Allergen Release Profiles of Fast-Dissolving Freeze-Dried Orodispersible Sublingual Allergy Immunotherapy Tablets.

Background: Sublingual allergy immunotherapy tablets (SLIT-tablets) provide a well-tolerated and clinically efficacious treatment for allergic disease such as allergic rhinitis and allergic asthma. In SLIT, uptake of allergen by immune-competent cells in the oral mucosa activates the immune system and leads to tolerance toward the sensitizing allergen. The ability to deliver the full allergen content into solution within the recommended sublingual holding time is therefore an essential quality of SLIT-tablets that must be supported by the tablet formulation for all relevant allergen sources. SLIT-tablets based on a fast-dissolving orodispersible freeze-dried formulation (Zydis) are currently available for 5 of the most prevalent allergens: tree (birch and related species from the birch-homologous group), grass, ragweed, Japanese cedar, and house dust mite. Objectives: The purpose of this study was to examine the allergen release properties of three freeze-dried SLIT-tablets containing tree, ragweed, and Japanese cedar extracts, respectively. The correlation between SLIT-tablet allergen release and the level of allergen-specific T-cell activation was examined for the tree SLIT-tablet. Methods: Allergen release kinetics and tablet disintegration times for the 3 freeze-dried SLIT-tablets were examined. For all 3 tablets, the magnitude of solubilized major allergen relative to time in solution was compared to external controls to achieve a measure of the total allergen release. Additional assessments of allergen release occurring after the initial timepoint (15 or 30 seconds in solution) were done independently of external controls by linear regression analyses. For the tree SLIT-tablet, the immunological potency of the released major allergen was assessed at each experimental timepoint by a Bet v-specific T-cell activation assay. Results: All 3 SLIT-tablets disintegrated within 1 second after contact with assay buffer without any detectible residue. Complete release of major allergens (Bet v 1, Amb a 1, and Cry j 1, respectively) was seen at the earliest experimental time points (15 or 30 seconds). For the tree SLIT-tablet, full T-cell activation was achieved at 30 seconds (earliest experimental time point). Conclusions: The freeze-dried SLIT-tablet formulation consistently provides rapid and complete release of allergen from a wide range of species in a standardized in vitro assay. Full release of the SLIT-tablet allergen content within the sublingual holding time is a prerequisite for maximal exposure of allergens to the sublingual mucosa immune system. The freeze-dried SLIT-tablet formulation examined here supports short sublingual holding times and furthermore offers a convenient administration form of allergy immunotherapy.

Treating allergies via skin - Recent advances in cutaneous allergen immunotherapy.

Subcutaneous allergen immunotherapy has been practiced clinically for decades to treat airborne allergies. Recently, the cutaneous route, which exploits the immunocompetence of the skin has received attention, which is evident from attempts to use it to treat peanut allergy. Delivery of allergens into the skin is inherently impeded by the barrier imposed by stratum corneum, the top layer of the skin. While the stratum corneum barrier must be overcome for efficient allergen delivery, excessive disruption of this layer can predispose to development of allergic inflammation. Thus, the most desirable allergen delivery approach must provide a balance between the level of skin disruption and the amount of allergen delivered. Such an approach should aim to achieve high allergen delivery efficiency across various skin types independent of age and ethnicity, and optimize variables such as safety profile, allergen dosage, treatment frequency, application time and patient compliance. The ability to precisely quantify the amount of allergen being delivered into the skin is crucial since it can allow for allergen dose optimization and can promote consistency and reproducibility in treatment response. In this work we review prominent cutaneous delivery approaches, and offer a perspective on further improvisation in cutaneous allergen-specific immunotherapy.

Novel Method for the Purification of House Dust Mite Allergen Der p 1 and Its Use in Structure-Based Chemical Design of Novel Inhibitors.

House dust mites are globally significant triggers of allergic disease. Notable among their extensive repertoire of allergens are the Group 1 cysteine peptidase allergens which function as digestive enzymes in house dust mites. Compelling evidence suggests that the proteolytic activity of these molecules plays a key role in the development and maintenance of allergic diseases through the activation of innate immune mechanisms which exploit genetic predispositions to allergy. Growing interest in this area creates a requirement for high-quality purified protein, whether natural or recombinantly expressed. It has also identified these allergens as therapeutic targets for a novel approach to allergy treatment through modulation of innate immune responses. The purpose of this chapter is to describe a new method for the purification of Der p 1 and use of the protein produced in a screening assay designed for the discovery of novel inhibitors of Group 1 house dust mite allergens.

The Importance of Tablet Formulation on Allergen Release Kinetics and Efficiency: Comparison of Freeze-dried and Compressed Grass Pollen Sublingual Allergy Immunotherapy Tablet Formulations.

PURPOSE: Efficient delivery of allergens to the sublingual mucosa is a prerequisite for successful sublingual immunotherapy (SLIT) for allergy, and in order to become available to immune-competent cells embedded in the sublingual mucosa, allergens need to be delivered in a soluble form. Delivery of solubilized allergens poses a particular challenge for tablet-based allergy immunotherapy, in which allergens are administered under the tongue in the form of dry tablets and need to be dissolved rapidly in a small volume of saliva, with little or no agitation. The purposes of this article were to compare the properties of 2 different pharmaceutical SLIT-tablet formulations, freeze-dried and compressed, and to examine how the tablet formulation affects the efficiency with which allergen is delivered from the dry state of the tablet into soluble form. METHODS: Two SLIT-tablet formulations, both indicated for grass pollen allergic rhinitis and containing grass pollen extract as the active ingredient, were examined with regard to tablet disintegration times, allergen dissolution kinetics, dependency on solvent volume and agitation, and the achieved recovery of the grass allergen content in soluble form with each tablet. FINDINGS: The freeze-dried and the compressed SLIT-tablet formulations differed markedly with respect to efficiency of allergen release. The freeze-dried tablet disintegrated faster and released grass allergen into solution with a release rate higher than that of the compressed formulation and, in contrast to the compressed formulation, achieved full recovery of the allergen content in soluble form in a small volume of solvent. IMPLICATIONS: Rapid and complete release of soluble allergen in a small volume of solvent, as demonstrated by the freeze-dried formulation, are key elements of efficient sublingual allergen delivery by SLIT-tablets. Complete allergen release means that the full allergen dose of the tablet is recovered from the tablet and made available to the sublingual immune system in soluble form, and rapid release ensures that the immune system becomes exposed to the highest possible dose of soluble allergen for the maximal duration before swallowing. In contrast, a SLIT-tablet formulation that provides incomplete and slower allergen release will likely require a higher allergen content compared to the more efficient formulation, in order to achieve the same dose of soluble allergen, consequently leading to an excess load of allergen that becomes swallowed without having been made immunologically available.

Cellular and Molecular Events in the Airway Epithelium Defining the Interaction Between House Dust Mite Group 1 Allergens and Innate Defences.

Serodominant group 1 allergens of house dust mites (HDMs) are cysteine protease digestive enzymes. By increasing the detection of any allergen by dendritic antigen presenting cells, upregulating inflammatory signalling molecules, and activating cells crucial to the transition from innate to acquired immune responses, the proteolytic activity of these HDM allergens also underlies their behaviour as inhalant allergens. The significance of this property is underlined by the attenuation of allergic responses to HDMs by novel inhibitors in experimental models. The group 1 HDM allergens act as prothrombinases, enabling them to operate the canonical stimulation of protease activated receptors 1 and 4. This leads to the ligation of Toll-like receptor 4, which is an indispensable component in HDM allergy development, and reactive oxidant-regulated gene expression. Intermediate steps involve epidermal growth factor receptor ligation, activation of a disintegrin and metalloproteases, and the opening of pannexons. Elements of this transduction pathway are shared with downstream signalling from biosensors which bind viral RNA, suggesting a mechanistic linkage between allergens and respiratory viruses in disease exacerbations. This review describes recent progress in the characterisation of an arterial route which links innate responses to inhaled allergens to events underpinning the progression of allergy to unrelated allergens.

Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens.

Group 1 allergens of house dust mites (HDM) are globally significant triggers of allergic disease. They are considered as initiator allergens because their protease activity enables the development of allergy to a spectrum of unrelated allergens from various sources. This initiator-perpetuator function identifies Group 1 HDM allergens as attractive drug design targets for the first small-molecule approach directed towards a non-human, root cause trigger of allergic disease. The purpose of this study was to: (i) identify exemplar inhibitors of these allergens using Der p 1 as a design template, and (ii) characterise the pharmacological profiles of these compounds using in vitro and in vivo models relevant to allergy. Potent inhibitors representing four different chemotypes and differentiated by mechanism of action were investigated. These compounds prevented the ab initio development of allergy to the full spectrum of HDM allergens and in established allergy they inhibited the recruitment of inflammatory cells and blunted acute allergic bronchoconstriction following aerosol challenge with the full HDM allergen repertoire. Collectively, the data obtained in these experiments demonstrate that the selective pharmacological targeting of Der p 1 achieves an attractive range of benefits against exposure to all HDM allergens, consistent with the initiator-perpetuator function of this allergen.

Chitosan-phenylalanine-mPEG nanoparticles: From a single step water-based conjugation to the potential allergen delivery system.

The chemical modification to obtain biocompatible chitosan (CS) nanoparticles for the application in biological system is still on expectation. By simply mixing CS with hydroxybenzotriazole (HOBt), the CS aqueous solution obtained allows a successful single step conjugation of both hydrophobic biomolecules, i.e. phenylalanine (Phe), and hydrophilic polymers, i.e. poly(ethylene glycol) methyl ether (mPEG), on CS, in water at room temperature. The CS-Phe-mPEG nanoparticles (20-50nm) exhibit positive charge leading to an entrapment of negatively charged house dust mite allergen (HDM) extract (Dermatophagoides pteronyssinus). The HDM-entrapped CS-Phe-mPEG shows biocompatibility as evidenced from the cell viability, the ROS (reactive oxygen species) reduction, and the HaCaTs proliferation. The clinical implementation on the healthy- and HDM-allergic volunteers indicates that the HDM-entrapped CS-Phe-mPEG stimulates cell-mediated immune response in peripheral blood mononuclear cells (PBMCs) and favors T cell immune response as seen from the reduction of interferon-(IFN)-γ and interleukin-(IL)-10 in the PBMCs of the HDM-allergic volunteers.

Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice.

The currently applied immunotherapy of type I allergy with aluminum hydroxide (alum) as adjuvant elicits - among other side effects - an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response toward Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles, and expression of stimulation and maturation markers as well as cytokines in supernatants was investigated. A favorable increase in Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy.