Immunomodulatory asthma therapy in the equine animal model: A dose-response study and evaluation of a long-term effect.

INTRODUCTION: Equine asthma represents a naturally occurring animal model for human allergic neutrophilic asthma. Inhalative nanoparticle-bound cytosine-phosphate-guanosine (CpG-GNP) immunotherapy, independent of specific allergens, has already shown promising clinical and immunological results in previous studies and offers the possibility to treat the underlying cause of the disease. This study analyses the relationship between dose and response, and evaluates a possible long-term effect. METHODS: In the prospective, randomised, double-blind clinical field study, 29 horses suffering from equine asthma received 10 inhalation treatments with either 187.5 µg CpG-GNP (CpG single dose [CpGsd]; n = 11), 375 µg CpG-GNP double dose (CpG double dose [CpGdd]; n = 9) (q48h for 20 days) or 1600 µg beclomethasone (n = 9) (q24h for 10 days). Each horse was examined three times: before the treatment (I), immediately after the 10 inhalations (II), and 8 weeks after the final inhalation (III). The three groups were compared according to clinical and laboratory parameters. The study examined the sustainability of the long-term effect of the treatment after 8 weeks, as well as the tolerability of the formula as a double dose. RESULTS: The CpGsd resulted in a significant improvement in 82% of the parameters, the CpGdd in 72%. In the long-term evaluation, the CpGsd showed a significant improvement in 100% of the parameters in comparison to the initial values, the CpGdd in 67%. On the immunological level, the bronchoalveolar lavage revealed a significant reduction of IL-4, IL-8, and interferon-γ. CONCLUSION: Both CpG groups displayed significant improvements in clinical and laboratory parameters, especially regarding the long-term effect of CpGsd. Doubling the CpG dose did not result in any improvement in comparison to the original single dose. On the immunological level, an anti-inflammatory, as well as an immunomodulatory effect, apart from a Th2-dominated immune response, could be observed. This immunomodulatory inhalation treatment could indicate a new possibility for human allergic asthma therapy.

Progress in formulation development and sterilisation of freeze-dried oligodeoxynucleotide-loaded gelatine nanoparticles.

Oligodeoxynucleotide (ODN)-loaded gelatine nanoparticles (GNPs) have proven their outstanding potential in the treatment of allergic diseases such as equine asthma and canine atopic dermatitis, which are appropriate models for the corresponding human diseases. To encourage the development of a marketable product, long term stability and sterility needs to be ensured. In this work, we aimed to advance freeze-drying options to stabilise ODN-loaded GNPs. Matrix-assisted laser desorption/ionisation mass spectrometry time-of-flight was implemented as a versatile tool to assess ODN stability. With this method long-term storage stability of lyophilised ODN-loaded GNPs formulated in sucrose or trehalose was achieved. Controlled nucleation was further introduced to optimise the lyophilisation approach. This allowed shortening of the process in comparison to standard freeze-drying procedures. Particle sizes, polydispersity indices, ODN stability, residual moisture and glass transition temperature were maintained upon storage. Excipient portfolio was enlarged by novel amino acid containing formulations for lyophilisates. His emerged as an excellent excipient in stabilising lyophilised ODN-loaded GNPs, whereas addition of Arg and Gly revealed to be inadequate at accelerated conditions. Lastly, gamma irradiation was evaluated as a suitable sterilisation method of ODN-loaded GNPs.

A comparison of nanoparticullate CpG immunotherapy with and without allergens in spontaneously equine asthma-affected horses, an animal model.

INTRODUCTION: New therapeutic strategies to modulate the immune response of human and equine allergic asthma are still under extensive investigation. Immunomodulating agents stimulating T-regulatory cells offer new treatment options beyond conventional symptomatic treatment or specific immunotherapy for human and equine allergic airway diseases, with the goal of a homoeostatic T-helper cell balance. The aim of this study was to evaluate the effects of a nebulized gelatin nanoparticle-CpG formulation (CpG-GNP) with and without specific allergens for the treatment of spontaneous allergic equine asthma as a model for human asthma. METHODS: Twenty equine asthma-affected horses were treated either with CpG-GNP alone or CpG-GNP with allergens. Two specific allergens were selected for each horse based on history and an in-vitro test. Each horse received seven administrations of the respective nebulized composition and was examined before treatment, immediately after and 6 weeks after the treatment course. RESULTS: Clinical parameters such as breathing rate, indirect interpleural measurement, arterial blood gases, amount of tracheal mucus and percentage of neutrophils and cytokines in tracheal washes and serum samples were evaluated. Treatment with CpG-GNP alone as well as in combinations with relevant allergens resulted in clinical improvement of nasal discharge, breathing rate, amount of secretion and viscosity, neutrophil percentage and partial oxygen pressure directly after and 6 weeks after treatment. There were no significant differences between the two treatments in clinical parameters or local cytokine profiles in the tracheal wash fluid (IL-10, IFN-g, and IL-17). IL-4 concentrations decreased significantly in both groups. CONCLUSION: Nonspecific CpG-GNP-based immunotherapy shows potential as a treatment for equine and possibly also human allergic asthma.

Optimisation of one-step desolvation and scale-up of gelatine nanoparticle production.

Gelatine nanoparticles (GNPs) are biodegradable and biocompatible drug delivery systems with excellent clinical performances. A two-step desolvation is commonly used for their preparation, although this methodology has several shortcomings: lack of reproducibility, small scales and low yields. A straightforward and more consistent GNP preparation approach is presented here focusing on the development of a one-step desolvation with the use of a commercially available gelatine type. Controlled stirring conditions and ultrafiltration are used to achieve large-scale production of nanoparticles of up to 2.6 g per batch. Particle size distributions are conserved and comparable to those determined for two-step desolvation on small scale. Additionally, a range of cross-linking agents is examined for their effectiveness in stabilising GNPs as an alternative to glutaraldehyde. Glyceraldehyde demonstrated outstanding properties, which led to high colloidal stability. This approach optimises the manufacturing process and the scale-up of the production capacity, providing a clear potential for future applications.