Biopharmaceutical Assessment of Mesh Aerosolised Plasminogen, a Step towards ARDS Treatment.

Acute respiratory distress syndrome (ARDS) is a severe complication of lung injuries, commonly associated with bacterial, fungal and viral infections, including SARS-CoV-2 viral infections. ARDS is strongly correlated with patient mortality and its clinical management is very complex, with no effective treatment presently available. ARDS involves severe respiratory failure, fibrin deposition in both airways and lung parenchyma, with the development of an obstructing hyaline membrane drastically limiting gas exchange. Moreover, hypercoagulation is related to deep lung inflammation, and a pharmacological action toward both aspects is expected to be beneficial. Plasminogen (PLG) is a main component of the fibrinolytic system playing key roles in various inflammation regulatory processes. The inhalation of PLG has been proposed in the form of the off-label administration of an eyedrop solution, namely, a plasminogen-based orphan medicinal product (PLG-OMP), by means of jet nebulisation. Being a protein, PLG is susceptible to partial inactivation under jet nebulisation. The aim of the present work is to demonstrate the efficacy of the mesh nebulisation of PLG-OMP in an in vitro simulation of clinical off-label administration, considering both the enzymatic and immunomodulating activities of PLG. Biopharmaceutical aspects are also investigated to corroborate the feasibility of PLG-OMP administration by inhalation. The nebulisation of the solution was performed using an Aerogen® SoloTM vibrating-mesh nebuliser. Aerosolised PLG showed an optimal in vitro deposition profile, with 90% of the active ingredient impacting the lower portions of a glass impinger. The nebulised PLG remained in its monomeric form, with no alteration of glycoform composition and 94% of enzymatic activity maintenance. Activity loss was observed only when PLG-OMP nebulisation was performed under simulated clinical oxygen administration. In vitro investigations evidenced good penetration of aerosolised PLG through artificial airway mucus, as well as poor permeation across an Air-Liquid Interface model of pulmonary epithelium. The results suggest a good safety profile of inhalable PLG, excluding high systemic absorption but with good mucus diffusion. Most importantly, the aerosolised PLG was capable of reversing the effects of an LPS-activated macrophage RAW 264.7 cell line, demonstrating the immunomodulating activity of PLG in an already induced inflammatory state. All physical, biochemical and biopharmaceutical assessments of mesh aerosolised PLG-OMP provided evidence for its potential off-label administration as a treatment for ARDS patients.

Effects of superoxide anion attack on the lipoprotein HDL.

High-density lipoprotein (HDL) is an anti-atherosclerotic lipoprotein. Thanks to the activity of apolipoprotein ApoA1, the principal protein component of HDL, this last is responsible for converting cholesterol into ester form and transporting excessive cholesterol to the liver ("reverse cholesterol transport" RCT). When HDL undergoes oxidation, it becomes dysfunctional and proatherogenic. ApoA1 is a target of oxidation, and its alteration affects RCT and contributes to atherosclerosis development. Until now, the mechanism of HDL oxidation is not fully understood and only hydroxyl radicals seem to induce direct oxidation of protein and lipidic components of lipoproteins. Here we demonstrate that superoxide radical, widely produced in early atherosclerosis, directly oxidizes HDL, and as a consequence, ApoA1 undergoes structural alterations impairing its anti-atherosclerotic functions. Our results highlight in an in vitro system the potential mechanism by which O2·- triggers atherosclerotic pathogenesis in vivo. Our study gets the basis for therapeutic approaches focused on the management of superoxide generation in early atherosclerosis onset.

Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutics.

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8-5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients.

Anti-A and anti-B hemagglutinin depletion during Cohn purification process of 5% immunoglobulin.

BACKGROUND: Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are widely used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. For high-quality products and to minimize adverse events related to the use of intravenous IgG (IVIG) it is very important to perform detailed analyses of their components. One of these components, that in rare cases can cause severe hemolytic conditions, is the amount of hemagglutinins, natural antibodies that bind A and/or B (anti-A or -B) antigens present in red blood cells (RBCs). STUDY DESIGN AND METHODS: To characterize different IgG batches and to monitor the efficacy of the production procedure in the hemagglutinin reduction, a direct agglutination test (DAT) and a new flow cytometry (FC)-based assay were used for measuring the activity and the content of hemagglutinins in IgG samples obtained at different stages of the purification process. RESULTS: A total of 113 batches of 5% IVIG, produced in 2013 by Kedrion Biopharma, were analyzed for the ability to agglutinate RBCs by DAT. All batches tested were within the limits set by the European Pharmacopoeia. Three batches of 5% IVIG were analyzed for their hemagglutinin levels. The finished products and the production intermediates were evaluated by the DAT and the FC assay. A significant decrease of anti-A and anti-B titer after the Fraction (F)III precipitation was observed in all batches tested and an evaluation of the results obtained by the two methods was performed. CONCLUSIONS: This study shows that the hemagglutinin titer, accurately measured in a high number of 5% IVIG batches, is within the allowed limits for the DAT method. The specific production process employed, in particular the FIII precipitation step, successfully removes IgM and significantly reduces IgG class hemagglutinins.

Apotransferrin inhibits interleukin-2 expression and protects mice from experimental autoimmune encephalomyelitis.

Transferrin (Tf) has a major role in T cell activation and proliferation. Here, we investigated whether Tf exerts immunomodulatory effects on T cells and in development of T-cell driven experimental autoimmune encephalomyelitis (EAE). While treatment of concanavalin A-stimulated splenocytes with apotransferrin (ApoTf) did not affect release of IL-1ß, TNF, IFN-γ, IL-17, IL-4, and IL-10, it markedly and dose-dependently down-regulated synthesis of IL-2 in these cells. ApoTf also inhibited IL-2 generation in purified CD3+ T cells and the effect was accompanied with down-regulation of MAPK p44/42 and NFκB signaling. Despite impeded IL-2 release, proliferation of splenocytes was not inhibited by ApoTf. Importantly, ApoTf ameliorated EAE in mice and significantly reduced ex vivo IL-2 production in proteolipid protein-specific lymphocytes. Thus ApoTf may be a promising beneficial agent for multiple sclerosis.

A simple method for large-scale purification of plasma-derived apo-transferrin.

We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo-Tf (apo-transferrin), preserving its physiological properties and functions. Apo-Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV-1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo-Tf produced was 80% on laboratory scale and 90% in scale-up lots, and the purity was higher than 95%. The purified protein preserves iron- and receptor-binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.