Digestibilnost alergoida polena pelina u simuliranim uslovima gastrointestinalnog trakta

Chemically modified allergens (allergoids) have found use in both traditional and novel forms of immunotherapy of allergic disorders. Novel forms of immunotherapy include local allergen delivery, via the gastrointestinal tract. This study conveys the gastrointestinal stability of three types of mugwort pollen allergoids under simulated conditions of the gut. Allergoids of the pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, succinic and maleic anhydride. Gastrointestinal tract conditions (saliva, and gastric fluid) were simulated in accordance with the EU Pharmacopoeia. The biochemical and immunochemical properties of the derivatives following exposure to different conditions were monitored by determining the number of residual amino groups with 2,4,6-trinitrobenzenesulfonic acid, SDS PAGE, immunoblotting and inhibition of mugwort-specific IgE. Exposure to saliva fluid for 2 min did not influence the biochemical and immunochemical properties of the derivatives. In the very acidic conditions of the simulated gastric fluid, the degree of demaleylation and desuccinylation, even after 4 h exposure, was low, ranging from 10 to 30 %. The digestion patterns with pepsin proceeded rapidly in both the unmodified and modified samples. In all four cases, a highly resistant IgE-binding protein the Mwof which was about 28-35 kD, was present. Within the physiological conditions, no new IgE binding epitopes were revealed, as demonstrated by immunoblot and CAP inhibition of the mugwort specific IgE binding. An important conclusion of this study is the stability of the modified derivatives in the gastrointestinal tract of patients, within physiological conditions. The means that they are suitable for use in much higher concentrations in local forms of immunotherapy than unmodified ones.U ovom radu su prikazani rezultati ispitivanja stabilnosti tri tipa alergoida polena pelina u simuliranom želudačnom soku. Koristeći kalijum-cijanat anhidrid ćilibarne i anhidrid maleinske kiseline, napravljeni su alergoidi polena pelina (Artemisia vulgaris). Saliva i želudačni sok su simulirani na osnovu evropske farmakopeje. Biohemijske i imunohemijske osobine derivata posle izlaganja različitim uslovima, praćene su: određivanjem broja slobodnih amino grupa u reakciji sa TNBS, SDS PAG elektroforezom, imunoblotom i određivanjem pelin-specifičnog imunoglobulina E (IgE). Izlaganje salivi u trajanju od 2 minuta ne utiče na biohemijske i imunohemijske osobine derivata. U kiseloj sredini želudačnog soka ne dolazi do značajnog demaleilovawa i desukcinilovanja. Čak i posle četvoročasovnog izlaganja, taj procenat je u opsegu 10-30 %. Alergoidi pelina se trenutno digestuju pepsinom, sa izuzetkom visoko rezistentne proteinske trake molekulske mase 28-35 kD, koja odgovara važnom IgE-vezujućem proteinu polena pelina. Imunoblotom i CAP-inhibicijom je pokazano da, u okviru fizioloških uslova, ne dolazi do stvaranja novih IgE-vezujućih epitopa. Hemijska stabilnost modifikovanih derivata u simuliranim uslovima želudačnog soka omogućuje da se tokom imunoterapije mogu primenjivati veće doze alergoida nego nemodifikovanog ekstrakta polena pelina

The influence of a residual group in low-molecular-weight allergoids of Artemisia vulgaris pollen on their allergenicity, IgE- and IgG-binding properties

Background: Reaction of epsilon-amino groups of lysine with potassium cyanate, maleic, or succinic anhydride leads to allergoids of low molecular weight. No study has been performed to compare their properties and investigate the influence of a residual group on allergenicity and human IgE- and IgG-binding of these derivatives. Methods: Allergoids of a pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, and succinic and maleic anhydride. Biochemical properties were investigated by determination of amino groups, enzyme activity, isoelectric focusing IEF and SDS-PAGE. IgE- and IgG-binding was determined using immunoblots and ELISA inhibition. Allergenicity was investigated by skin prick tests (SPT) on a group of 52 patients, of which 6 were control subjects, 30 were patients with no previous immunotherapy (IT), and 16 were patients undergoing immunotherapy. Results: The same degree of amino-group modification (more than 85%), residual enzyme activity (less then 15%), IEF, and SDS-PAGE pattern were noted. In the immunoblots of IgE-binding, there was more pronounced reduction in the succinyl and maleyl derivatives than in the carbamyl one. IgG-binding was less affected by carbamylation than by acid anhydride modification. The SPT showed that the succinylated derivative had the most reduced allergenicity (98% showed a reduced wheal diameter when tested with the succinyl derivative, 87% with the maleyl allergoid, and 83% with the carbamyl allergoid). The most significant difference among allergoids could be seen in the group of patients with high skin reactivity (83% of patients showed no reaction to the succinyl derivative when compared to the value of 28% for the carbamyl derivative or 22% for the maleyl derivative). Conclusions: According to our results, all three modification procedures yielded allergoids with a similar extent of modification. No single biochemical parameter investigated in the study could predict the degree of reduced allergenicity in vivo. The most reduced allergenicity was seen in the succinyl derivative while the preservation of IgG binding epitopes was of the highest degree for the carbamyl derivative