RU2279888C1 - Bee venum allergoid for allergen-specific therapy of patients with allergic response for bee stinging and method for its preparing - Google Patents

Abstract

FIELD: medicine, allergology, chemical-pharmaceutical industry.

SUBSTANCE: invention proposes an allergoid for carrying out the allergen-specific immunotherapy of patients with allergic responses for stinging by bees that comprises allergen fraction of molecular mass 3-12; 16-31; 38-46; 20 and 60 kDa. Allergoid is prepared by polymerization of the total allergen fraction with formaldehyde and by stabilization with NaBH₄. Also, invention proposes a method for preparing allergoid and a method for carrying out the allergen-specific immunotherapy of patients with allergic response for stinging by bees by using the proposed allergen. Invention provides preparing allergoid showing high specific activity and safety in using.

EFFECT: improved preparing method, valuable medicinal properties of allergoid.

5 cl, 2 tbl

Description

The invention relates to the pharmaceutical industry and relates to the creation of a safe highly effective hyposensitizing allergoid with an immunostimulating effect for allergen-specific immunotherapy of patients suffering from allergies to bee venom.

An allergy that forms when stinging bees, wasps, is recognized as the most dangerous type of insect allergy.

Bee venom contains a number of pharmacologically and biochemically active substances: biogenic amines (norepinephrine, histamine, serotonin, etc.), peptides (melittin, Mc peptide, MSP peptide, secapin, minimin, terciatin, etc.), enzymes (phospholipase A, B, hyaluronidase, acid and alkaline phosphatases, etc.). The following allergens of honey bees are currently described and presented in accordance with the requirements of the modern International Nomenclature (Table 1).

It was also noted that the poison of bees, wasps and bumblebees has common antigenic determinants, with which the effect of a cross-allergic reaction can be associated with the bites of these insects. To bee stings, toxic reactions are formed both local and systemic. Allergic reactions are based on immunological mechanisms, including extensive local (local) reactions, anaphylactic, like serum sickness. The nature of the allergic response to Hymenoptera venom is characterized by the production of specific IgE and IgG antibodies. The latter can be detected in patients within weeks and months from the time of the bite. Most authors believe that an increase in IgG antibodies is associated with an increase in "blocking" (protective) antibodies in the patient's body. The clinically protective effect of these antibodies has not always occurred. The presence of IgE antibodies is associated with the presence of positive skin tests in patients. IgE antibodies specific for bee venom were detected in the sera of patients with a general and strongly pronounced local reaction to stinging hymenoptera. Significant levels of specific IgG antibodies were detected in beekeepers who were repeatedly stung, which, apparently, indicates the possible protective functions of IgG antibodies, the titers of which sharply increase in patients after specific immunotherapy.

From the well-known development of the allergoid form of the allergen from bee venom, it follows that when bees sting people with sensitization to hymenoptera venom, acute allergic reactions can occur up to anaphylactic shock. The only reliable method for the prevention and treatment of these conditions is specific immunotherapy (SIT) with an allergen from bee venom (AP). However, in the process of SIT with the introduction of small doses of allergens, the risk of obtaining anaphylactic type reactions in patients remains.

An allergoid preparation (ALD) from Apis mellifera bee venom was developed based on the modification of a native allergen with formaldehyde using immunochemical (gel chromatography, immunoblotting) and clinical laboratory analysis (ELISA, chemiluminescence). Based on the main allergens (fractions with MM = 3-12 kD, 16-31 kD, 38-46 kD), the main allergen fraction (OAF) is formed. In animal experiments, the dynamics of IgG “block” antibodies was studied according to A.D. Ado et al., 1991 (V.N. Fedoseeva, A.I. Martynov, I.A. Orlova, T.G. Fedoskova. Development the treatment form of an allergoid from bee venom. - "Allergology and Immunology, Volume 4, No. 2, 2003, p. 84. Materials of the V Congress of Immunologists and Allergists of the CIS, St. Petersburg, Russia, July 8-11, 2003).

The objective of the present invention is to provide a drug for allergen-specific immunotherapy of patients with allergic reactions to stings by bees, which has specific activity and is safe.

To solve this problem, a group of inventions is proposed, united by a single inventive concept:

Allergoid for allergen-specific immunotherapy of patients with allergic reactions to bee stings, containing allergenic fractions with a molecular weight of 3-12, 16-31, 38-46, as well as 20 and 60 kD and obtained by polymerization of the total allergenic fraction with formaldehyde and stabilization of NaBH ₄ .

A method for producing an allergoid is proposed, which consists in extracting bee venom, dissolving the venom in saline, stabilizing human serum albumin, clarifying, purifying with dialysis for no more than 3 hours and isolating the total allergen fraction followed by formaldehyde treatment for protein polymerization when the temperature changes from + 4 ° From + 22 ° С, at the end of polymerization dialysis is carried out, centrifuged at 6000 rpm for 30 minutes, stabilized with NaBH ₄ , freeze-dried.

Clarification of bee venom is carried out by centrifugation.

The polymerization is carried out within 30 days.

A method of allergen-specific immunotherapy of patients with allergic reactions to bee stings is also proposed, which uses the above allergoid.

To obtain an allergoid, carry out:

1) Dissolution of bee venom in physiological saline,

2) Stabilization of bee venom with dry HSA (human serum albumin),

3) Clarification of the stabilized bee venom by centrifugation,

4) pH correction (up to 7.0),

5) dialysis treatment,

6) Chromatography and isolation of major and minor allergens,

7) Obtaining the total allergenic fraction for conversion to the allergic form (fractions with MM = 3-12, 16-31, 38-46 kD - main; 20 and 60 kD - small),

8) The study of immunochemical properties,

9) The study of biological properties,

10) Processing the total allergen fraction with formaldehyde, in which the polymerization of proteins occurs, within 30 days with a change in temperature conditions from 4 to 22 ° C,

11) Dialysis

12) Centrifugation (6000 rpm for 30 min),

13) Stabilization of NaBH ₄ ,

14) Freeze drying.

The immunochemical properties of the allergoid were studied, the ASIT scheme (allergen-specific immunotherapy) was developed, the biological properties of the allergoid, specific activity were studied, and the safety of the drug was proved.

To treat patients with allergic reactions to bee stinging, symptomatic therapy of an acute reaction to bee stinging and allergen-specific immunotherapy with bee venom allergens was performed.

An animal hyposensitization scheme has been developed that can be used in a clinic. The principle of introducing increasing doses according to a certain scheme is shown in table 2.

The above experimental studies and tests on volunteers showed that the claimed allergoid is harmless.

Table 1
Honey Bee Allergens (Apis mellifera) Title Allergen Mm Apis mellifera (Honey bee) Api m 1 (Phosfolipase A2) 16,000 Api m 2 (Hyaluronidase) 44,000 Api m 4 (Melittin) 3,000

Claims (5)

1. Allergoid for allergen-specific immunotherapy of patients with allergic reactions to bee stings, characterized in that it contains allergenic fractions with a molecular weight of 3-12; 16-31; 38-46, as well as 20 and 60 kD and obtained by polymerization of the total allergenic fraction with formaldehyde and stabilization of NaBH ₄ . 2. A method for producing an allergoid, characterized in that the bee venom is extracted, the venom is dissolved in saline, then stabilized with human serum albumin, clarified, then purified by dialysis for no more than 3 hours and the total allergenic fraction with a molecular weight of 3-12 is isolated; 16-31; 38-46, as well as 20 and 60 kD, which is then treated with formaldehyde to polymerize proteins when the temperature changes from 4 to 22 ° C, dialysis is carried out at the end of polymerization, centrifuged at 6000 rpm for 30 minutes, the resulting allergoid is stabilized with NaBH ₄ and freeze-dried. 3. The method according to claim 2, clarification of bee venom is carried out by centrifugation. 4. The method according to claim 2, the polymerization is carried out within 30 days. 5. The method of allergen-specific immunotherapy of patients with an allergic reaction to stings by bees, characterized in that they use the allergoid according to claim 1.