Development of new in vitro models of lung protease activity for investigating stability of inhaled biological therapies and drug delivery systems.

Proteases play a vital role in lung health and are critically important to the metabolic clearance of inhaled protein-based therapeutics after inhalation. Surprisingly little is known about lung fluid protease composition and there is a consequent lack of biorelevant experimental models, which limits research and development in the burgeoning field of inhaled biologics. The aim of this study was to quantify proteases in human lung fluid and to use this data to design novel in vitro experimental models of lung lining fluid possessing biorelevant lung protease activity for use in biopharmaceutical stability studies. As a proof of concept, these novel models were used to investigate the effect of proteolytic activity on the stability of albumin nanoparticles, a biologic nanoparticle formulation widely investigated as a pulmonary drug delivery system. Bronchoalveolar lavage fluid was collected from healthy human volunteers and proteomic analysis was used to quantify the predominant proteases. Based on these data, four new lung protease models were constructed based on: (i) trypsin as a sole protease, (ii) dipeptidyl peptidase IV, cathepsin D, cathepsin H, and angiotensin converting enzyme in ratio and concentration to mimic the protease concentration in healthy lungs. Neutrophil elastase was used to model protease activity in inflammation. Albumin nanoparticles of 100 nm diameter remained intact over 48 h in phosphate buffered saline, but were degraded more rapidly in trypsin (50% reduction in 10 min) compared to the healthy lung protease model (50% reduction in 150 min). The addition of neutrophil elastase to the healthy lung protease model resulted in a similar, but more variable degradation profile. Nanoparticle degradation was associated with concomitant appearance of small fragments and aggregates. In conclusion, we have characterised the protease concentration in the lungs of healthy humans, designed models of lung protease activity and demonstrated their utility in studying albumin nanoparticle degradation. These methods and models have wide application to study the influence of proteases in lung disease, expression of proteases in respiratory cell culture models, stability of peptide and protein-based drugs and inhaled drug delivery systems.

Differences in the coronal proteome acquired by particles depositing in the lungs of asthmatic versus healthy humans.

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.

A Biocompatible Synthetic Lung Fluid Based on Human Respiratory Tract Lining Fluid Composition.

PURPOSE: To characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays. METHODS: The structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF. RESULTS: SLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble's solution. CONCLUSION: The SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

A case of uncontrolled severe asthma patient with coexisting carcinoid tumor presenting as pneumomediastinum.

INTRODUCTION: Patients with inadequately controlled or uncontrolled asthma are at a greater risk of attacks for asthma requiring emergency room visits or hospital admissions. There is a significant correlation between the severity of the disease and the severity of exacerbations. Patients with poorly controlled asthma are at a higher risk for complications. CASE STUDY: We present a 24-year-old aspirin-intolerant, uncontrolled asthma patient with the complication of pneumomediastinum. RESULTS: Severe symptoms persisted after the resolution of the pneumomediastinum despite intense anti-inflammatory and anti-obstructive therapy. A bronchoscopy revealed an endobronchial lesion and she was diagnosed with a carcinoid tumor. CONCLUSION: This case is an example of the importance of re-evaluating asthma patients who do not respond to standard medical treatment. Clinicians should be aware of the complications associated with asthma attacks such as pneumomediastinum and the possibility of a differential diagnosis that worsen asthma symptoms such as a carcinoid tumor.

Identification of vitamin C transporters in the human airways: a cross-sectional in vivo study.

OBJECTIVES: Vitamin C is an important low-molecular weight antioxidant at the air-lung interface. Despite its critical role as a sacrificial antioxidant, little is known about its transport into the respiratory tract lining fluid (RTLF), or the underlying airway epithelial cells. While several vitamin C transporters have been identified, such as sodium-ascorbate cotransporters (SVCT1/2) and glucose transporters (GLUTs), the latter transporting dehydroascorbate, knowledge of their protein distribution within the human lung is limited, in the case of GLUTs or unknown for SVCTs. SETTING AND PARTICIPANTS: Protein expression of vitamin C transporters (SVCT1/2 and GLUT1-4) was examined by immunohistochemistry in endobronchial biopsies, and by FACS in airway leucocytes from lavage fluid, obtained from 32 volunteers; 16 healthy and 16 mild asthmatic subjects. In addition, antioxidant concentrations were determined in RTLF. The study was performed at one Swedish centre. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was to establish the location of vitamin C transporters in the human airways. As secondary outcome measures, RTLF vitamin C concentration was measured and related to transporter expression, as well as bronchial epithelial inflammatory and goblet cells numbers. RESULTS: Positive staining was identified for SVCT1 and 2 in the vascular endothelium. SVCT2 and GLUT2 were present in the apical bronchial epithelium, where SVCT2 staining was predominately localised to goblet cells and inversely related to RTLF vitamin C concentrations. CONCLUSIONS: This experimental study is the first to demonstrate protein expression of GLUT2 and SVCT2 in the human bronchial epithelium. A negative correlation between SVCT2-positive goblet cells and bronchial RTLF vitamin C concentrations suggests a possible role for goblet cells in regulating the extracellular vitamin C pool.

Live/Real time three-dimensional transthoracic echocardiographic assessment of the involvement of cardiac valves and chambers in carcinoid disease.

We present an adult with metastatic carcinoid disease affecting the heart, in whom live/real time three-dimensional transthoracic echocardiography (3DTTE) provided incremental value over two-dimensional transthoracic echocardiography (2DTTE). Initial 2DTTE was able to demonstrate severe pulmonic and tricuspid regurgitation, but was unable to visualize the posterior leaflet of the tricuspid valve or the right (right anterior) leaflet of the pulmonic valve. Further analysis with 3DTTE demonstrated thickening, restricted mobility, and noncoaptation of all three leaflets of both the tricuspid and the pulmonary valves. En face viewing of tricuspid and pulmonary regurgitation vena contractas permitted more reliable quantification of regurgitation severity. In addition, localized, linear, echogenic areas consistent with carcinoid deposits were noted along the inner walls of the right atrium, atrial septum, and inferior vena cava. To the best of our knowledge, endocardial carcinoid deposits have never been reported by 2D or 3D echocardiography. En face viewing of these deposits by 3DTTE enabled measurement of their dimensions and areas. Subcostal examination also identified large circumscribed hepatic lesions consistent with metastatic disease. Neither the carcinoid deposits nor the metastatic lesions were detected by 2DTTE. This case demonstrates the usefulness of 3DTTE as a supplement to 2DTTE in more comprehensively assessing carcinoid involvement of the heart.

Atrial septal occluder device embolization to an iliac artery: a case highlighting the utility of three-dimensional transesophageal echocardiography during percutaneous closure.

Percutaneous closure of secundum atrial defects has become an accepted treatment in part because it is minimally invasive and relatively low risk. Despite recent advances in implantation technique and device improvements, complications occur. Here, we report a case of device embolization during percutaneous repair of an atrial septal defect (ASD) with multiple fenestrations. We highlight the value of using live/real time three-dimensional transesophageal echocardiography to help plan the percutaneous procedure and detect complications.

Incremental value of live/real time three-dimensional transesophageal echocardiography over the two-dimensional technique in the assessment of aortic aneurysm and dissection.

We compared findings from intraoperative live/real time three-dimensional transesophageal echocardiography (3DTEE) and two-dimensional transesophageal echocardiography (2DTEE) with surgery in 67 patients having aortic aneurysm and/or aortic dissection. Of these, 20 patients had aortic aneurysm without dissection, 21 aortic aneurysm and dissection, and 26 aortic dissection without aneurysm. 3DTEE diagnosed the type and location of aneurysm correctly in all patients unlike 2DTEE, which missed an aneurysm in one case. There were four cases of aortic aneurysm rupture. Three of them were diagnosed by 3DTEE but only one by 2DTEE, and one missed by both techniques. The mouth of saccular aneurysm, site of aortic aneurysm rupture, and communication sites between perfusing and nonperfusing lumens of aortic dissection could be viewed en face only with 3DTEE, enabling comprehensive measurements of their area and dimensions as well as increasing the confidence level of their diagnosis. In all patients with aortic dissection, 3DTEE enabled a more confident diagnosis of dissection because the dissection flap when viewed en face presented as a sheet of tissue rather than a linear echo seen on 2DTEE which can be confused with an artifact. 2DTEE missed dissection in one patient. In six cases the dissection flap involved the right coronary artery orifice by 3DTEE and surgery. These were missed by 2DTEE. Aortic regurgitation severity was more comprehensively assessed by 3DTEE than 2DTEE. Aneurysm size by 3DTEE correlated well with 2DTEE and surgery/computed tomography scan. In conclusion, 3DTEE provides incremental information over 2DTEE in patients with aortic aneurysm and dissection.

Incremental role of three-dimensional transesophageal echocardiography in the assessment of mitral-aortic intervalvular fibrosa abscess.

We report a case of an elderly patient in whom live/real time three-dimensional transesophageal echocardiography (3DTEE) provided definitive diagnosis of mitral-aortic intervalvular fibrosa abscess. This could not be done by two-dimensional transthoracic echocardiography (2DTTE) and two-dimensional transesophageal echocardiography (2DTEE). 3DTEE was also helpful in ruling out associated mitral valve endocarditis, which was initially suspected by 2DTEE leading to a mitral valve sparing surgery. Thus, 3DTEE provided incremental information over 2DTTE and 2DTEE in this patient.

Live/real time three-dimensional transthoracic echocardiographic assessment of the involvement of cardiac valves and chambers in carcinoid disease.

We present an adult with metastatic carcinoid disease affecting the heart, in whom live/real time three-dimensional transthoracic echocardiography (3DTTE) provided incremental value over two-dimensional transthoracic echocardiography (2DTTE). Initial 2DTTE was able to demonstrate severe pulmonic and tricuspid regurgitation, but was unable to visualize the posterior leaflet of the tricuspid valve or the right (right anterior) leaflet of the pulmonic valve. Further analysis with 3DTTE demonstrated thickening, restricted mobility, and noncoaptation of all three leaflets of both the tricuspid and the pulmonary valves. En face viewing of tricuspid and pulmonary regurgitation vena contractas permitted more reliable quantification of regurgitation severity. In addition, localized, linear, echogenic areas consistent with carcinoid deposits were noted along the inner walls of the right atrium, atrial septum, and inferior vena cava. To the best of our knowledge, endocardial carcinoid deposits have never been reported by 2D or 3D echocardiography. En face viewing of these deposits by 3DTTE enabled measurement of their dimensions and areas. Subcostal examination also identified large circumscribed hepatic lesions consistent with metastatic disease. Neither the carcinoid deposits nor the metastatic lesions were detected by 2DTTE. This case demonstrates the usefulness of 3DTTE as a supplement to 2DTTE in more comprehensively assessing carcinoid involvement of the heart.