosaccharide OligoG CF-5/20 to evaluate their structural and antimicrobial properties. Overnight microbial cultures were adjusted to 2.8 × 109 colony-forming units (cfu) mL–1 in Mueller Hinton broth and growth rates of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 1061A monitored for 24 h in CNF dispersions sterilized by γ-irradiation. Two CNF formulations were prepared (20 g m–2) with CNF as air-dried films or freeze-dried aerogels, with or without incorporation of an antimicrobial alginate oligosaccharide (OligoG CF-5/20) as a surface coating or bionanocomposite, respectively. The materials were structurally characterized by scanning electron microscopy (SEM) and laser profilometry (LP). The antimicrobial properties of the formulations were assessed using single- and mixed-species biofilms grown on the materials and analyzed using LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) and COMSTAT software. OligoG–CNF suspensions significantly decreased the growth of both bacterial strains at OligoG concentrations >2.58% (P < 0.05). SEM showed that aerogel–OligoG bionanocomposite formulations had a more open three-dimensional structure, whereas LP showed that film formulations coated with OligoG were significantly smoother than untreated films or films incorporating PEG400 as a plasticizer (P < 0.05). CLSM of biofilms grown on films incorporating OligoG demonstrated altered biofilm architecture, with reduced biomass and decreased cell viability. The OligoG–CNF formulations as aerogels or films both inhibited pyocyanin production (P < 0.05). These novel CNF formulations or bionanocomposites were able to modify bacterial growth, biofilm development, and virulence factor production in vitro. These data support the potential of OligoG and CNF bionanocomposites for use in biomedical applications where prevention of infection or biofilm growth is required.

and 5.9% respectively.For BI 1265162, application of 0.01 mg/kg caused maximum inhibition of 32.9%.Doses of 0.003, 0.0003 and 0.00003 mg/kg showed an inhibition of 31.3, 26.9, 26.2 and 5.9% respectively.For comparison, with 3 mg/kg amiloride, a mean of only 15% inhibition was achieved.Calculated with the GraphPad Prism software package, BI 443651 and BI 1265162 reduced the fluid absorption from rat lungs with an ED 50 of 0.6 μg/kg or 0.09 μg/kg respectively.Conclusion: Both BI 443651 and BI 1265162 showed inhibition of fluid absorption from rat lungs.Therefore, ENaC inhibition might be a good target for clinical studies in CF.

Alginate oligomers retain most of the chemical and physical properties of the higher molecular weight commercial alginates, retaining affinity towards monovalent and divalent ions, which is dependent on the chemical composition of the oligomer. However, due to their low molecular weight, they will normally not form gels in the presence of divalent cations. This property is exploited in biological systems to chelate multivalent ions and disrupt Ca2+-mediated cross-linking. Studies have also identified interactions between alginate oligomers and complex mucin polymer systems, bacteria and extracellular polymeric substance (EPS), which suggests that these interactions are not simply the result of cationic chelation. By virtue of their low molecular weight, alginate oligomers stay in solution at high concentration without significant increase in viscosity and can be tailor-made to precisely defined chemical composition and molecular weight. This affords the opportunity to design effective formulations with precisely defined properties and biological effects. The properties now being identified for alginate oligomers represent a promising new approach in the management of chronic lung diseases, biofilm infections and antibiotic use. This chapter outlines the research performed to date, highlighting the excellent safety profile and novel chemical characteristics of alginate oligomers that emphasize their potential in multiple therapeutic applications.

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol −1 ) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca 2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height ( P < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA ( P < 0.05) with a corresponding increase in nanoparticle diffusion ( P < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca 2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca 2+ -DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections.

diagnosis and other quality measures.