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Allergoids Derived from Allergens

a technology of allergoids and allergens, applied in the field of preparation of allergoids derived from allergens, can solve the problems of not giving encouraging results on the reduction of allergenic potential, poor reduction of allergenic activity, etc., and achieves the effects of reducing the desensitization effect, reducing the risk of possible undesired effects, and reducing the risk of undesired effects

Inactive Publication Date: 2012-05-10
LOFARMA SPA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]An object of the present invention is to provide a preparation for specific immunotherapy (ITS) that is provided with a higher tolerability, and particularly further minimizes the risk of possible undesired effects shown by the prior art allergoids, without thereby decreasing the desensitizating effect which is intrinsic of the conventional allergenic therapy.

Problems solved by technology

It has been noticed that the chemical modification of allergenic extracts or single proteins carried out by reaction with phenylglyoxal alone induces a poor reduction of the allergenic activity thereof.
Similarly, the inversion of the modification sequence, phenylglyoxal and subsequently potassium cyanate, did not give encouraging results on the reduction of the allergenic potential.

Method used

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  • Allergoids Derived from Allergens
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  • Allergoids Derived from Allergens

Examples

Experimental program
Comparison scheme
Effect test

example 1

Chemical Modification Procedure of an Extract of Mites of the Genus Dermatophagoides pteronissinus by KCNO, i.e., a KCNO / PGO Combination in Sequence

[0054]The extract of mites of the genus Dermatophagoides pteronissinus (Greer Labs, Lenoir, N.C., USA) has been prepared, after defatting with ethyl ether, by combining 100 mL of PBS (0.015 M phosphate buffer, 0.135 M NaCl, at pH 7.2) containing 0.05% azide (PBS-A) to 5 g of dehydrated mites bodies, and then subjecting the mixture to an ultrasonic treatment for 1 minute (Branson Ultrasonics, Sonifier 450, Darbury Conn., USA) in order to break the mites exoskeleton and to promote the extraction of the allergenic proteins contained therein. At the end, the preparation was put under stirring at 4° C. overnight. After centrifugation at 14000 rpm for 30 min and removal of the insoluble pellet, the surnatant was dialyzed against distilled H2O 2 and freeze dried.

[0055]The freeze dried extract is then taken up in a volume of 20 mM sodium phospha...

example 2

Chemical Modification Procedure of the Major Allergen Der p1 purified with KCNO, i.e., by a KCNO / PGO combination

[0069]a) Purification Step of the Der p1 Allergen from the DP Extract

[0070]The Der p1 allergen was purified from the DP extract by affinity chromatography, using a specific monoclonal antibody (isotype IgG1, produced at the Lofarma laboratories) covalently linked to a suitable matrix, such as CNBr-Sepharose (GE Helthcare, Milan), according to the procedure suggested by the manufacturer.

[0071]The Der p1 allergen, hold in a column, is eluted therefrom by using a buffer of 5 mM glycine, 50% ethylene glycol, pH 10.0. The purified allergen was quantified by spectrophotometric reading at 280 nm, by considering the molar extinction coefficient thereof (E280) as equal to 47330, hence an absorbance value, at a concentration of 1 mg / mL, equal to 1.89. Finally, the Der p1 was freeze dried in the presence of 1% saccharose.

[0072]The freeze dried Der p1 sample is then taken up in a volu...

example 3

Chemical Modification Procedure of the Major Allergen Ovalbumin (OVA) with KCNO, i.e., a KCNO / PGO Combination

[0083]A suitable amount of commercial OVA allergen (Sigma Aldrich, Milan), purified from egg albumen, is weighted and dissolved in a volume of 20 mM sodium phosphate buffer, pH 6.86, so as to reach a protein concentration of 2 mg / mL according to Lowry. For the modification with KCNO, 50.25 mg sodium tetraborate decahydrate and 101 mg potassium cyanate are added to 2.5 mL of OVA solution. The salts were brought to solution by slow stirring and the pH optionally adjusted to 9.3 with 1 M NaOH. The resulting solution was kept under slow stirring for 16 hours in a bath thermostated at 40° C. in a sealed flask. During the first hours, the pH was monitored and optionally adjusted by the addition of 1 M phosphoric acid. The thus-obtained preparation was gel-filtered again on G-25 column to remove the excess reagent, and sterilized on Millipore 0.22 micron membranes. A minimal part th...

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Abstract

Modified allergens having reduced allergenicity compared to corresponding native allergenic material, wherein all or a part of the primary amine groups of the lysine and arginine residues of the allergenic molecules are functionalized and the modified allergens have the structurewherein R and R2 are independently selected from H, C1-C5 alkyl, phenyl, phenyl substituted in ortho, meta, or para with a hydroxy, C1-C4 alkoxy, halogen, amino, alkylamino, dialkylamino, mercapto, C1-C4 alkylmercapto group; X represents O, S, or NR3, where R3 is H, alkyl with 1-6 carbon atoms, phenyl, or CN; R1 represents H, alkyl with 1-8 carbon atoms, phenyl or arylalkyl with up to 8 carbon atoms, or alkyl containing a heterocyclic ring; prot represents the protein residue of the allergen; n is the number of functionalized arginine groups and ranges between 1 and the number of arginine groups present in the allergen; m is the number of functionalized lysine groups and ranges between 1 and the number of lysine groups present in the allergen.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the preparation of allergoids derived from allergens by chemical functionalization, in order to reduce the risk to induce side-effects when employed as antiallergic vaccines in the immunotherapy of the allergic diseases.BACKGROUND ART[0002]The allergic diseases are caused by an abnormality of the immune system and are caused by the production of particular antibodies of the IgE class, specific towards ubiquitary substances (referred to by the term allergens), per se completely harmless, such as mainly pollens, mites, the epithelial derivatives, the poison of hymenoptera, fungal spores, and several foods. Such IgE antibodies are capable of linking to a specific receptor that is, present on the membrane, for example, on the membrane of the mucosal mastocytes, i.e., of the basophiles, and by subsequently reacting with the allergens which they are directed to, are capable of inducing the release of mediators (among which, hist...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/35C07K14/77A61P37/08C07K1/34C07K1/107C07K14/435C07K14/415
CPCA61K39/35A61K39/36C07K14/435C07K14/415A61K2039/55566A61P37/00A61P37/04A61P37/08C07K1/1077C07K17/02
Inventor MISTRELLO, GIOVANNIRONCAROLO, DANIELAZANONI, DARIOZANOTTA, STEFANIAFALAGIANI, PAOLOFALAGIANI, PIETRO
Owner LOFARMA SPA
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