The Cystic Fibrosis-Like Airway Surface Layer Is not a Significant Barrier for Delivery of Eluforsen to Airway Epithelial Cells.

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

The Use of Magnetic Resonance Imaging for Non-Invasive Assessment of Venofer® Biodistribution in Rats.

PURPOSE: The aim of this study was to determine the potential of magnetic resonance imaging to evaluate the biodistribution of exogenous iron within 24 h after one single injection of Venofer® (iron sucrose). METHODS: Venofer® was evaluated in vitro for its ability to generate contrast in MR images. Subsequently, iron disposition was assessed in rats with MRI, in vivo up to 3 h and post mortem at 24 h after injection of Venofer®, at doses of 10- and 40 mg/kg body weight (n = 2 × 4), or saline (n = 4). RESULTS: Within 10-20 min after injection of Venofer®, transverse relaxation rates (R2) clearly increased, representative of a local increase in iron concentration, in liver, spleen and kidney, including the kidney medulla and cortex. In liver and spleen R2 values remained elevated up to 3 h post injection, while the initial R2 increase in the kidney was followed by gradual decrease towards baseline levels. Bone marrow and muscle tissue did not show significant increases in R2 values. Whole-body post mortem MRI showed most prominent iron accumulation in the liver and spleen at 24 h post injection, which corroborated the in vivo results. CONCLUSIONS: MR imaging is a powerful imaging modality for non-invasive assessment of iron distribution in organs. It is recommended to use this whole-body imaging approach complementary to other techniques that allow quantification of iron disposition at a (sub)cellular level.

Evaluation of the suitability of a Sprague Dawley rat model to assess intravenous iron preparations.

The aim of the study was to examine the reproducibility of a rat model to assess the preclinical similarity in safety profiles and tissue accumulation of iron products. Accordingly, the effect of several doses of intravenously administered Venofer® and of Ferrlecit® on blood parameters, and on kidney and particularly liver toxicity were examined in non-anemic Sprague Dawley rats. The different analysis showed neither a clear treatment nor a dose effect after multiple injections. The parameters measured in this rat strain showed some iron induced adverse effects, but these could not be correlated to treatment specific differences. The findings presented in this paper indicate the difficulty to define a useful preclinical model to evaluate iron-based nano-colloidal preparations.

A novel oral iron-complex formulation: Encapsulation of hemin in polymeric micelles and its in vitro absorption.

Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac2). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac2) followed by rapidly heating the mixture at 50°C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac2) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37°C resulted in ferritin levels of 2500ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery.

The effects of dodecyl maltoside and sodium dodecyl sulfate surfactants on the stability and aggregation of recombinant interferon Beta-1b.

Aggregation often occurs during manufacturing and storage of protein drugs. Detergents such as sodium dodecyl sulfate are commonly used to prevent aggregation but need to be eliminated before final formulation for safety reasons. We studied the ability of dodecylmaltoside (DDM), a nontoxic alkyl saccharide surfactant, to reduce aggregation and increase the stability of interferon beta-1b (IFN)-ß-1b. An increase of 8°C in the Tm of IFN-ß-1b was observed when 0.1% of DDM was present in the protein solution. The absorption of DDM on hydrophobic surfaces of IFN-ß-1b enables the surface to become hydrophilic and non-ionic, and increases the stability of the protein. 0.1% DDM also results in a 62% increase in helical and a 25% decrease in ß-sheet structures. 0.1% DDM not only suppresses aggregate formation but also improves IFN-ß-1b solubilization. Furthermore, we have showed the protective effect of DDM on the anti-viral activity of IFN-ß-1b in solution.

Antibody response against Betaferon® in immune tolerant mice: involvement of marginal zone B-cells and CD4+ T-cells and apparent lack of immunological memory.

PURPOSE: The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNß) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNß. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. METHODS: Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNß (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. RESULTS: Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. CONCLUSION: The immune response against rhIFNß in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).