Conjugates of hydroxyalkyl starch and allergen

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to conjugates of hydroxyalkyl starch and allergen, in which at least one hydroxyalkyl starch is covalent bonded with allergen for hyposensitisation. Conjugates HAS-allergen provide epitope profile comparable to natural, that enables to increase immunotherapy efficiency.

EFFECT: increasing of immunotherapy efficiency.

14 cl, 5 dwg

 

DESCRIPTION

The present invention relates to compounds that include conjugate of hydroxyalkylated (HAS) and allergen, and HAS either directly or through a linker covalently bound to the allergen. The invention relates further to methods for the respective conjugates and their use as pharmaceuticals.

Excess specific immune response against exogenous substances currently the notion allergies. According to the classification Coombus and Gell allergic reactions can be divided into types I-IV, which, in particular, can be differentiated by class of antibodies involved in the reaction, recognizable antigens and induced effector mechanisms.

Allergens, accordingly, referred to as compounds that can induce an allergic immune response, in a more narrow sense of the allergic immune reaction of immediate type (type I), on the skin and mucous membranes. As a rule, in the case of allergens talking about polypeptides or proteins with a molecular weight of from about 5,000 Da to about 80000 Yes. The polypeptides can be of vegetable, animal or microbiological origin. The polypeptides may be in the form of parts of household dust.

Allergens induce IgE antibodies,which through its constant part of the contact surface of the fat cells and cause mast cell degranulation. The released fat cells substances (histamines, proteolytic enzymes and inflammatory mediators) is called directly or indirectly Allergy symptoms, usually rhinitis, conjunctivitis and/or asthma.

Mediated by IgE allergies of the immediate type (type I) are the strong allergic response. Up to 20% of people in industrialized countries suffer from allergic symptoms of type I. currently available treat along with drug therapy by specific immunotherapy, the so-called hyposensitization (Kleine-Tebbe and others, Pneumologie, 5, 438-444 (2001)).

In the case of classical hyposensitization subcutaneously injected in increasing quantities specific allergen extract until you reach an individual maintenance dose. With continued treatment, this dose is administered repeatedly, and use a variety of treatment protocols (Klimek and others, Allergologie und Umweltmedizin, Schattauer Verlag, c. 158 and later).

The effect of the treatment at that, apparently, is closely connected with used during the continuation phase quantities of the allergen. With the increase of input quantities of the allergen, however, in principle, increases the risk of IgE mediated reactions are suffering from allergies of the patient. In other words, therapy is also limited by allergic reaction patient and emerging instead is E. this patient has a risk of anaphylactic shock.

The effect of the treatment is to reduce allergic symptoms, which leads to an individual to reduce the need for drugs, respectively, to increase tolerance to the allergen.

Was offered the specific allergenic polypeptides by recombinant expression and use for hyposensitization (patent Germany 10041541).

For more allergens with reduced binding properties of IgE modify them using polyethylene glycol (PEG), and used for hyposensitization. Accordingly, in many publications describes obtaining conjugates PEG-allergen, which are formed by the covalent bond of the allergen with polyethylene glycol. Mosbech and others (Allergy, 45 (2), 130-141 (1990)) reported, for example, on the treatment of adult Allergy, asthma, using conjugates of PEG-home dust and immunological reactions after treatment. The authors found clinical improvement actions, if the dosage of the allergen is sufficient to reduce the amount of specific IgE and/or to induce IgG reactions, particularly IgG4.

Similarly, Schafer and others (Ann. Allergie, 68 (4), 334-339 (1992)) reported a study in which the use of allergenic composition of a mixture of grass pollen-modified PEG, for hyposensitization adults. The results sravnivats such received by hyposensitization when using partially purified mixture of pollen of grasses. The treatment is carried out in the framework of the double-blind study. Due to the modification with PEG the frequency and magnitude of side effects can be reduced by about 50%. In both exposed treatment groups was found to be significant improvement in allergic reactions.

The conjugates of PEG-based, however, are not naturally occurring patterns, which describe ways to cleavage in vivo.

Along with conjugates of PEG-based also received and investigated other derived allergens. The well-known modified dextran allergens, which are due to conjugation with carboxymethylcysteine. Some studies with β-lactoglobulin showed that the reaction of antibodies against dextranomer the conjugates significantly weakened compared to non-modified compounds (Kobayashi and others, J. Agric. Food Chem., 49 (2), 823-831 (February 2001); Hattori and others, Bioconjug. Chem., 11 (1), 84-93 (January-February 2000)).

Then get cross stitched high molecular weight allergens, the so-called allergodil. These products can be obtained, for example, by modification of allergens with formaldehyde or glutaraldehyde. The products can be produced Allergopharma, Joachim Ganser KG, 21462, Reinbek; HAL Allergie GmbH, 40554 düsseldorf and SmithKline Beecham Pharma GmbH, Benckard, 80716 Munich.

A broad overview of the range of various methods of obtaining bioconjugates, in General, presents G.T. Hermanson (Bioconjugate Techniques, Academic Press, San Diego, 1996). The binding of oligo - and polysaccharides with proteins occurs at least through lysine (-NH2) or cysteine (-SH) side chains and less through the side chain of aspartic or glutamic acid (-COOH), or through the tyrosine (aryl-OH) side chains.

Derivatives of starch, however, still have not been used for modification of allergens.

Gidroxiatilkrahmal (HES), for example, represents a substituted derivative of a carbohydrate polymer, and amylopectin, amounting to 95% of corn starch. HES has a favorable rheological properties and currently clinically used as a blood substitute and hemodilutional therapy (Sommermeyer and others, Krankenhauspharmazie, 8 (8), 271-278 (1987); and Weidler and others, Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

The amylopectin consists of chains of glucose, and in the main chains are α-1,4-glycosidic bonds,however, in areas of branching α-1,6-glycosidic bonds. Physico-chemical properties of this molecule are determined essentially by the type of glycosidic linkages. Because of the curved α-1,4-glycosidic bonds are formed helical structure with about 6 parts of glucose on stage.

Physico-chemical is the cue, and biochemical properties of the polymer HES can be changed by substitution. Introduction hydroxyethylene group can be achieved by alkaline gidroksietilirovaniya. Due to the reaction conditions it is possible to use different reactivity of the corresponding hydroxyl group unsubstituted Monomeric glucose in relation to gidroksietilirovaniyu so that you can influence the type of substitution.

So HES characterized, essentially, molecular weight distribution and degree of substitution. The degree of substitution can be pointed to as a DS (degree of substitution"), which refers to the proportion of the substituted Monomeric glucose in the calculation at all levels of glucose, or as MS ("molecular replacement"), which denotes the number hydroxyethylene groups on the level of glucose.

The HES solutions are in the form of a polydisperse compositions in which the individual molecules differ from each other in degree of polymerization, the number and location of branching, as well as on the type of substitution. HES thus is a mixture of compounds with different molecular weight. Accordingly, certain HES solution is characterized by an average molecular weight with the help of statistical quantities. While Mncalculated as a simple average based on the number of molecules (srednikov what I molecular weight), while Mw, mass-average molecular weight, is a dependent on the mass of the measured value.

Thus, the task of the invention to provide an improved derivative of allergens, in particular derivatives of allergens, which provide a depot effect and therefore they need less often to insert.

The problem is solved by the conjugates of hydroxyalkylated (HAS) and the allergen, in which at least one hydroxyalkylated covalently linked to the allergen.

According to the invention unexpectedly found that conjugates HAS-allergen especially preferably can be used for specific immunotherapy. Reliability hyposensitization enhanced by the application of the proposed according to the invention conjugates. Proposed according to the invention, the conjugates at the same time have a higher periodhalf-life in vivo, and therefore by conjugation with HAS achieved depot effect, positive impact on clinical effectiveness. Especially compared to water extracts of allergens depot effect is proposed according to the invention conjugates has the advantage, that is, to achieve therapeutic actions they need less often to insert.

Compared to non-modified allergens proposed according to the invention to jugate HAS-allergen can be obtained thereby they have reduced binding with specific allergen IgE. Conjugates HAS-allergen according to a particularly preferred variant implementation may have only very little or not at all to have a specific relationship with a specific allergen IgE. Proposed according to the invention, the conjugates thus you can enter in higher doses, which again increases the chance of a successful hyposensitization.

Compared with crosslinked allergoids conjugates HAS-allergen according to the present invention have the advantage that they can provide comparable with the natural allergen epitope profile. This way you can increase the effectiveness of immunotherapy. In contrast, the copolymerization of allergens with formaldehyde or glutaraldehyde leads to poorly defined macromolecular compounds (Crit. Rev. Ther. Drug Carrier Syst., 6 (4), 315-365 (1990)), which can create non-natural epitopes, so that in case of their action to investigate.

In the conjugate is at least one hydroxyalkylated associated with the allergen. Under the scope of the invention fall, of course, also the products of the binding, which have several molecules hydroxyalkylated and one allergen molecule or multiple molecules of an allergen and one mole is kulai hydroxyalkylated.

Hydroxyalkylated in the conjugate can be directly associated with the allergen, or is associated with allergen through a linker. Next hydroxyalkylated can be linked to the polypeptide chain or with one or more sharedname chains allergenic glycoprotein.

Hydroxyalkylated (HAS)

In the framework of the present invention the term "hydroxyalkyl" is used to denote derivatives of starch, substituted hydroxyalkyl group. Hydroxyalkyl group preferably contains from 2 to 4 carbon atoms. Referred to as "hydroxyalkyl" thus preferably consists of hydroxyethylamine, hydroxypropylmethyl and hydroxybutyryl. Especially preferred is the use of hydroxyethylamine (HES) as a component binding in the case of all embodiments of the invention.

According to the invention, preferably, gidroxiatilkrahmal used to obtain conjugates, has an average molecular mass (mass-average molecular mass) 1-300 kDa, and especially preferred average molecular weight of from 5 kDa to 200 kDa. Gidroxiatilkrahmal further may have a molecular degree of substitution of 0.1 to 0.8 and the ratio of substitution With2:C6within 2-20, in each case calculated on hydroxyethylene group.

Allergens

In the framework of the present invention allergens, primarily referred to as compounds that can induce an allergic immune response, in a more narrow sense IgE mediated allergic reactions (type I). Next covers produced from the sequence of the allergen peptides, such as, for example, the cleavage products obtained by enzymatic splitting. Relevant allergens used for specific immunotherapy and they are available for sale.

Allergens can be isolated from natural sources. For example, in the case of pollen allergens receive extracts of allergens from the corresponding pollen. Further, the allergens can be obtained, for example, by recombinant.

In the case of allergens, it is preferably the compounds that are selected from the group consisting of polypeptides, proteins and glycoproteins.

Ways to get

According to one aspect of the invention relates to methods of producing conjugates HAS-allergen, which HAS either directly or through a linker covalently bound to the allergen. Linking can be accomplished in a number of ways. The General structure of the synthesis of neoglycoproteins when using the linker is represented in figure 1.

According to one variant of implementation of the present invention relates to the persons receiving conjugates HAS-allergen, in which case HES associated with ε-NH2group, with α-NH2group, SH group, COOH group or-C(NH2)2group allergen.

The invention relates further to a method, in which case HES associated with ε-NH2-group protein via reductive amination. An alternative to this, the invention relates to a method in which the allergen is associated with the reducing end groups of hydroxyethylamine.

According to the following variant implementation of the invention relates to methods in which to bind to the allergen in HAS introduced the active group. The active group may be, for example, aldehyde group, Tolna group or amino group.

Allergen and oligo - or polysaccharide can communicate with each other directly or through the use of a linker. As the linker, you can use any crosslinking agent. The linker can be, for example, bifunctional linker, or Homo - or heterobifunctional cross-linker.

Commercially available and known specialist numerous cross-linking agents that can be used within the present invention, such as SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (see list in alphabetical order under the title "Cross-linking Reagents" in the catalogue of products Perbio www.piercenet.com).

The present invention according to another variant implementation is to get the methods of the conjugates HAS-allergen.

Below outlines some of the ways of synthesis of conjugates HAS-allergen. Specialist in the field bioconjugates will not have problems with the choice of the described methods are particularly suitable in respect of tasks (choosing allergen, HAS chosen and so on).

Direct linking of unmodified HAS with allergenic proteins by reductive amination

A simple and gentle way that you can implement without modification HAS is a direct link HAS with ε-allergenic amino groups of the protein by reductive amination in the presence of NaCN/BH3(G.R. Gray, Arch. Biochem. Biophys., 163, 426-428 (1974)) (Fig. 2.1a).

As reducing agents can also be used pyridineboronic complex and other aminoborane complexes that are more stable and more easily manipulated (J.C. Cabacungan and other, Anal. Biochem., 124, 272-278 (1982)). In contrast to the acylation modified amino group of the protein, in addition, under physiological conditions remains positively charged. Therefore, impacts on the tertiary structure of the protein during the restoration aminating less. In the case of this method, one is about, disappears cyclic structure reducing sugar.

Way linking HAS modified.

The oxidation of the reducing end with getting aldonova acids

In the case of rarely used by oxidation with iodine (or bromine) to the appropriate aldonova acid (G. Ashwell, Methods Enzymol., 28, 219-222 (1972)) vanishes cyclic structure reducing sugars (Fig. 2.1b), in addition, requires careful control of the reaction to avoid nonspecific oxidation. The resulting carboxyl group in the presence of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (J. Lunngren, I.J. Goldstein, Methods Enzymol., 247, 116-118 (1994)) can be associated with ε-lysine amino groups of the side chains of allergenic protein or via a hydrazide linker (see figure 3). Similarly for the binding, you can also use the existing polysaccharide structures carboxyl group, for example, mannurone, glucuronic or sulinowo acids.

According to a particularly preferred variant implementation of the present invention receives compounds consisting of conjugate HES-allergen, in which the allergen is specifically linked to the reducing end groups of hydroxyethylamine. For this purpose, the reducing end groups previously subjected to selective oxidation, for example, according to the method described by Hashimoto and others (Kunstoffe, Kautschuk, Device, 9, 1271-1279 (1992)) for the oxidation of the reducing end aldehyde group of the saccharide.

Activating the hydroxyl group HAS

One of the most common methods of non-specific activation of polysaccharides is an introduction into interaction with bromine cyan (CNBr) (C. Chu and others, Infect. Immun., 40, 245-256 (1983)) (Fig. S). Activated hydroxyl group acelerou lysine, cysteine and his-tag side chains of the protein. This method of binding, however, may have disadvantages, which are reduced to a high pH value, as well as toxicity and poor manipuliruemoy.

Alternative CNBr is CDAP (1-cyano-4-dimethylamino-pyridinylmethyl) (A. Lees and others, Vaccine, 14, 190-198 (1996); D.E. Shafer and others, Vaccine, 18, 1273-1281 (2000)) with high reactivity of ceanography that allows to carry out the reaction in a much more mild conditions.

In General, non-specific activation of polysaccharides can lead to multiple substitution and thus to cross-links between polysaccharide and protein. By selecting the reaction conditions to this, however, can largely be discouraged.

The introduction of aldehyde groups

Also in nereguliruemyi polysaccharides can be entered aldehyde group by cleavage of vicinal hydroxyl groups using NaIO4(J.M. Bobbit, Ad. Carbohydr. Chem., 11, 1-41 (956)) (Fig. 2.1d), and due to the concentration of the solution periodate sodium can achieve sufficient selectivity. Particularly easily oxidized is sulinova acid (S.M. Chamov and others, Biol. Chem., 267, 15916-15922 (1992)).

Increase the reaction rate in the case of a direct reductive amination using reducing polysaccharides can be achieved by introducing aldehyde groups, which are not cyclists to polyacetale. This can be achieved by restoring the reducing end to receive the reduced sugar and subsequent selective oxidation of vicinal diols in the open reduced sugar (Y.C. Lee, R.T. Lee, Neoglycoproteins: Preparation and Application, Academic Press, San Diego, 1994) (Fig. 2.1d).

Along with direct linking modified with aldehyde groups of the polysaccharides with the amine groups of the protein due to reductive amination in this way it is also possible modification of the polysaccharide using bifunctional hydrazide groups of the linkers (see below).

Introduction of amino groups

Compared with polysaccharides, in the case of oligosaccharides (with the amount of carbohydrate monomer unit up to 20) on the basis of a slightly higher reactivity created the best possible conversion of reducing sugars due to reductive amination in glucamine or glycosylamine with intact qi is symbolic structure (Fig. 2.2).

To bind modified with amino groups of proteins with different functional groups of the side chains of the protein provides the use of a bifunctional linker (see below).

Introduction of amino groups by reductive amination

In contrast to the synthesis of glycemia by reductive amination with NH3or aliphatic amines (B. Kuberan, etc., Glycoconj. J., 16, 271-281 (1999)), when using aromatic amines as, for example, benzylamine (.Yoshide, Methods Enzymol., 247, 55-64 (1994)), 2-(4-AMINOPHENYL)ethylamine (AREA) (H.D. Grimmecke, H. Brade, Glycoconj. J., 15, 555-562 (1998)) or 4-triftoratsetofenona (E. Kallin, Methods Enzymol., 247, 119-123 (1994)), under comparable conditions it is possible to achieve higher yields (Fig. A).

While in the case of AREA for selective reactions using different reactivity of aliphatic and aromatic amino groups, using 4-triftoratsetofenona get monoamine connection (alternative also use benzyloxycarbonylamino (M Barström and others, Carbohydr. Res., 328, 525-531 (2000)), and due to the subsequent removal of trifluoracetyl group again released an aromatic amino group. In addition, we discovered that glucamine to off trifluoracetyl protective groups can be stabilized by a simple N-acetylation using uksosn the th anhydride.

Introduction of amino groups by N-glycosylation

N-Glycosylation (Fig. 2.2b) allows you to save a cyclic structure reducing sugar. Produced by the interaction with the ammonium bicarbonate unstable β-glycosylamine (I.D. Manger and others, Biochemistry, 31, 10724-10732 (1992); I.D. Manger and others, Biochemistry, 31,10733-10740 (1992); S.Y.C. Wong and others, Biochem. J., 296, 817-825 (1993); E. Meinjohannes etc., J. Chem. Soc., Perkin Trans., 1, 549-560 (1998)) can be stabilized by subsequent acylation with Chloroacetic anhydride acid and by aminolysis be turned into a l-N-glycerine compound with a free amino group. Similarly, N-glycosylation can be achieved through allylamine and after stabilization at the expense of N-acetylation by photochemical join the group probably facilitates double bond (D.Ramos and others, Angew. Chem., 112, 406-408 (2000)).

Obtaining amino groups of aldonova acids

In aldonova acid obtained by oxidation of the reducing polysaccharides, due to the interaction with diamines, you can enter a free amino group. This is possible by reaction of the acid with carbodiimide and diamines. Alternative lactones derived from dehydration aldonova acids, you can enter into interaction with the diamines (S.Frie, Thesis, Special school, Hamburg, 1998).

Binding assays modified and HES allergic Proteus is s using bifunctional linkers

As diverse as the functional group of the modified HES and protein side chains that need to be connected to each other via a linker, are current opportunities reactions (figure 3 presents the conventional activated using linkers).

In the case of reactive groups can be the difference between reactivity towards amino groups (complex NHS-esters, complex imidiately and arylazide), aldehyde groups, and (in the presence of EDC) groups of carboxylic acids (hydrazides) or SH-groups (maleinimide, halogenated or pyridylsulfonyl).

Reagents reactivity towards amino groups

Of binding reagents most commonly used are reactive towards amino groups of the cross-linkers. The complex N-hydroxysuccinimidyl (complex NHS-esters) (Fig. 3.1a) represent the most common form of activation. When the removal of the NHS are formed acylated compounds. Another possible modification of primary amines are difficult imidiately (F.C. Hartman, F. Wold, Biochemistry, 6,2439-2448 (1967)) (Fig. 3.1b), and are formed amidoamine (amidine). Complex imidiately repeatedly used as a protein cross-linkers and they have minimal reactivity towards others is nucleophiles connections. Then there are various arylazide linkers (photoreactive cross-linkers), in which case by photolysis formed a short-lived nitrene. Due to the expansion cycle of them (instead of non-specific insertions) are formed dehydroalanine, which preferentially react with nucleophilic compounds, particularly amines (Fig. S).

Due to the variety of manufactured reagents binding activity in relation to the amino groups and a variety of linkers are other possible reactions, which, for example, in cooperation with isocyanates and isothioscyanates increasingly losing value.

Reagents with reactivity against carbonyl or carboxyl groups

Hydrazide linkers are used to link compounds with carbonyl or carboxyl groups (D.J. O Shanessy, M. Wilchek, Anal. Biochem., 191, 1-8 (1990)) (Fig. 3.2). While aldehydes are converted to hydrazones, which can be stabilized by restoring using NaCN/BH3, carboxyl groups in the presence of EDC reacts with the education imenik links. Activated hydrazide linkers are a versatile alternative to reductive amination and enable carboxyla using "zero-length" cross-linkers, as carbonyldiimidazole (CDI).

Reagents R the promotional ability in relation to sulfhydryl groups

Reagents binding reactivity against SH represent the second large class of cross-linkers. Binding assays initially include two paths reactions: alkylation (Fig. A-b) or disulfide exchange (Fig. 3.3s). In the formation of stable simple thioester communications, along with alkylation using α-halogenated double bond of maleimide selectively reacts with SH groups by reaction joining Michael. Thiol-disulfide exchange is another specific sulfhydryl groups reaction. Especially preferred is the reaction with pyrimidinylidene (J. Carlsson and others, Biochem. J., 173, 723-737 (1978)), since the removal of 2-pyridone is possible to attain complete transformation in mixed disulfides.

Cross-linkers

For the synthesis proposed according to the invention bioconjugates HAS-allergen using the above binding assays due to a variety of Homo - and heterobifunctional cross-linkers.

Homobifunctional cross-linkers

Symmetric homobifunctional linkers (see, for example, shown in Fig. 4.1) on both ends contain the same reactive group suitable for binding of the compounds with the same functional groups. According to reactions with whom Azania commercially available bifunctional linkers, for example, with complex bis-amidoamine, bis-operations, bis-hydrazide groups and bis-maleinimide functions.

The drawback to the use of homobifunctional of linkers is that even when using a large excess of cross-linker by activating the first connection can't fully prevent cross-stitching (S. Bystrick and others, Glycoconj. J., 16, 691-695 (1999)). You must complete separation before being associated with the second connection and this can be difficult due to instability of the activated intermediate product (for example, sensitivity to hydrolysis activated with NHS connections). As reactivity towards amino groups and hydrolysis of complex NHS-esters increase with increasing pH value, resulting in reactions under physiological conditions (pH 7) is carried out in buffered solutions (half-time complex NHS-ester DSP at a temperature of 0°and the pH value of 7 is 4-5 hours, at pH 8,6 only another 10 minutes; A.J. Lomant, G. Fairbanks, J. Mol. Biol., 104, 243-261 (1976)).

Heterobifunctional cross-linkers

Using heterobifunctional reagents binding (see, for example, shown in Fig. 4.2) can be related to each other in connection with various functional groups. Linkers containing two different reactive groups, and the and by combining different coupling reaction can be selectively engaged with one end of the cross-linker. For example, one side of the linker possesses activity against amino group, and the other in respect of sulfhydryl groups, which makes compared to homobifunctional the linkers achieve the best possible control of the reaction.

First, the interaction becomes more reactive or more unstable side heterobifunctional linker. Since the complex NHS-esters can react not only with amino groups with the formation of a stable amide bond, but with sulfhydryl and hydroxyl groups, heterobifunctional the first linker interacts with aminoguanidinium. Maleinimide group, in contrast, not only has greater selectivity, but also more stable in aqueous solution, so that the activated intermediate product can be cleaned and then selectively enter into interaction with the connection with sulfhydryl activity.

When selecting the cross-linker are guided not only native functional groups that you want to use for binding, but also the desired length and composition, the so-called cross a bridge, spacer. So, some spacers, particularly those with a rigid cyclic structure, such as SMCC or MBS, lead to the emergence of a specific antibody response (J.M. Peeters and others,J. Immunol. Methods, 120, 133-143 (1989)), and thus may be less suitable for immunogene the hapten-carrier and application in vivo.

For the selection of linkers according to figure 4 are produced specifically degradable linkers, which may be disclosed by cleavage of the disulfide (e.g., DSP, DTME or DTBP) or splitting periodate (diols, as BMDB or DST) and which are used for studying biospecific intermediate reactions or for the purification of unknown structures of target.

Abbreviations manufactured binding reagents are produced from the systematic names of the compounds, as, for example, DMA (Dimethyladipimidat=dimethylacetamide), DMS (Dimethylsuberimidat=dimethylsuberimidate), GMBS (N-(γ-Maleimidobutyryloxy)siccinimidester=complex N-(γ-maleimidomethyl)Succinimidyl) etc.

Review various heterobifunctional cross-linkers, which, for example, can be used to bind sulfhydryl groups are presented in figure 5.

Great variety in this case are activated by maleinimide linkers, often in combination with activation in the form of a complex NHS-ester. These linkers with reactivity against sulfhydryl groups and amino groups are water-insoluble linkers forming linear alkylene the bridges, as, for example, AMAS, GMBS and EMCS, or, as SMCC, SMPB, or MBS, has a rigid cyclic structure. Both UV-active linker SMPB and MBS are usually used for immunochemical methods, such as enzyme-linked immunosorbent assay.

Using M2C2H moreover get a linker with the same rigid bridge connection, as in the case of SMCC, however, with the activation of the hydrazide to bind compounds with sulfhydryl and carbonyl or carboxyl activity.

In contrast to the water-insoluble linkers that before the reaction must first be dissolved in an organic solvent, such as dimethylformamide or dimethylsulfoxide, there are linkers in the form of complex NHS-esters with hydrophilic sulfo (J.V. Staros, Biochemistry, 21, 3950-3955 (1982)), as, for example, sulfo-GMBS, sulfo-EMCS, sulfo-SMCC, in addition, water soluble versions of some linkers.

Along with activated maleinimide heterobifunctional the linkers to bind sulfhydryl groups are different halogenated, as, for example, SIA (and promoterami similar), and fairs are forthcoming-Siab SBAP (Fig. 5.2), and pyridylsulfonyl as SPDP and LC-SPDP and Sulfo-LC-SPDP (Fig. 5.3), in combination with activation in the form of a complex NHS-ester binding amino groups. Halogenoacetyl group you can enter in aminirovanie polysaccharides by reaction with the free acid and in rastvorimym a carbodiimide (N.J. Davies, S.L. Flitisch, Tetrahedron Lett., 32, 6793-6796 (1991)) or with an appropriate anhydride (I.D. Manger and others, Biochemistry, 31, 10733-10740 (1992); S.Y.C. Wong and others, Biochem. J., 300, 843-850 (1994)) (see Fig. 2.2b).

Regarding the binding of synthetic oligosaccharides with side SH-chains of proteins using heterobifunctional maleinimide of linkers in the literature there are various examples (V. Fernandez-Santana and others, Glycoconj. J., 15, 549-553 (1998); G. Ragupathi, etc., Glycoconj. J., 15, 217-221 (1998); W. Zou and others, Glycoconj. J., 16, 507-515 (1999); R. Gonzalez-Lio, J. Thiem, Carbohydr. Res., 317, 180-190 (1999)). In addition, also use direct linking iodoacetamide derived oligosaccharides for specific glycosylation of proteins (N.J. Davies, S.L. Flitsch, Tetrahedron Lett., 32, 6793-6796 (1994); S.Y.C. Wong and others, Biochem. J., 300, 843-850 (1994)).

Modification of glycoproteins in the carbohydrate part by using poly - and oligosaccharides

Alternative amino acid side chains of the protein, in the case of glycoproteins also linked oligosaccharides have other sites linking to education proposed according to the invention conjugates (J.J. Zara and other, Anal. Biochem., 194, 156-162 (1991)).

The introduction of aldehyde groups by oxidation with periodate sodium

By oxidation with periodate sodium in nereguliruemyi oligosaccharides can introduce aldehyde groups. Depending on the choice of oxidation conditions can selectively oxidize available sulinova acid sludge is less selectively oxidize also fucose residues, mannose, galactose and N-acetylglucosamine (S.M. Chamov, etc., J. Biol. Chem., 267, 15916-15922 (1992)). As a side reaction, the formation of aldehyde groups of the N-terminal serine, cysteine or threonine (D.J. O Shanessy, M. Wilchek, Anal. Biochem., 191, 1-8 (1990)).

Enzymatic introduction of aldehyde groups

Oxidation of glycoproteins using galactosidase leads to the formation of C6aldehyde groups in end galactose or N-acetylgalactosamine. Especially in the glycoproteins of animal cells, these sugars are not the end, so they need to make available only at the previous stage (D.J. O Shanessy, M. Wilchek, Anal. Biochem., 191, 1-8 (1990)).

The pharmaceutical composition

The present invention further relates to pharmaceutical compositions containing proposed according to the invention, the conjugate HAS the allergen. Proposed according to the invention, the conjugates are particularly preferably suitable for the preparation of pharmaceutical compositions that can be used to hyposensitization Allergy sufferers. Pharmaceutical compositions particularly suitable for the treatment of allergies, which detect IgE mediated sensitization and see the corresponding clinical symptoms.

Accordingly, proposed according to the invention, the conjugates can be used in particular for preparing Pharma is efticiency compositions which are suitable for specific immunotherapy of patients with clinically relevant reactions to allergens, immediate type, as, for example, allergies are allergic to pollen, mites, hair (saliva) mammals, fungi, insect bites, food, and natural rubber/latex. Thus, immunotherapy particularly suitable for the treatment of asthmatics and patients suffering from hay fever.

Proposed according to the invention compositions can be used in various forms of specific immunotherapy, particularly in the case of hyposensitization. Thus, the desensitization can be accomplished by introducing through the mucous membrane and subcutaneous, oral, oral or sublingual proposed according to the invention HES-conjugates. Next hyposensitization can take the form of various treatment protocols (pre-season/regular).

Especially in the case of Allergy sufferers are allergic to insect bites can be proposed implementation of treatment Rush-method or ultra-Rush-method (see Kleine-Tebbe and others, Pneumologie,5,438-444 (2001)).

For the preparation of pharmaceutical compositions proposed according to the invention, the conjugates are mixed with suitable for hyposensitization carriers and/or auxiliary substances.

Conjugate of HES and allergenic glycopro theine

Upon receipt of conjugates of HES-glycoprotein for binding can be used, for example, the following types of functional groups glycoprotein:

And: Tolna group of the cysteine side chain;

In: aldehyde group of the oxidized galactose residue.

In the case of proteins, which deglycosylation, accordingly, there is no alternative to the Century

HES differs only reducing end. On the basis of this structural characteristic, in the framework of the present invention is particularly suitable targeted regioselective binding.

For the synthesis of conjugate HES-protein, you can use the concept of chemical ligation, which was developed for the design of proteins greater magnitude of unprotected peptide fragments. These concepts are based on selecting a single reactive functional groups in the stitching fragments, which in the presence of many other functional groups in natural proteins selectively react with each other with formation of a stable final product.

In General, HES a drug is first transferred to a specific, highly purified and well-characterized intermediate product (reactive HES), which then spontaneously and regioselective in physiological conditions can react with the functional group of the target allergen.

Pre is respectful is a selective transfer of the reducing end HES in primary amino group (1-amino-HES). This "1-amino-HES" then flexibly adapt to binding assays with protein, and you can do different ways of synthesis, and the reaction due to prior reagents (linkers) can be combined into one stage.

HS-reactive HES

Below schematically describes and evaluates alternative ways to get HS-reactive HES:

1. Reductive amination HES using a bifunctional linker M2With2N (Fig. 5.1.b) to HS-reactive HES (A);

- purification by dialysis and freeze-drying;

- binding protein HS by joining Michael.

This synthesis has special advantages, as it is very simple (one step) and the reaction with the protein target is very selective. If the toxicity of hydrazine derivatives causes problems, then they need to fix due to the known from the prior art cleaning methods.

2. The transformation of HES-lactone (oxidized HES) using a bifunctional linker M2With2N (Fig. 5.1.b) in HS-reactive HES ();

- purification by dialysis and freeze-drying;

binding of HS-protein by reaction of accession to Michael.

This reaction differs from that described above in paragraph 1 of the additional costs of obtaining HES-lactone.

3. The transformation HES using ammonium hydrogen carbonate in 1-amino-HES ();

- cleaning is here freeze-drying;

acylation of 1-amines using bromine/iodoacetonitrile without basic catalysis with obtaining bromine/iodoacetamide (HS-reactive HES (D));

- purification by dialysis and freeze-drying; the binding of HS-protein by alkylation.

This is the preferred method; it includes only two stages and requires only very simple reagents. The method thus requires a very small cost. The scale of synthesis is legalacsonyabb. The reaction with the protein target is very selective.

4. The transformation of HES-lactone (oxidized HES) with the diamine (1,4-diaminobutane) Frie (S. Frie, Thesis, Special high school, Hamburg, 1998) amino - HES (E);

the acylation of amino-HES using bromine/iodoacetonitrile without basic catalysis to bromine/iodoacetamide (HS-reactive HES (F));

- purification by dialysis and freeze-drying; the binding of HS-protein by alkylation.

This path synthesis differs from that described above in paragraph 3 additional costs of obtaining HES-lactone.

SNO-jet HES

Below schematically describes and evaluates alternative ways to get SNO-jet HES:

1. The use of amino-HES (E) as SNO-jet HES (G);

- linking with SNO-protein by reductive amination.

This synthesis is very about the t and is inexpensive. Competition internal lysine can cause problems that can be easily controlled by choosing the reaction conditions.

2. The transformation HAS-lactone (oxidized HES) using hydrazine hydrazide (SNO-jet HES (N));

- purification by dialysis and freeze-drying;

- linking with SNO-protein through the formation of hydrazone at pH 5-6; binding reaction preferably should be performed in situ during the oxidative formation of hydrazone (enzymatic or chemical) of the galactose residues; optionally perform subsequent reductive stabilization using NaCN/NR3;

- enzymatic oxidation of galactose preferably should be implemented using linked polymer enzyme to facilitate purification of the enzyme.

This synthesis is very simple and selective (no competition internal lysine). Problems may arise because of the toxicity of hydrazine derivatives.

3. Further transformation of D or F using ammonium hydrogen carbonate in glycinamide (SNO-jet HES (H) and (I));

- purification by dialysis and freeze-drying;

- linking with SNO-protein by reductive amination.

This method is carried out in three stages, however, required a very simple reagents and, therefore, it is inexpensive. The scale of the synthesis of the C is easily extensible. However, you can face the competition of the internal lysine (see above).

4. The acylation of amino-HES (C) or (e) of cBz-aminouksusnoy acid, followed by hydrogenation to aminooxy-HES (SNO-jet HES ());

- linking with SNO-protein due to oximes communication at pH 5-6; preferably the binding reaction should be performed in situ during the oxidation of education (enzymatic or chemical) of the galactose residues;

- enzymatic oxidation of galactose preferably should be implemented using linked polymer enzyme to facilitate purification of the enzyme.

This synthesis is costly, however, binding to the protein target is the same selective, as in the case described in paragraph (2 reactions (no competition internal lysine).

1. The conjugate of hydroxyalkylated and allergen, in which at least one hydroxyalkylated covalently linked to the allergen, for hyposensitization.

2. The use according to claim 1, and hydroxyalkylated associated with the allergen directly or through a linker.

3. The use according to claim 1, wherein hydroxyalkenals is gidroxiatilkrahmal, hydroxypropylmethyl or hydroxybutyryl.

4. The use according to claim 1, and gidroxiatilkrahmal has an average molecular weight of from 1 to 300 K Yes, preferably the average mo is coolaroo weight of from 5 to 200 kDa.

5. The use according to claim 1, and gidroxiatilkrahmal has a molecular degree of substitution of 0.1 to 0.8 and the ratio of substitution With2:C6in the range of 2 to 20, in each case calculated on hydroxyethylene group.

6. The use according to claim 1, the allergen is selected from the group consisting of polypeptides or proteins.

7. The use according to claim 6, wherein the allergen is a glycoprotein.

8. The use according to claim 7, and hydroxyalkylated linked to the polypeptide chain or with one or more sharedname chain glycoprotein.

9. The use according to claim 1 for hyposensitization Allergy, in which case the detected IgE mediated sensitization or have observed clinical symptoms.

10. The use according to claim 1, and specific immunotherapy is used to treat allergies to pollen, mites, hair (saliva) mammals, fungi, insect bites, food, and natural rubber/latex.

11. The use according to claim 1, and therapy used to treat asthmatics suffering from hay fever patients and patients who have other clinically relevant reactions to allergens, immediate type.

12. The use according to claim 1, and introduced subcutaneously, through the mucous membrane, oral, oral or sublingual.

13. The use according to claim 1, and immunotherapy in case the e aeroallergens is pre-season or permanently.

14. The use according to claim 1, and immunotherapy in the case of Allergy sufferers are allergic to insect bites is Rush-method or ultra-Rush-method.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: activated polymer derivatives of bicin are described as well as conjugates obtained with the aid of the derivates. Also modes of obtaining and application of the bicin derivatives are described.

EFFECT: obtaining of bicin derivatives.

24 cl, 11 dwg, 2 tbl, 21 ex

FIELD: medicine.

SUBSTANCE: composition contains: a. core composition including therapeutically active agent, swelling agent and capillary agent; b. coating composition including water-insoluble polymer and water-soluble polymer.

EFFECT: composition releases therapeutically active agent in pulse mode at preset time.

21 cl, 15 tbl, 7 ex

FIELD: medicine, polymers, pharmacy.

SUBSTANCE: invention relates to a copolymer or its pharmacologically acceptable salt that comprises the following components as elemental links forming their: (a) one or some structural elemental links describes by the formula (I) given in the invention description, and (b) one or some structural links describes by the formula (II) given in the invention description. Disposition of these structural elements represented by the formulae (I) and (II) is chosen from the following sequences: (i) sequence with alternation "head-to-head"; (ii) sequence with alternation "head-to-tail"; (iii) mixed sequence with alternation "head-to-head" and "head-to-tail"; (iv) random sequence and taking into account that the ratio between structural links of the formula (I) and structural links of the formula (II) in indicated copolymer is in the range from 10:1 to 1:10. Also, the invention relates to a copolymer or its pharmacologically acceptable salt synthesized by addition of one or some links of carboxylic acid anhydride described by the formula (III) given in the invention description that comprises as elemental links: (a) one or some structural elemental links described by the formula (I), and (b) structural link comprising carboxylic acid anhydride link described by the formula (III) for one or some reactions chosen from the group consisting of: (i) hydrolysis; (ii) ammonolysis; (iii) aminolysis, and (iv) alcoholysis. Also, invention relates to a pharmaceutical composition used for prophylaxis or treatment of osseous metabolism disorder and comprising an acceptable excipient or carrier, at least one of above indicated copolymers or their pharmaceutically acceptable salts and at least one protein representing osteoclastogenesis inhibition factor (OCIF) or its analogue, or variant. Also, invention relates to a modifying agent comprising above said copolymers, to a complex between of one of above said copolymers and protein or its analogue, or variant, to a pharmaceutical composition comprising this complex. Also, invention relates to a method for time prolongation when OCIF is retained in blood stream after intake by a patient a complex between protein and at least one of above said copolymers. Also, invention relates to a method for treatment or prophylaxis of disorders of osseous metabolism involving intake by a patient the effective amount of complex comprising complex including OCIF or its analogue or variant and bound with at least one of the claimed copolymers. Also, invention relates to use of the complex comprising OCIF bound with at least one of the claimed copolymers designated for preparing a drug designated for prophylaxis or treatment of disorder of osseous metabolism and showing sensitivity to the protein effect. Modifying the protein, namely OCIF, by the claimed copolymers results to formation of complex possessing uniform properties being especially characterizing by reduced formation of disordered structure cross-linked with protein, improved retention of the protein activity and the excellent retaining protein in blood after intake of the indicated complex.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agents.

110 cl, 13 tbl, 3 dwg, 40 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to drugs and concerns sustained-release oral compositions. Agent comprises micelle-forming water-soluble main drug showing a positive charge at physiological pH value, polymer showing an opposite charge chosen from group consisting of polyacrylic acid, carboxymethylcellulose, xanthane gum, hellane gum, guara gum, dextran-sulfate and carragheenan, polyethylene oxide and if necessary a hydrophilic base. Also, invention proposes a method for sustained-release of micelle-forming drug.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agent.

20 cl, 4 tbl, 24 dwg, 13 ex

FIELD: pharmacy, peptides.

SUBSTANCE: invention relates to a pharmaceutical composition that comprises an oligopeptide preparation and comprising oligopeptide of the formula (I): cyclo-(n-Arg-nGly-nAsp-nD-nE) and esterified β-cyclodextrin, and to a method for preparing an aqueous pharmaceutical preparation. Invention provides significant increasing solubility of oligopeptide and stability of the preparation for relatively prolonged time.

EFFECT: improved and valuable pharmaceutical properties of preparation.

13 cl, 6 tbl, 5 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention relates to drugs and concerns an antitumor composition for injection. Agent comprises derivative of anthracycline possessing the antitumor activity as an active component, and block-copolymer formed by derivative of polyethylene oxide and derivative of polyaspartic acid. The composition comprises additionally saccharide and a base represented by sodium hydrocarbonate, sodium hydrophosphate, sodium citrate and sodium hydroxide with pH value in the range 4-9. Also, invention proposes a composition based on block-copolymer micelles, solution of block-copolymer micelles with pH value in the range 4-9, and a solid composition for injection prepared by drying a micellar solution. Compositions show stability for a prolonged period and possess the improved capacity for repeated dissolving.

EFFECT: improved and valuable pharmaceutical properties of preparation.

15 cl, 2 tbl, 10 ex

FIELD: chemical-pharmaceutical industry and technology, pharmacy.

SUBSTANCE: invention relates to a composition in form of microemulsion concentrate designated for oral using, for example, in gelatin capsule. Proposed pharmaceutical composition provides high bioavailability of preparation. Pharmaceutical composition in form of microemulsion concentrate comprises ciclosporin and accessory components: (a) propanediol monocaprylate as a solvent; (b) monocaprylate glyceryl; (c) polyethylene glycol 40, hydrogenated castor oil and linoleic acid glycerides polyethylene glycol as surfactants; (d) 1,2-propylene glycol as a hydrophilic component; (e) alpha-tocoferol acetate as an antioxidant wherein in dilution microemulsion concentrate forms microemulsion to be easily dosed with particles size 15-20 nm.

EFFECT: improved and valuable properties of pharmaceutical composition.

1 tbl, 5 ex

FIELD: organic chemistry, pharmacy.

SUBSTANCE: invention relates to polyalkylene glycolic acids and their binding with therapeutically effective biopharmaceutical preparations, such as polypeptides, sugars, proteins and therapeutically active low-molecular compounds in aims for preparing therapeutically active conjugates of polyalkylene glycol with indicated biopharmaceutical preparations. Invention claims compounds of general formulae (I-A) and (I-B) wherein X means -NH-, and compounds of the general formula (I-C) wherein X means -O- or -NH-. Also, invention claims methods for synthesis of these compounds that involve the condensation reaction of compound of the formula: RO-PAG-V (for compounds of formulae (I-A) and (I-B)) or compound of the formula: RO-PAG1-O-(CH2)k-V (for compound of the formula (I-C)) wherein V means -OH or -NH2 with corresponding halide, hydrolysis of prepared ester to form a corresponding acid and, if necessary, interaction of indicated free acid with halogen in an activated leaving group in the presence of a condensing agent to form indicated activated ester. Also, invention claims conjugates of the general formula that are given in the invention description. Indicated reagents show enhanced stability in aqueous medium and they can be used for site-specific modification of protein. Claimed compounds possess higher activity and prolonged biochemical half-life index.

EFFECT: improved method of synthesis, valuable properties of additives.

19 cl, 9 ex

FIELD: pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a method for preparing the inclusion compound of piroxicam with β-cyclodextrin. Method involves freezing process of aqueous solution of two components before drying at very high rate. Prepared products show physicochemical properties and technological and biopharmaceutical properties that display advantage properties as compared with properties prepared by methods of preceding practice. Solubility of piroxicam in tablets prepared by the proposed method is ≥90% for 10 minutes. Prepared products are useful for preparing pharmaceutical compositions for oral administration.

EFFECT: improved preparing method, improved properties.

13 cl, 2 dwg, 2 ex

FIELD: medicine, endocrinology, pharmacology.

SUBSTANCE: invention describes a method for preparing the insulin preparation. Method involves adding insulin to 1-50% aqueous solution of polyethylene oxide of molecular mass 0.4-40 kDa irradiated preliminary by ionizing radiation in the dose 1.0-5.0 Mrad to its final concentration 1-10 mg/ml and in the ratio polyethylene oxide : insulin = (1-500):1. Prepared mixture is stirred up to preparing a homogenous, clear or slightly opalescent solution. Method provides simplifying the process for preparing the insulin preparation and to enhance its therapeutic activity.

EFFECT: improved preparing method.

2 cl, 3 tbl, 3 ex

FIELD: veterinary microbiology.

SUBSTANCE: the innovation deals with intracutaneous injection of tuberculin at the volume of 0.2 ml followed by analyzing the reaction 72 h later. Animals at increased skin fold by 3 mm and more should be considered to be sick ones. Moreover, tuberculin should be injected at the dosage ranged 0.6-0.8 thousand IU. The innovation excludes the impact upon final result in predicting the factors of tuberculosis-free etiology and provides the liquidation of groundless slaughtering in cattle.

EFFECT: higher accuracy of diagnostics.

2 ex, 2 tbl

FIELD: medicine, in particular production of diagnostic allergen from cockroach.

SUBSTANCE: claimed method includes alkali extraction of raw materials followed by centrifugation, filtering, and sterilizing filtering of target product, wherein as raw material mixture of frozen cockroaches is used. Said mixture is crushed, then ether is added, mixture is shacked, filtered, dried to full ether evaporation, grated to produce powder. In obtained powder ether is added again, powder is dried to full ether evaporation, and alkali extraction is carried out. Extracting material is agitated and for 3 days is shacked three times daily for 30 min with 1.5 h interval and stored for these intervals at 2-10°C. After extraction supernatant is discharged, centrifuged, and after sterilizing filtering sterile allergen solution is held for 1-3 months at 2-10°C.

EFFECT: high effective preparation for diagnosis of diseases associated with sensitizing to cockroach.

1 ex

FIELD: medicine.

SUBSTANCE: method involves applying alkaline extraction from raw material. Daphnias are used as initial raw material, preliminary degreased with ethylic ether, dried up and crushed up to powder condition. The fat-free powder is extracted with 0.1 mole/l sodium hydroxide solution during 3 days at temperature of 4-8°C. Supernatant liquid is poured out after having finished extracting and protein is sedimented from alkaline extract with sodium benzoate as follows: 20 g of sodium benzoate is available in 1 l of alkaline extract, alkaline extract рН is reduced to 3.7. Sedimented benzoic acid is precipitated with the adsorbed allergen by centrifuging within 5 min at 2000 rpm. Allergen desorbed with benzoic acids by adding to acetone deposit. The mixture is stirred to completely dissolve benzoic acid and then it is places for 12-18 h into refrigerator at -20°С temperature. The solution is filtered, waiting 18-20 hours for full evaporation of acetone traces. Allergen powder is extracted during 3 days. Allergenic extract is centrifuged at 5000-6000 rpm within 30-40 minutes with sterilizing filtration following until ready form of diagnostic allergen being obtained. The produced preparation has been studied according to methodical instructions to primary experimentally-clinical approbation of new forms of allergens.

EFFECT: reduced risk of adverse side effects; high specific activity; no animal sensitization when given in the diagnostic doses.

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

FIELD: medicine, immunology, allergology.

SUBSTANCE: one should detect the parameters of immune state and objective anamnesis, before carrying out specific immunotherapy (SIT) with allergens one should detect IgM level (g/l), the quantity of monocytes (Mon), lymphocytes (Lymph) in total blood analysis in %, the quantity of B lymphocytes in % (CD19), immunoregulatory index (IRI) (CD4/CD8), phagocytic value in % (PhV), IgE serumal level in IU/ml, the quantity of points in evaluating accompanying nonallergic anamnesis (Point) {one point in case of one chronic disease, 2 points in case of two, etc.}, then one should calculate the changes in immunity parameters on the 7th d of SIT-therapy, obtained values should be applied in the following equation of regression (Y= -195.4 + 117.3·IgM + 10.8·Mon - 0.35·IgE - 17.1·Point + 7.8·Dlymph + 20.4·Dmon + 3·Dbl + 22.8·Diri) and calculating the possibility of complications it is necessary to perform by the following formula: P = 100/(1+Exp(-Y)).

EFFECT: higher accuracy of prediction and improved quality of therapy.

3 ex

FIELD: medicine.

SUBSTANCE: allergen is taken as aqueous salt solution of protein polysaccharide complex of 10000 PNU activity produced by extracting dry degreased gnat of Aedes aegypti with alkaline buffer solution at pH of 8.0-8.2 with following cleaning as means for specific immunotherapy means applicable to allergic disease patients having sensitization to gnat stings. Method involves applying allergen of 10000 PNU concentration in 10-fold dilution introduced to a patient beginning from a dose of 0.2 ml with 1-2 days long pause. Supporting doses of the allergen are administered to the patients before gnat flight season with 10-12 days long pauses.

EFFECT: high effectiveness of the allergen and treatment method.

3 cl

FIELD: medicine, radiation biology.

SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.

EFFECT: improved preparing method, improved method for diagnosis.

1 tbl, 3 ex

The invention relates to medicine, in particular to pulmonology and Allergology, and for the treatment of atopic bronchial asthma
The invention relates to the production of drugs for research in the field of Allergology and can be used in dermatology to determine the cause of contact allergic reactions and irritation reactions in dental practice for determining sensitivity to an orthopedic ingredients and medicines, surgery and traumatology at the use of synthetic grafts in cosmetology to identify portability cosmetic and ointment preparations and their components, in pathology to determine the contact dermalive different nature
The invention relates to medicine, namely to Allergology, and can be used to identify sensitization to allergens in adults and children

FIELD: medicine, radiation biology.

SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.

EFFECT: improved preparing method, improved method for diagnosis.

1 tbl, 3 ex

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