LETI Pharma (Germany)

Quantifying major allergens is essential for evaluating the quality and efficacy of allergenic extracts. They are usually measured in non-polymerized extracts using immunoassays. However, the direct measurement of allergens in allergoids is currently not supported. This study set out to develop a method for quantifying Bet v 1 in polymerized birch extracts using mass spectrometry-based targeted analysis. Three isotopically labelled peptide sequences of Bet v 1 were synthetized and used as internal standards for the development of a mass spectrometry-based targeted analysis. The calibration curves of the three peptides to assess the linearity and limit of detection, as well as reverse calibration curves with a constant amount of sample, were constructed. The Bet v 1 content was determined and measured in 18 batches of depigmented (native extracts purified by a mild acid treatment) and depigmented-polymerized extracts. Bet v 1 isoforms were identified in both type of extracts by mass spectrometry. According to mass spectrometry-targeted analysis depigmented and depigmented-polymerized extracts exhibited mean values of 70.5 and 73.5µg Bet v 1/mg of lyophilized extract, respectively. A statistically significant correlation between the allergen content of both extracts was identified. Statistically significant differences were observed when the Bet v 1 content in non-polymerized extracts was measured via mass spectrometry (70.5±11.6µg/mg) or immunoassay (83.7±19.8µg/mg). These results represent the first direct quantification of Bet v 1 in allergoids using mass spectrometry-based targeted analysis. The proposed method demonstrates robustness and reliability and constitutes a promising alternative for detailed characterization of polymerized allergenic extracts.

Although many patients with allergic rhinoconjunctivitis have symptoms due to sensitization with more than one pollen allergen, and mixed pollen extracts are widely used for allergen immunotherapy in practice, there are few published trials. We performed a 2-year multicentre, double-blind, placebo-controlled trial of subcutaneous immunotherapy with mixed depigmented-polymerized birch and grass pollen extract in 285 patients with allergic rhinoconjunctivitis symptomatic during both birch and grass pollen seasons. Primary outcome was combined symptom and medication score (SMS) assessed by daily visual analogue scales (VAS). Analysis included a placebo-based analysis examining the effect of treatment only on days when placebo patients were symptomatic. There was a significant reduction in median SMS for actively treated patients (median 5.70 (interquartile range 2.62-10.02) compared with 7.07 (3.47-10.71) for placebo, P=0.0385). Rhinitis quality-of-life scores were significantly better for active compared with placebo, and other secondary endpoints were not significantly different. Placebo-based analysis showed a 33.7% reduced SMS at year 2 for active treatment compared with placebo on days when placebo patients were symptomatic. Both birch pollen- and grass pollen-specific IgG4 increased with active treatment. This study shows efficacy of mixed pollen extracts for immunotherapy for patients symptomatic to both birch and grass pollen allergens. The relatively modest effect may reflect 50% dose reduction for each allergen in the mixture. It supports VAS for symptom assessment and placebo-based analysis as useful for the analysis of immunotherapy trials. The safety of modified extracts may allow study of mixed extracts without dose reduction to improve efficacy.

Abstract Sowohl organische Nitrate als auch Hemmstoffe der Phosphodiesterase 5 (PDE5) sind vasodilatatorische Substanzen. Während die zunächst als Sprengstoffe konzipierten organischen Nitrate als Antianginosa hauptsächlich zur Behandlung der koronaren Herzkrankheit (KHK) genutzt werden, finden die ursprünglich zur Angina‐pectoris‐Therapie entwickelten PDE5‐Hemmer Anwendung als Therapeutika der erektilen Dysfunktion sowie der pulmonalen Hypertonie.

Oral mite anaphylaxis is a new syndrome characterized by severe allergic manifestations occurring in atopic patients shortly after the intake of foods made with mite-contaminated wheat flour. This clinical entity, observed more frequently in tropical/subtropical environments, is more often triggered by pancakes and for that reason it has been designated "pancake syndrome". Because cooked foods are able to induce the symptoms, it has been proposed that thermoresistant allergens are involved in its production. A novel variety of this syndrome occurs during physical exercise and therefore has been named dust mite ingestion-associated exercise-induced anaphylaxis. To prevent mite proliferation and the production of anaphylaxis, it has been recommended that wheat flour be stored at low temperatures in the refrigerator.

The physicochemical modification of allergen extracts provides a chance for administering higher doses of allergen vaccines. To evaluate the safety of a chemically modified (depigmented-glutaraldehyde polymerized) therapeutic vaccine of Phleum pratense administered at doses that are 10 times higher than those used in clinical practice, in comparison with conventional doses of the corresponding non-modified alum-adsorbed vaccine. The design of the study was randomized, double-blind, parallel and included two groups of patients. Twenty-three patients were treated weekly during nine visits for the build-up phase, followed by two weekly maintenance doses (a total of 11 injections per patient). Twelve patients received a vaccine containing the standardized unmodified extract, at a maximum concentration of 308.5 mcg of freeze dried material/mL (Group A). Eleven patients received a standardized modified allergen extract (Group B). The maximum dose used was 2400 mcg/mL. Safety was evaluated recording all adverse events. Skin test results and specific antibody levels were evaluated at the beginning and at the end of the study. Group A patients experienced three local immediate (two clinically irrelevant and one with a diameter>5 cm) and 18 delayed reactions (15 irrelevant and three with a diameter>10 cm), while Group B experienced six local immediate and 12 delayed reactions (all clinically irrelevant). Nine Group A patients experienced 12 systemic reactions (one immediate of grade 1, one of grade 2; and one delayed of grade 1; four of grade 2 and three of grade 3), while Group B patients experienced one immediate systemic reaction of grades 1, and 1 delayed reaction of grade 1. The modified extract of P. pratense is safe to treat sensitive patients, even at concentrations that are 10 times higher than those regularly administered in clinical practice. The majority of the local reactions were clinically irrelevant. No systemic reactions of grade 2, 3 or 4 were reported using the modified extract.

Background: The physiochemical modification of allergens to reduce allergenicity, while retaining immunogenicity, provides a chance for the administration of higher doses of immunotherapy, with a decreased risk of systemic reactions. Objective: To evaluate the safety of doses of depigmented glutaraldehyde-polymerized vaccine of Dermatophagoides pteronyssinus increasing those used in normal clinical conditions in comparison with regular doses of a non-modified vaccine. Materials and Methods: The study was double-blind, parallel and included two patient groups. Twenty-three patients were treated weekly during 9 visits for the build-up phase, followed by 2-weekly maintenance doses (a total of 11 injections per patient). Eleven patients (mean age 22 years) received immunotherapy with the standardized modified vaccine. The maximum dose used was the result of depigmenting and polymerizing 100 times the maximum dose used with the native extract. The maximum concentration used was 990 µg/ml of freeze-dried material. Twelve patients (mean age 24 years) received a standardized, unmodified allergenic extract. The maximum concentration used was 70 µg of freeze-dried material/ml, with a potency of 10 HEP_L/ml. The tolerance was evaluated recording all the side reactions related to immunotherapy and graded following the recommendations of the European Academy of Allergology and Clinical Immunology. Results: In both groups, all immediate local reactions were clinically not relevant. Patients treated with the native extract experienced 42 local immediate and 24 delayed reactions (1 of them had a diameter >10 cm), whereas patients treated with the modified extract experienced 30 local immediate and 21 delayed reactions (2 of them had a diameter >10 cm). Four systemic reactions in 2 patients, 1 immediate of grade 1 and 3 delayed of grade 2 were reported in the group treated with native extract and 1 delayed of grade 2 in the group treated with the modified preparation. Conclusions: The modified extract of D. pteronyssinus, used at concentrations which are 10 times higher than those regularly administered in clinical practice, demonstrated to be safe to treat D. pteronyssinus-sensitive patients. The major ity of the local reactions (immediate and delayed) were clinically not relevant (diameter <5 cm). No systemic reactions of grade 3 or 4 were noted.