Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Electrically induced transport of macromolecules through oral buccal mucosa.

OBJECTIVE: To investigate the feasibility of iontophoretic delivery of large molecules across buccal mucosa, and to establish its potential for enhanced drug delivery. METHODS: Qualitative (6h) and quantitative (8 and 36 h) assessment of porcine buccal mucosa, using a diffusion cell in vitro model, was carried out by fluorescent microscopy and UV/Vis spectroscopy respectively, with four fluorescently-labeled model species (3 and 10 kDa dextrans, 12 kDa parvalbumin and 66 kDa bovine serum albumin, BSA). Passive and iontophoresis parameters were obtained. The experimental iontophoresis data were compared with theoretical predictions. RESULTS: The two dextrans and parvalbumin showed enhanced permeation through buccal mucosa after anodal iontophoresis (1-6h). Passive diffusion and cathodal iontophoresis resulted in minimal permeation. BSA could not be measured by either mode. Iontophoretic delivery profiles compared to passive delivery, had reduced time lags (30-50 versus ~270 min) and increased flux (~37 times faster). Time lag factor/enhancement ratio (TLF/ER) data confirmed that iontophoresis significantly enhanced permeation. The diffusion coefficients (D, passive) for dextrans were significantly higher than for parvalbumin, with the converse obtained for solubility (C0); permeability coefficients (P) were similar for all three species. Potential differences (V) for the two higher kDa species were significantly higher than for the lowest kDa species. Experimental and theoretical data were in reasonable agreement. SIGNIFICANCE: The experimental and theoretical data, confirming enhanced delivery of the model species via iontophoresis, gave a suitable basis for its potential application in the mouth, in a clinical setting and opens pathways to further research for delivering precious drugs topically and systemically.

Antacid medication inhibits digestion of dietary proteins and causes food allergy: a fish allergy model in BALB/c mice.

BACKGROUND: Digestible proteins were supposed to be irrelevant for oral sensitization and induction of food allergy. Approximately 10% of the adult population uses antacids for the treatment of dyspeptic disorders, drugs that hinder peptic digestion. In these patients, proteins that are normally degradable might act as food allergens. OBJECTIVE: We aimed to study the influence of antacid intake on the allergenicity of dietary proteins, taking sturgeon caviar and parvalbumin, the major fish allergen, as examples. METHODS: Caviar proteins and recombinant parvalbumin from carp, rCyp c 1, were applied for intragastric feedings with or without the antacids sucralfate, ranitidine or omeprazole, using a Balb/c mouse model. RESULTS: Both caviar proteins and parvalbumin were rapidly degraded in an in vitro digestion assay at pH 2.0, but not at pH 5.0, imitating the effect of antacids. The groups fed with caviar in combination with ranitidine hydrochloride intramuscularly or sucralfate orally had significant levels of caviar-specific IgE antibodies (P <.01), T-cell reactivity, and elevated counts of gastrointestinal eosinophils and mast cells. Food allergy in these groups was further evidenced by oral provocation tests and positive immediate-type skin reactivity. In contrast, feedings with caviar alone led to antigen-specific T-cell tolerance. None of the groups showed immune reactivity against the daily mouse diet. As a proof of the principle, feeding mice with parvalbumin in combination with ranitidine or omeprazole intramuscularly induced allergen-specific IgE antibodies (P <.05). CONCLUSIONS: When antacid medication impairs the gastric digestion, IgE synthesis toward novel dietary proteins is promoted, leading to food allergy.

Calcium-calmodulin-stimulated phosphorylation of rat parotid secretion granule proteins.

In studies designed to determine the mechanism by which Ca++ and calmodulin stimulate the fusion of parotid secretion granules with plasma membrane vesicles, the hypothesis tested was that Ca++ and calmodulin act by stimulating protein phosphorylation. It was earlier found that Ca++ and calmodulin, but neither alone, stimulated the phosphorylation of four secretion granule proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the degree of phosphorylation of a 36-kDa protein. Further studies have shown that in the presence of an optimal concentration of calmodulin (2.4 microM), half-maximal activation of phosphorylation of the four proteins occurred at approx. 8 microM Ca++, and at a maximally effective Ca++ concentration (10(-4) M), half-maximal stimulation occurred at calmodulin concentrations between 0.13 and 1.1 microM for the different proteins. The studies now described also demonstrate that the need for calmodulin for stimulating the phosphorylation, but not the dephosphorylation, is specific; two other Ca(++)-binding proteins, parvalbumin and troponin, could not replace calmodulin in stimulating phosphorylation of the four secretion granule proteins, but either one could substitute for calmodulin in stimulating dephosphorylation of the 36-kDa protein. Additionally, the phosphorylated proteins appear to be located on the granule surface. When secretion granules were subjected to mild treatment with a concentration of trypsin that did not lyse the granules, the 31-, 36-, 55-, 58- and 64-kDa proteins were no longer observed. In the presence of optimal concentrations of Ca++ and calmodulin, a dose-dependent inhibition of the phosphorylation of the various proteins by two calmodulin antagonists, trifluoperazine and calmidazolium, was observed; 50% inhibition of phosphorylation of the different proteins was obtained at approx. 20-40 microM trifluoperazine and at about 2.5-3.0 microM calmidazolium. Inhibition of the dephosphorylation of the 36-kDa protein required greater concentrations of trifluoperazine and calmidazolium; 128 microM and 50 microM, respectively. These results are consistent with the hypothesis that the phosphorylation of one or more of the 31-, 55-, 58- and 64-kDa proteins, but not the dephosphorylation of the 36-kDa protein, may be involved in the action of Ca++ and calmodulin in secretion granule-plasma membrane fusion.