Wasp venom allergen with diminished reaction capability ige and method for its preparing (variants)

FIELD: medicine, allergology, toxins, pharmacy.

SUBSTANCE: invention relates to recombinant allergens of insect venom and to specific methods for their preparing, in particular, antigen 5 of wasp venom allergen. Recombinant antigen 5 is prepared in bacterial cells as insoluble aggregates followed by their denaturation and transfer to a soluble monomeric allergen. Transfer is carried out by dialysis using acid buffer solution (pH = 3.5-6.5) that can comprise guanidine hydrochloride, or by using a cysteine-containing solvent. Based on describes methods the practically pure recombinant antigen 5 of wasp venom allergen is isolated and used in pharmaceutical composition for hyposensibilization of body to wasp venom allergen. Invention provides preparing protein with reduced reaction capability JgE owing to it can be used in immunotherapy in treatment of allergy.

EFFECT: improved preparing method, valuable properties of allergen.

8 cl, 1 dwg

 

The present invention relates to recombinant allergens poison insects and how they are targeted obtaining, and mentioned allergens may take a form similar natural structures, or different from them depending on the way of obtain.

Application forms, which correspond to the natural molecule is nonallergenic diagnostics (in vitro or in vivo) disorders in persons with allergies, especially those with allergies to insect venom.

Artificial forms may be used as a therapeutic agent for a specific (individual) immunotherapy, which has minor side effects. These recombinant forms, thus, can provide a more effective treatment than the natural product. The developed method allows to implement biotechnological production in the conditions required in the pharmaceutical industry (GMP).

Allergies from stinging insects is caused predominantly by wasps and honey bees and can cause serious systemic symptoms or even potentially fatal anaphylaxis (Müller, U.R., in: Insect sting allergy, Gustav Fischer Verlag; 1990). Substances that cause allergies type 1, are proteins, glycoproteins or polypeptides of insect poison. After injection susceptible people, these allergens react with IgE molecules SV is related to the surface of the support cells. If associated with FcεRI IgE molecules of this type are sewn with each other by means of the allergen, it causes the release of mediators (e.g. histamine, leukotrienes) and cytokines, effector cells and, thus, to the corresponding clinical symptoms.

In addition to the peptide Meletina as allergenic components of bee venom are the enzymes hyaluronidase and phospholipase A2 (Habermann, E., 1972, Science 177, 314-322). In the case of wasps main antivaccinia allergens similar hyaluronidase, which is similar to the one that is included in the composition of bee venom (Hoffmann, D.R., 1986, J. Allergy Clin.Immunol. 78, 337-343), and the phospholipase A1. The most important major allergen of venom of wasps is an antigen 5, which has hitherto enzymatic activity was not detected (King and others, 1978, Biochemistry 17, 5165-5174). All of these allergens have already been characterized in molecular biology and have already cloned the corresponding cDNA molecules (including Fang and others, 1988, PNAS, 895-899; Soldatova and others, 1993, FEBS, 145-149; Kuchler and others, 1989, Eur. J. Biochem. 249-254). Using the cDNA sequence can be obtained recombinant allergens, which can be used in the diagnosis and treatment of allergies (Scheiner and Kraft, 1995, Allergy 50, 384-391).

In this invention the primary antigen 5 allergen is particularly important, below this molecule is used as a sample. This molecule represents deglycosylation is a protein of about 25 kDa. The primary sequence contains 8 cysteine residues, which indicates the presence of four disulfide bridges (Hoffman, D.R., 1993, J. Allergy Clin Immunol. 92: 707-716). The classical approach to effective therapy for allergies to insect venom is specific immunotherapy or desensitization (MÜller, U.R., in: Insect sting allergy, Gustav Fisher Verlag; 1990). While the natural extracts of the allergen is administered to the patient subcutaneously increasing doses. However, this method entails the risk of allergic reactions or even anaphylactic shock. Because you can expect the emergence of strong reactions, especially if hyposensitization insect bites, the treatment is carried out exclusively in the hospital setting.

Optimization of treatment with the help of allergens produced by recombinant methods can be particularly significant in the case of allergies to insect bites. The allergen extracts of natural origin can be replaced by certain cocktail of allergens with high purity are obtained recombinant methods (Scheiner and Kraft, 1995), which may be appropriate to the individual sensitization of patients. Real prospects safer hyposensitization opened in connection with the possibility of deliberate modification of recombinant allergens, in which antigenic determinants of IgE removed from elficiency way without weakening the paired T-cell antigenic determinants, being receptive to therapy (Schramm and others, 1999, J. Immunol. 162, 2406-2414).

From heterologous expression in E. coli it is known that the majority of eukaryotic proteins are not produced with a similar structure to natural structures, or are produced in very small extent. The result of these incorrect forms is often the insolubility of these proteins. In particular, it is observed in cysteinaemia proteins (Kuchler et al., 1989, Eur. J. Biochem. 184, 249-254). About the antigen 5 reported that the expression in bacteria leads to the formation of insoluble aggregates that do not meet the natural structure (Monsalve et al., 1999, Protein Express Purif. 16(3): 410-416). Insoluble aggregates of this type cannot be used either for diagnosis or for therapy.

Proteins that are insoluble in E. coli often prepared for research purposes in the system of eukaryotic gene expression, such as, for example, yeast or insect cells (Monsalve et al., 1999, Protein Express Purif. 16(3): 410-416; Soldatova et al., 1998, J Allergy Clin Immunol 101: 691-698). However, the disadvantages of systems of eukaryotic expression are, in particular, the possibility of leakage processes hyperglycinaemia (Grobe et al., 1999, Eur. J.Biochem 263: 33-40) and proteolytic degradation, as well as a relatively small output (Glover and Hames (eds.), 1995, Expression Systems, IRL Press, Oxford-New York-Tokyo). Therefore, proteins of this type are generally unsuitable the La allergic applications in the fields of pharmaceutical and medical diagnosis and therapy.

The products obtained by the method according to this invention, which have a natural structure, mainly you can apply in-vitro and in-vivo for diagnosis of allergic disorders, especially allergies to insect bites. This natural form suitable for detection of IgE antibodies by known methods.

On the other hand, the variants obtained by this invention, which differ completely inactive or only partially active structures IgE, can be used as hypoallergenic ingredients in products for specific immunotherapy. In the context of this invention, the above mentioned term "hypoallergenic" means reduced up to the absence, preferably from 5 to 95%, in particular from 20 to 85%, the ability to cause an allergic reaction in comparison with the natural allergen) due to the low IgE response.

The object of the present invention is a method by which recombinant allergens can be obtained in bacteria (E. coli). The first stage of purification is carried out by using a significant enrichment of insoluble aggregates of protein. Then these units are denatured without the addition of reducing agents. Depending on conditions subsequent dialysis get various forms. Importantly, these molecules are Monomeric and soluble. The activity of IgE in the first var is ante soluble form comparable to the activity of natural allergen and, consequently, it can be applied for diagnostic purposes. The product of this type is obtained by dialysis with systemsterrain solution.

Other alternatives soluble form structurally different from the natural allergen and identifies a reduced or absent reactivity of IgE. For this reason, products of this type are suitable for promoting improved immunotherapy. Hypoallergenic product of this type receive this invention by dialysis with acidic buffer solutions, preferably in an environment with a pH between 3.5 and 6.5, especially between 4.0 and 5.5.

Thus, the present invention relates to the recombinant allergen insects, which differs in that it has a lower activity of IgE or reduced ability to cause an allergic reaction. In this invention the ability of these proteins to cause an allergic reaction is reduced up to 95% compared with the natural allergen.

In particular, the invention relates to appropriate the recombinant allergen insect, wasps, namely hot vulgaris and hot germanica.

In addition, this invention relates to a method of allocation of sufficient pure recombinant allergen insect venom, which differs in that allergenic proteins get in insoluble form, the quality is TBE "implementation bodies" in the bacterial cells; next, the above-mentioned insoluble aggregates of denatured and these denatured products transform by dialysis in a soluble Monomeric allergens of different structural forms and then exhale. Specified denaturing preferably carried out using chloride guanidine without the addition of reducing agents.

This invention relates particularly to a method for isolation of recombinant allergens insect venom with reduced allergenicity or reactivity of IgE, in which the dialysis is carried out with the use of acidic buffer solution; preferably this buffer is sodium acetate with a pH between 4.5 and 5.0.

In addition, the invention relates also to a method for isolation of recombinant allergens insect venom with the usual allergenicity or reactivity of IgE; in this case, the dialysis is carried out with application sistemstartado solution.

The invention relates to the recombinant allergen poison Stingers, which can be obtained by using the appropriate method described above and below.

In addition, the present invention relates to pharmaceutical preparations containing the appropriate recombinant allergen with low or reduced reactivity of IgE and appropriate ancillary medicinal substance and Vitek is.

Finally, this invention relates to the use of allergens insect venom obtained by using the appropriate method described above and below, for the diagnosis of Allergy to insect stings in-vivo and in-vitro.

Below is a detailed description of this method.

In specific examples, the antigen 5 allergens poison wasps from hot vulgaris (Ves v 5) and antigen 5 from hot germanica (Ves 5 g) were cloned in the expression vector pSE420 and transformed into strain M15 pREP4 K12 bacteria. The sequence of operations when carrying out this method is shown on the drawing.

Recombinant allergens are produced by pre-growing strain for inoculation of the culture of expression. Expression induced by IPTG, produced in a rotating container with stirring at 37°C in LB medium with limited access of oxygen (90 rpm). Bacteria are concentrated by centrifugation (5000 × g for 10 min at 20°) 5 hours after expression. Bacterial theriofauna carried out after re-suspension of the cells in a buffer solution (50 mm Tris/HCl, 25% (weight/volume) sucrose, pH 8.0) by adding lysozyme (10 mg/g wet weight). Next, add the same volume of a solution of detergent (0.2 M NaCl, 1% (weight/volume) DOC, 1% (wt/vol) Nonidet P40). This digeriyse the solution is subsequently treated with ultrasound (3 min on ice, 130 watts, pulse the pulse 0.5 seconds). Because the products of expression are initially in the form of insoluble aggregates (inclusion body), due to their high density, they can be separated from most other components (fragments of cell walls, ribosomes, etc.) by centrifugation at 3000 × g. Further purification is performed in three successive stages using petersenstrasse solutions (1% Triton X-100). Purified inclusion body subsequently digeridoo the addition of denaturing buffer (6 M chloride guanidine, 20 mm Tris/HCl, pH 8.0) and shaken for 2 hours at RT.

In order to isolate the forms of structures with active IgE, denaturing the mixture is introduced into a dialysis tube (limit thererofre 12-14 kDa) and deleteroute to 100 volumes of a solution of cysteine (5 mm cysteine) for 12 hours at room temperature, with stirring. Further, removal of cysteine, dialysis to distilled water.

In order to obtain structures with reduced reactivity of IgE, first dialysis to 20 mm buffer solution of sodium acetate (pH 5.0). In this case also required to conduct subsequent dialysis to distilled water. After removal of water-soluble allergens separated from precipitated by centrifugation units. The supernatant contains the target soluble recombinant allergen is. Instead of a buffer solution of sodium acetate can also use other acidic buffer solutions, with the ability to be buffered in the range from 3.5 to 6.5, preferably from 4.0 to 5.5. Examples of buffer systems of this type are sufficiently described in the literature.

Precipitated recombinant allergens obtained in both ways, can be reused are denatured and purified following the same pattern. This greatly increases the output.

After stages of dialysis products have a purity of about 95%. Further clean-up steps basic allergens insect venom produced by cation-exchange chromatography (buffer solution pH of 7.2), using, for example, the Source is set to S (Pharmacia, Freiburg, Germany) and gel chromatography. In addition to removing present in a small amount of high molecular weight and low molecular weight impurities by gel-chromatography is also used for desalination.

The quality control carried out on the basis of a number of characteristic properties, which are presented below in tabular form for antigen 5:

p-antigen=natural antigen

PropertyThe form with the natural activity of IgEThe form with the reduced activity of IgE
The install. MW in SDS-page (non conditions) 26-27 kDa
The salt concentration for elution in Source S320 mm NaCl400 mm NaCl
Cleavage by protease V8The 15 kDa fragment + peptidespeptides <10 kDa
Monoclonal antibodies specific to the antigen 5Registered through US, ERegister only with LUCK
The frequency of manifestations of the activity of IgE in the serum of allergic>95%<10%
Allergenic powerSimilar to the p-antigen 5More than 10 times less than that of the p-antigen 5

The method according to this invention is suitable for all types of allergens, insect venom. The method of purification, recombinant cloning and methods of expression known in the art and may be replaced by other similar methods.

1. The way to obtain almost pure recombinant antigen 5 allergen wasp venom, characterized in that the allergenic protein get into bacterial cells in the form of insoluble aggregates, insoluble aggregates of denatured in the absence of reductants derived denatured products transferred to soluble Monomeric antigen 5 dialysis using acidic buffer rastv the RA and secrete antigen.

2. The method according to claim 1, characterized in that the denaturation is done with the use of buffer solution, which includes the guanidine hydrochloride.

3. The method according to claim 1, characterized in that during dialysis using a buffer solution with a pH value between 3.5 and 6.5.

4. The method according to any one of claims 1 to 3, characterized in that the use of allergen wasp species hot sp., mostly hot vulgaris and hot germanica, Paravespula sp. and Apis mellifera.

5. A method of obtaining a recombinant antigen 5 allergen wasp venom, characterized in that the antigen 5 get in bacterial cells in the form of insoluble aggregates, insoluble aggregates of denatured in the absence of reductants derived denatured products transferred to soluble Monomeric allergen dialysis solvent containing cysteine.

6. The method according to claim 5, characterized in that the use of allergen wasp species hot sp., mostly hot vulgaris and hot germanica, Paravespula sp. and Apis mellifera.

7. Recombinant antigen 5 allergen wasp venom obtained by one of the methods according to claims 1-6.

8. Pharmaceutical composition for hyposensitization of the body to the allergen wasp venom containing recombinant antigen according to claim 7, obtained by the method according to any of pp.5-6, as well as the excipient and media.



 

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5 cl, 12 dwg

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to benzimidazole derivatives or their salts useful in medicine of the general formula (1): wherein R1 and R2 can comprise similar or different values and represent independently of one another hydrogen atom, halogen atom, cyano-group, hydroxyl group, alkyl group comprising 1-4 carbon atoms, alkoxy-group comprising 1-4 carbon atoms, trifluoromethyl group; A represents unsubstituted, linear alkylene group comprising 1-7 carbon atoms; E represents group -COOR3 comprising 1-6 carbon atoms; G represents unsubstituted, linear alkylene group comprising 1-6 carbon atoms; M represents a simple bond or -S(O)m- wherein m represents a whole number in the range 0, 1 or 2; J represents substituted or unsubstituted heterocyclic group comprising 4-10 carbon atoms and one heteroatom in ring taken among the group consisting of nitrogen atom or sulfur atom excluding unsubstituted pyridine ring; a substitute in indicated aromatic heterocyclic group is taken among halogen atom, cyano-group, linear alkyl group comprising 1-6 carbon atoms, linear alkoxy-group comprising 1-6 carbon atoms, trifluoromethyl group and trifluoromethoxy-group wherein one or more indicated substituted can be replaced by random positions in ring; X represents methane group (-CH=). Also, invention relates to a pharmaceutical composition used in inhibition of human chymase activity based on these compounds. Invention provides preparing new compounds and pharmaceutical composition based on thereof in aims for prophylaxis and/or treatment of inflammatory disease, cardiovascular disease, allergic disease, respiratory disease or osseous either cartilaginous metabolic disease.

EFFECT: valuable medicinal properties of compounds and composition.

14 cl, 3 tbl, 20 ex

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