Cystic Fibrosis Mucus Model to Design More Efficient Drug Therapies.

Mucus represents a strong barrier to tackle for oral or pulmonary administered drugs, especially in mucus-related disorders. This study uses a pathological cystic fibrosis (CF) mucus model to investigate how mucus impacts the passive diffusion of 45 ad hoc commercial drugs selected to maximize physicochemical variability. An in vitro mucosal surface was recreated by coupling the mucus model to a 96-well permeable support precoated with structured layers of phospholipids (parallel artificial membrane permeability assay, PAMPA). Results show that the mucus model was not a mere physical barrier but it behaves like an interactive filter. In nearly one-half of the investigated compounds, the diffusion was reduced by mucus, while other drugs were not sensitive to the mucus barriers. We also found that permeability can be enhanced when drug-calcium salts are formed. This was confirmed with cystic fibrosis sputum as a rough ex vivo model of CF mucus. Since the drug discovery process is characterized by a high rate of failure, the mucus platform is expected to provide an efficient support to early reduce the number of poor-performing drug candidates.

Thiolated polyglycerol sulfate as potential mucolytic for muco-obstructive lung diseases.

Increased disulfide crosslinking of secreted mucins causes elevated viscoelasticity of mucus and is a key determinant of mucus dysfunction in patients with cystic fibrosis (CF) and other muco-obstructive lung diseases. In this study, we describe the synthesis of a novel thiol-containing, sulfated dendritic polyglycerol (dPGS-SH), designed to chemically reduce these abnormal crosslinks, which we demonstrate with mucolytic activity assays in sputum from patients with CF. This mucolytic polymer, which is based on a reportedly anti-inflammatory polysulfate scaffold, additionally carries multiple thiol groups for mucolytic activity and can be produced on a gram-scale. After a physicochemical compound characterization, we compare the mucolytic activity of dPGS-SH to the clinically approved N-acetylcysteine (NAC) using western blot studies and investigate the effect of dPGS-SH on the viscoelastic properties of sputum samples from CF patients by oscillatory rheology. We show that dPGS-SH is more effective than NAC in reducing multimer intensity of the secreted mucins MUC5B and MUC5AC and demonstrate significant mucolytic activity by rheology. In addition, we provide data for dPGS-SH demonstrating a high compound stability, low cytotoxicity, and superior reaction kinetics over NAC at different pH levels. Our data support further development of the novel reducing polymer system dPGS-SH as a potential mucolytic to improve mucus function and clearance in patients with CF as well as other muco-obstructive lung diseases.

Squaraine Dyes as Fluorescent Turn-on Probes for Mucins: A Step Toward Selectivity†.

Mucins are a family of long polymeric glycoproteins which can be overexpressed in several types of cancers, and over recent years, great attention was addressed to identify mucins as an important biomarker of adverse prognosis. Fluorometric detection mediated by fluorescent probes could represent a winning strategy in the early diagnosis of different pathologies. Among promising biological fluorescent probes, squaraines are gaining particular attention, thanks to their sharp and intense absorption and emission in the NIR region. In this contribution, three squaraine dyes bearing different substituents and with different lipophilicity have been investigated for their ability to detect mucin. The turn-on response upon the addition of mucin has been investigated by means of absorbance and fluorescence spectroscopy. After a preliminary screening, the squaraine (S6) bearing bromine as a substituent and C4 aliphatic chains showed the highest fluorescence turn-on and highest affinity for mucin than albumin. To further highlight the selectivity of S6 for mucin, the fluorescence response has been evaluated in the presence of serum and site-specific proteins different than albumin. Absorption spectroscopy was used to characterize the binding mechanism of squaraine to mucin.

Permeability Assessment of a High-Throughput Mucosal Platform.

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling.

Commercially Available Cell-Free Permeability Tests for Industrial Drug Development: Increased Sustainability through Reduction of In Vivo Studies.

Replacing in vivo with in vitro studies can increase sustainability in the development of medicines. This principle has already been applied in the biowaiver approach based on the biopharmaceutical classification system, BCS. A biowaiver is a regulatory process in which a drug is approved based on evidence of in vitro equivalence, i.e., a dissolution test, rather than on in vivo bioequivalence. Currently biowaivers can only be granted for highly water-soluble drugs, i.e., BCS class I/III drugs. When evaluating poorly soluble drugs, i.e., BCS class II/IV drugs, in vitro dissolution testing has proved to be inadequate for predicting in vivo drug performance due to the lack of permeability interpretation. The aim of this review was to provide solid proofs that at least two commercially available cell-free in vitro assays, namely, the parallel artificial membrane permeability assay, PAMPA, and the PermeaPad® assay, PermeaPad, in different formats and set-ups, have the potential to reduce and replace in vivo testing to some extent, thus increasing sustainability in drug development. Based on the literature review presented here, we suggest that these assays should be implemented as alternatives to (1) more energy-intense in vitro methods, e.g., refining/replacing cell-based permeability assays, and (2) in vivo studies, e.g., reducing the number of pharmacokinetic studies conducted on animals and humans. For this to happen, a new and modern legislative framework for drug approval is required.

Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection.

Pseudomonas aeruginosa (PA) is a highly, if not the most, versatile microorganism capable of colonizing diverse environments. One of the niches in which PA is able to thrive is the lung of cystic fibrosis (CF) patients. Due to a genetic aberration, the lungs of CF-affected patients exhibit impaired functions, rendering them highly susceptible to bacterial colonization. Once PA attaches to the epithelial surface and transitions to a mucoid phenotype, the infection becomes chronic, and antibiotic treatments become inefficient. Due to the high number of affected people and the severity of this infection, CF-chronic infection is a well-documented disease. Still, numerous aspects of PA CF infection remain unclear. The scientific reports published over the last decades have stressed how PA can adapt to CF microenvironmental conditions and how its surrounding matrix of extracellular polymeric substances (EPS) plays a key role in its pathogenicity. In this context, it is of paramount interest to present the nature of the EPS together with the local CF-biofilm microenvironment. We review how the PA biofilm microenvironment interacts with drugs to contribute to the pathogenicity of CF-lung infection. Understanding why so many drugs are inefficient in treating CF chronic infection while effectively treating planktonic PA is essential to devising better therapeutic targets and drug formulations.

Synthetic Mucin Gels with Self-Healing Properties Augment Lubricity and Inhibit HIV-1 and HSV-2 Transmission.

Mucus is a self-healing gel that lubricates the moist epithelium and provides protection against viruses by binding to viruses smaller than the gel's mesh size and removing them from the mucosal surface by active mucus turnover. As the primary nonaqueous components of mucus (≈0.2%-5%, wt/v), mucins are critical to this function because the dense arrangement of mucin glycans allows multivalence of binding. Following nature's example, bovine submaxillary mucins (BSMs) are assembled into "mucus-like" gels (5%, wt/v) by dynamic covalent crosslinking reactions. The gels exhibit transient liquefaction under high shear strain and immediate self-healing behavior. This study shows that these material properties are essential to provide lubricity. The gels efficiently reduce human immunodeficiency virus type 1 (HIV-1) and genital herpes virus type 2 (HSV-2) infectivity for various types of cells. In contrast, simple mucin solutions, which lack the structural makeup, inhibit HIV-1 significantly less and do not inhibit HSV-2. Mechanistically, the prophylaxis of HIV-1 infection by BSM gels is found to be that the gels trap HIV-1 by binding to the envelope glycoprotein gp120 and suppress cytokine production during viral exposure. Therefore, the authors believe the gels are promising for further development as personal lubricants that can limit viral transmission.

Mucosomes: Intrinsically Mucoadhesive Glycosylated Mucin Nanoparticles as Multi-Drug Delivery Platform.

Mucus is a complex barrier for pharmacological treatments and overcoming it is one of the major challenges faced during transmucosal drug delivery. To tackle this issue, a novel class of glycosylated nanoparticles, named "mucosomes," which are based on the most important protein constituting mucus, the mucin, is introduced. Mucosomes are designed to improve drug absorption and residence time on the mucosal tissues. Mucosomes are produced (150-300 nm), functionalized with glycans, and loaded with the desired drug in a single one-pot synthetic process and, with this method, a wide range of small and macro molecules can be loaded with different physicochemical properties. Various in vitro models are used to test the mucoadhesive properties of mucosomes. The presence of functional glycans is indicated by the interaction with lectins. Mucosomes are proven to be storable at 4 °C after lyophilization, and administration through a nasal spray does not modify the morphology of the mucosomes. In vitro and in vivo tests indicate mucosomes do not induce adverse effects under the investigated conditions. This study proposes mucosomes as a ground-breaking nanosystem that can be applied in several pathological contexts, especially in mucus-related disorders.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Unveiling the interaction between PDT active squaraines with ctDNA: A spectroscopic study.

Squaraine dyes are potential photosensitizers in photodynamic therapy (PDT) due to their ability to release reactive oxygen species (ROS) and cause DNA damage. For this reason, the evaluation and determination of the type of interaction between squaraines and DNA is of the utmost importance. In this study different spectroscopic techniques such as UV-Vis and fluorescence spectroscopies were used to investigate the type of interaction that occurs between two photosensitizers (halogenated squaraines, i.e. Br-C4 and I-C4) and calf thymus DNA (ctDNA). Squaraines were found to bind ctDNA externally following a minor groove binding as they were able to replace Hoechst (a classic groove binder) from the groove of DNA. This binding mode was further supported by iodide quenching studies, ionic strength assay and Florescence Resonance Energy Transfer. Moreover, association (KA) and dissociation (KD) constants were obtained and compared with constants of well-known groove binders.

Squaraine dyes as fluorescent turn-on sensors for the detection of porcine gastric mucin: A spectroscopic and kinetic study.

Mucin-type glycoproteins are the principal components of mucus which cover all the mucosal surfaces of the human body. The mucus and mucins are essential mediators of the innate immune system, however in the last decades mucins have been identified even as an important class of cancer biomarkers. Luminogenic materials with fluorescence turn-on behavior are becoming promising materials because of their advantages of label free, relatively inexpensive and simple to use properties for biological detection and imaging. Squaraines are luminogens characterized by high fluorescence in organic media but poor emission in aqueous environments due to their tendency to self-aggregate. Herein we investigate the interaction between porcine gastric mucin (PGM) and several squaraines in aqueous media. While squaraine dyes showed low fluorescence intensity and quantum yield in water, as a result of the formation of aggregates, an enhancement of fluorescence up to 45-fold was achieved when PGM was added. PGM was detected in a linear range of 10-300 µg/mL with a limit of detection of 800 ng/mL. The assay was used to quantify mucin in diluted human serum samples and recoveries of 94.9-116.2% were achieved. To the best of our knowledge, this is the easiest and convenient method for mucin detection in the reported literature.

Mucin binding to therapeutic molecules: The case of antimicrobial agents used in cystic fibrosis.

Mucin is a complex glycoprotein consisting of a wide variety of functional groups that can interact with exogenous agents. The binding to mucin plays a crucial role in drug pharmacokinetics especially in diseases, such as cystic fibrosis (CF), where mucin is overexpressed. In this study, we have investigated the interaction between mucin and several drugs used in CF therapy. Protein-drug interaction was carried out by UV-Vis and fluorescence spectroscopy; quenching mechanism, binding constants, number of binding sites, thermodynamic parameters and binding distance of the interaction were obtained.

Squaraine Dyes: Interaction with Bovine Serum Albumin to Investigate Supramolecular Adducts with Aggregation-Induced Emission (AIE) Properties.

Bovine serum albumin (BSA)-squaraine supramolecular adducts with aggregation-induced emission (AIE) properties were prepared and characterized by spectroscopic methods. While squaraine dyes showed a very low fluorescence quantum yield in water, a great enhancement in the fluorescence of the aggregated BSA adducts was achieved due to the abnormal aggregation-induced emission properties of squaraines. The adducts formation was studied from a kinetic point of view showing unexpected structure-properties relationships.

Disassembling the complexity of mucus barriers to develop a fast screening tool for early drug discovery.

Mucus is a natural barrier with a protective role that hinders drug diffusion, representing a steric and interactive barrier to overcome for an effective drug delivery to target sites. In diseases like cystic fibrosis (CF), pulmonary mucus exhibits altered features, which hamper clearance mechanisms and drug diffusion, ultimately leading to lung failure. Effectively modelling the passage through mucus still represents an unmet challenge. An airway CF mucus model is herein proposed to disassemble the complexity of the mucus barrier following a modular approach. A hydrogel, mainly composed of mucin in an alginate (Alg) network, is proposed to specifically model the chemical-physical properties of CF mucus. The steric retention of pathological mucus was reproduced by targeting its mesh size (approximately 50 nm) and viscoelastic properties. The interactive barrier was reproduced by a composition inspired from the CF mucus. Optimized mucus models, composed of 3 mg ml-1 Alg and 25 mg ml-1 mucin, exhibited a G' increasing from ∼21.2 to 55.2 Pa and a G'' ranging from ∼5.26 to 28.8 Pa in the frequency range of 0.1 to 20 Hz. Drug diffusion was tested using three model drugs. The proposed mucus model was able to discriminate between the mucin-drug interaction and the steric barrier of a mucus layer with respect to the parallel artificial membrane permeability (PAMPA) that models the phospholipidic cell membrane, the state-of-the-art screening tool for passive drug diffusion. The mucus model can be proposed as an in vitro tool for early drug discovery, representing a step forward to model the mucus layer. Additionally, the proposed methodology allows to easily include other molecules present within mucus, as relevant proteins, lipids and DNA.