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.

Hypersensitivity reactions to metronidazole.

BACKGROUND: Hypersensitivity reactions to metronidazole are infrequently described. However, we believe that such reactions are increasing due to growing use of the drug for the treatment of amebiasis and anaerobe infections combined with other antibiotics. The present study assesses the need for oral provocation in patients with probable hypersensitivity reactions to metronidazole. METHODS: We performed cutaneous prick tests with spiramycin and metronidazole as well as epicutaneous tests with metronidazole at different concentrations in four patients with cutaneous reactions to Rhodogil (metronidazole plus spiramicyn). Controlled oral challenges were then carried out with placebo using erythromycin, spiramycin and metronidazole except in the last patient due to a positive prick test. RESULTS: Only one patient showed a positive metronidazole prick test. The epicutaneous tests were negative. All patients tolerated erythromycin and spiramycin up to therapeutic doses. Oral provocation with metronidazole proved positive, the first patient presenting a delayed exanthema and the other two early erythema and itching. CONCLUSIONS: We present four cases of cutaneous exanthemas caused by metronidazole (two early and two delayed) and probably mediated by an immune mechanism which we have only been able to demonstrate in one case. Taking into account the low sensitivity of the cutaneous tests (prick tests and epicutaneous tests), oral provocation must be considered the "gold standard" for establishing the diagnosis in many cases of hypersensitivity reactions to metronidazole.