Microparticles containing carbohydrate spheres covalently bonded with allergen

FIELD: medicine.

SUBSTANCE: present invention refers to medicine and concerns microparticles containing sphere mainly consisting of cross-linked agarose carbohydrate and allergen covalently bonded with sphere, applied for immune system disturbance treatment. Used allergen is produced of plant pollen, specifically of timothy grass pollen. Application of specified microparticles provides effective treatment for patients suffering from allergy, as well as reduces by-effects of parenteral introduction.

EFFECT: development of effective method of allergy treatment and prevention.

9 cl, 4 dwg, 1 tbl, 5 ex

 

The present invention relates to the field of immunotherapy and especially to the treatment of patients suffering from allergic reactions to allergens, particularly for allergens originating from plant pollen.

Allergen-specific immunotherapy, which is carried out mainly by injection of extracts of allergens to patients with allergies, was introduced many years ago. The manifestation of severe anaphylactic side effects caused by the injection of aqueous extracts of allergens, and the need to introduce a large number of injections over long periods of time led to the creation of safe and effective compositions allergens. More than sixty years ago, the extracts of allergens adsorbed on aluminum hydroxide was introduced for depot-vaccination, demonstrating enhanced immunostimulatory and weakened anaphylactic properties. Even at the present time, the aluminum hydroxide is certainly the most common adjuvant used for injection immunotherapy.

Allergen-specific immunotherapy is one of the few known therapies aimed at the cause of the disease, IgE mediated allergies, and numerous clinical studies confirm its clinical efficacy. Well-known clinical practice includes under ojou injection of extracts of allergens, adsorbed on aluminum hydroxide, with gradually increasing doses to maintain levels and treatment periods up to 5 years or more. Aluminum hydroxide is preferred other adjuvants (for example, oil emulsions, liposomal dosage forms for injection immunotherapy in humans because it causes relatively weak tissue reaction. However, the aluminum hydroxide may cause the formation of local granuloma at the injection site. Other major disadvantages of aluminum hydroxide are unpredictable efficiency of adsorption of some antigens/extracts antigens, unpredictable stability of the adsorbents, the possibility that allergens change during adsorption, and difficulties in assessing the quality and quantity of allergens, which had once adsorbiroval on aluminum hydroxide.

The object of the present invention is the provision of improved forms of antigens that can be used in particular for the treatment of patients suffering from allergic reactions.

The present invention discloses microparticles, comprising: (a) sphere, essentially consisting of a carbohydrate, and (b) the allergen that is covalently bound to the field. Carbohydrate sphere consists mainly of three-dimensional linked polymer, which may be polyacrylamide, vinyl polymer, the code is ran, or preferably agarose. Can also be used a mixture of polymers. Carbohydrate-containing particle consists of appropriate polymeric carbohydrates, preferably agarose, which is connected in three directions. Respective spheres commercially available, e.g. under the trademark of sepharose. Spheres are small particles consisting of a gel-like carbohydrate, which forms the substrate for the antigen. The particle consists of a carbohydrate-containing sphere which is covalently linked to the antigen at a high density without drastically changing its immunological properties.

Microparticles of the present invention have a certain size, which varies in the range from 0.1 μm to 10 μm and preferably from 0.5 μm to 5 μm. The size of the microparticles is essential. The size distribution means that the largest percentage, usually at least 80% of all spheres, is in this range. Of course, there are a number of areas outside of this range, because the diameter distribution is statistically. However, applying a special technique, you can be sure that more than 99% of the spheres are in this range.

Microparticles of the present invention contain at least one antigen, which is covalently attached to the carbohydrate field. The antigen is a compound against which immune is the first system of the animal or human, being immunized, forms antibodies. The antigen may be any structure that forms the epitope. The antigen may be a polypeptide, carbohydrate, such, for example, Picatinny remains attached to the polypeptide or nucleic acid. In a preferred variant embodiment of the invention the antigen is an allergen, which comes from plant pollen. Surface patterns of plant pollen are the causative agents of allergic reactions. In preferred embodiments of the present invention allergens are structures derived from grass pollen. In a particularly preferred embodiment of the present invention the allergen is derived from the pollen of Timothy grass meadow Phleumpratense.

Condensation derived from carbohydrate spheres with antigen based on the principle of formation of the covalent bond between the carbohydrate skeleton sphere and the reactive group antigen. Covalent bond can be formed, for example, the activation of bromine cyan, leading to stable formation of amide bonds, which with high efficiency can be applied to most proteins and peptides. Alternative methods of binding, which are well known in the art, may also be used depending on the antigen, which must be connected with the sphere.

In experiments the Ah for this application investigated the antigen was purified recombinant Phi p 5b, the major pollen allergen of Timothy grass meadow, which are condensed from the carbohydrate particles (SVR). In the examples the same antigen was only mixed with SVR (as a comparative sample), and in another comparative test antigen was absorbed on aluminum hydroxide.

In the examples used song Phi p 5b for immunization of mice and analyzed the levels, kinetics and profiles of antibody formation.

In another series of experiments investigated the production of cytokines in cell cultures of mouse spleen and injection sites were then analyzed using histopathology. Induced antigen SVR-rPhl p 5b mouse antibodies were also examined for cross-reactivity to natural group 5 allergens of various herbs and their ability to inhibit binding of IgE to study allergen in patients allergic to grass pollen.

It was found that the microparticles of the present invention cause a comparable immune response, but a weaker response granulomatous tissue than aluminum hydroxide. Therefore, microparticles should have fewer side effects. In addition, the antibodies induced by microparticles of the present invention, blocked from allergic patients IgE binding to rPhl p 5b, which shows that the microparticles can be successfully applied to cured what I'm allergic patients.

The present invention discloses also a drug for the treatment of the immune system, which comprises microparticles of the present invention. This drug can be entered nasal, rectal or preferably parenterally. Microparticles can be included in the relevant pharmaceutical composition as solutions for injection, rectal foam or nasal sprays. You can also prepare the appropriate ointments or patches. The present invention also provides a diagnostic test system for measurement of released cell mediators, which include microparticles of the present invention.

One system is described in detail in example 2 and method 6.

The results of these experiments show that microparticles can provide suitable carrier and adjuvant for allergen-specific immunotherapy, leading to comparable to aluminum hydroxide immune reactions. Experiments show that the purified allergen rPhl p 5b, which is covalently linked to the obtained from the carbohydrate field induces in mice intensive formation of antibodies IgGI, IgG2a/b and IgG3. These antibodies cross-react with the natural group 5 allergens from all 5 species of grass, containing the group 5 allergens, and perhaps more important compete with the binding of the antibody to IgE with Pi p 5b in patients with Allergy to grass pollen. This finding suggests that the antibodies induced by microparticles have the required characteristics of the blocking antibodies. Such blocking antibodies that arise in the course of immunotherapy, have a positive impact, because they can suppress allergen-induced effect of cell activation and IgE mediated presentation of allergens to T cells.

Microparticles of the present invention exhibit several advantages compared to alternative used forms of immunotherapy, as, for example, the use of aluminum hydroxide.

Because condensation antigen realm uses are described in detail and reproducible technique, precisely predictable, what amount of antigen is loaded onto the sphere. Therefore, the areas covered by the antigen at a high density, high yield and in a reproducible number.

Another advantage can be seen in the fact that the antigens are covalently conjugated to a realm, presents a very efficient way by antigen-presenting cells involved in the immune system cells. We can conclude from the experiments that show that the cytokine response is much stronger in the group treated with microparticles of the present invention, compared with the control group, where the antigen is was b absorbed on aluminum, what cellular immune response is stimulated.

Another advantage is that carbohydrate spheres, particularly sepharose, possess high biocompatibility. Therefore, dosage forms for vaccination can be entered in different ways, resulting is preferred parenteral route of administration. However, you can also enter a medicinal product according to the present invention for oral, nazalnam, rectal or intravenous way. However, it is preferable for subcutaneous or intramuscular use.

The following experiments show that these microparticles are quite tolerant when exposed to different types of cells in tissue culture, and as a single-column matrix in clinical studies ex vivo. This is especially confirmed by experiments that show that immunization of mice with microparticles of the present invention induces less granulomatous reactions than the use of aluminum hydroxide in the comparative processing.

Method 1. Serum of patients.

Nine patients with confirmed medical history - allergies to pollen of Timothy grass meadow sensitized to rPhl p 5b and the pollen of Timothy grass meadow with a value of class 2 or higher radioallergosorbent test (RAST), were included in the study along with the control saw what rukami.

2. Recombinant allergen

cDNA encoding the major allergen rPhl p 5b, got polymerase chain reaction (PCR) in accordance with the published sequence rPhl p 5b (Bufe and other Major allergen Phi p 5b in timothy grass is novel pollen Rnase". FEBS Lett., 1995; 363:6-12). Was subclinically Phi p 5b cDNA in plasmid pet 17b (Novagen, Madison, WI), expressed in E. coli BL-21 (DE3) and purified to a homogeneous state, as described (Vrtala, etc. "Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phi p 1, Phi p 2, Phi p 5)". J. Allergy Clin. Immunol. 1996; 97:781-787).

3) Obtaining conjugates and adsorbed substances.

Spherical particles sepharose activated with bromine cyan (SVR), i.e. spherical agarose with an average diameter of 2.1 μm provided by the company Pharmacia Diagnostics, Uppsala, Sweden. Areas were activated with bromine cyan, as described previously (Ahip and other Chemical coupling ofpeptides and proteins to polysaccharides by means ofcyano halides". Nature, 1967; 214:1302-1304). Before conjugation 4 mg of liofilizirovannogo rPhl p 5b was dissolved in 8 ml of 0.1 M carbonate buffer, pH 8.0 was added to 110 mg of activated particles in 2.0 ml of 0.1 M carbonate buffer, pH 8.0. Allergen conjugatively particles by mixing from bottom to cover for 1 h at room temperature. Particles SVR-rPhl p 5b was centrifuged at 1000 g for 5 minutes, it Was found that the efficiency of condensation is >95% in the measurement of protein concentration by UV absorption at 280 nm in supernate is the before and after conjugation. Remaining active groups were blocked by re-suspension of the particles in 5 volumes of 0.1 M glycine at pH 8.5 and incubated by mixing from bottom to cover for 1 h Then the gel was washed with 0.1 M sodium acetate, 1.0 M NaCl, pH 4.0 and 0.1 M Tris-buffer, 1.0 M NaCl, pH 8.0 by alternating 5 volumes each. At the end of the SVR-rPhl p 5b was transferred into a 50 mm phosphate buffer, 0.15 M NaCl, 10 mm ethylenediaminetetraacetic acid (EDTA), 0.02% of NaN3, 0.05% tween-20, pH 7.5 and stored at +4°s to use. The stability of the covalent bond between rPhl p 5b and SVR was confirmed after three months of storage at +4°analysis rPhl p 5b in the supernatant. Absorbent material based on aluminum hydroxide (Alum) was obtained for comparative examples immediately before injection, as described (Vrtala and other T cell an epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v.1, induce blocking antibodies". J. Immunol., 2000; 165:6653-6659). In short, aluminum hydroxide, AluGel-S (Serva, Heidelberg; Germany) was diluted 1:1 in SFR and mixed with rPhl p 5b, receiving 5 μg in 100 μl of gel.

Immunization of mice

Three groups, each consisting of five female mice of line BALB/c mice aged 6-8 weeks (Charles River, Kissleg, Germany), were immunized with covalently bound CBP-rPhI p 5b (group I), rPhl p 5b-Alum (group II) as a control and the second control rPhl p 5b was mixed with SVR (group III).

Group processing and the time of immunization is given in the next table.

GroupProcessingImmunization days
Group ISVR-rPhl p 5b0, 28, 63, 121, 128
Group IIAlum-rPhl p 5b0, 28, 63, 121, 128
Group IIICBP+rPhl p 5b0, 28, 63, 121, 128

Mice were immunized subcutaneously in the neck with 5 µg rPhl p 5b 100 μl of the suspensions from each of the three drugs. Immunization was performed, and 30 blood was collected on days 0, 28 and 63. Day 121 they were re-injection and after 7 days the animals were killed and spleen cells were prepared for measurement of cytokines. The animals were kept in the vivarium of the Department of pathophysiology, University of Vienna, in accordance with local standards of care for the animals.

5) Determination of enzyme-linked immunosorbent assay (ELISA) specific for the allergen antibodies

All 15 mice were bled, and IgGI antibody, 2a/b, 3 and IgE specific to rPhl p 5b, were analyzed separately in an ELISA test as described Vrtala, etc. "Immunization with purified natural and recombinant allergens dosage mouse IgGI antibodies that recognize similar epitopes as human IgE inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation". J. Immunol, 1998; 160:6137-6140. Covered hole 96 tablets for micrometrology (Nunc, Roskilde, Denmark) with a layer of 100 μl, 5 μg/ml rPhl p 5b in SFR, overnight at +4°C. the Tablets were washed 2 times with 0.05% tween-20 is SFR (wash buffer (WB)). To reduce nonspecific binding was added 200 μl of the mixture WB-1% bovine serum albumin (BSA) for 2.5 h at room temperature. In each cell was added 100 μl of mouse serum diluted in WB-0,5% BSA 1:1000 for IgGI, 1:100 for IgG2a/b and 3 and 1:20 for IgE, respectively, and incubated over night at +4°followed by washing 5×250 ál WB. Incubated with 100 ál of rat artemisinin IgGI, 2a/b, 3 and IgE (PharMingen, San Diego, CA), respectively, diluted 1:1000 WB-0,5% BSA, overnight at +4°followed by washing. Incubated with 100 ml labeled with horseradish peroxidase sheep Anticriminal IgG, diluted 1:1000 WB-0,5% BSA for 2 h at room temperature followed by washing 5×250 ml WB. Used 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate, and the color reaction was read at 405 nm with a device for reading microplates Dynateck (Denkendorf, Germany).

The group 5 allergens of different types of grass pollen were determined using ELISA. Extracts of pollen allergens in SFR from Phleum pratense, Lolium perenne, Poa pratensis, Anthoxantum odoratum, Triticum sativum, Avena sativa, Cynodon dactylon, Zea mays and Phragmites antralis (Allergon AB, Valinge, Sweden) were placed in 96-well tablets ELISA (Nunc). After blocking and washing associated with the tablet extract was subjected to mouse serum pool from group I, diluted 1:100 in SFR, 0,5% BSA and 0,0% tween-20. For targets used prelimanry serum pool. Bound antibodies to IgG was determined using labeled with horseradish peroxidase sheep antimachine anticorodal IgG (Amersham, Buckinghamshire, UK)diluted 1:1000 in a mixture of 0.05% tween-20 - STR.

6) Measurement of cytokine production in cultures of spleen cells

To measure the production of IL-4, IL-5 and INF-γ suspension of the spleen cells of immunized mice were cultured in 48-hole plates (Costar, Cambridge, MA) with an extract of pollen of Timothy grass meadow (25 μg/well) or without it, at a concentration of 5×106cells/well. Supernatant were collected after 24 h for IL-4 and IL-5 and after 48 h for INFγ after stimulation by antigen and kept at -20°until analysis. The levels of IL-4 and IL-5 was measured using ELISA (Endogen, Cambridge; Mass). The sensitivity of the measurements was <5 PG/ml Levels of INF-γ measured in 96-well plates (Nunc, Maxisorp)coated with rat antimisting INFγ (Endogen, Woburn, MA) at a concentration of 0.5 μg/ml in carbonate buffer, pH 9,6, for 6 h at room temperature. Then labeled with Biotin rat antimurine antibodies to the INFγ (Endogen) was used at a concentration of 0.1 μg/ml with subsequent processing of conjugated horseradish peroxidase-streptavidin (1:10000 in SFR/4% BSA; Endogen) for 30 minutes To obtain a color used as the substrate tetramethylbenzidine (TMB) (Chemicon, Temecula CA), and measured the absorbance at 450 nm. Sensitivity analysis was <15 PG/ml Results reflect measured the levels of cytokines in PG/ml after subtraction of baseline levels of unstimulated cultures.

7) Histopathological analysis of skin sections

The skin was dissected from the injection areas (1.5 cm2), cut into strips 4 mm thick, were fixed with 7.5% formalin, pH 7.5, and were filled with wax. Packiclone the sections were stained with a mixture of hematoxylin-eosin or Giemsa.

8) Competitive ELISA

Tablets for ELISA connected with rPhl p 5b (0.1 µg/well), blocked and washed as described above for ELISA analysis. The tablets were subjected to United sera of mice immunized or SVR-rPhl p 5b (group I), or Alum-rPhl p 5b (group II), or for control purposes - pools corresponding preimmune serum, diluted 1:100, overnight at 4°C. After washing the plates were incubated with sera from patients allergic to grass pollen, diluted 1:5. Related human antibodies were detected with conjugated alkaline phosphatase mouse monoclonal antibody (PharMingen). Inhibition of binding of IgE allergic patients with rPhl p 5b using mouse immune sera was calculated using the formula 100-(OD of the second descent of blood/OD preimmune serum)×100.

9) Statistical the rd analysis

Nonparametric test of Kruskal-Wallis analysis of variance ANOVA was used to assess responses specific to the allergen antibody and cross-reactivity of allergen group 5 between grass species. Used U-Mann-Whitney test to assess blocking mouse serum binding of human IgE. Confidence interval p<0,05 was considered statistically significant.

Example 1

CBP-bound rPhl p 5b induces a strong response specific to the allergen antibodies

In order to compare the levels and kinetics of reactions specific to rPhl p 5b antibodies, sera from all groups of mice fees and blood were analyzed separately for IgE and each IgG subclass in one tablet ELISA. All three groups showed amplification reactions specific to rPhl p 5b antibodies in the course of immunization, which reached a maximum value after the second immunization. The results of example 1 is shown in figure 1. Figure 1 shows the responses of mice in the form of IgE and IgG subclass an associated tablet ELISA rPhl p 5b. The values of optical density (OD 405 nm), depicted on the Y-axis correspond to the levels rPhl p 5b-specific antibodies to IgE, IgGi, IgG2 and serum IgGa three mouse groups (group I: SVR-rPhl p 5b; group II: Alum-rPhl p 5b; group III: SRV+rPhl p 5b). In the group III rPhl p 5b was only mixed with SVR. The results are depicted for pridumannoj serum (B), first (1) and second (2) slope kr is VI in the form of a rectangular charts, where 50% of the values are within the rectangles and not anomalous values between columns. Filled squares indicate mean values, open circles - anomalous values and sprocket - limit values of each group, respectively.

Fed mice injected together rPhl p I and SVR without covalent binding (group III)showed significantly lower levels of antibodies after the first immunization, than mice treated with CBP or Alum-bound allergen. As the levels and features of the responses rPhl p 5b-specific antibodies were similar in mice treated SVR-rPhl p 5b (group I) and Alum-rPhl p 5b (group II), demonstrating the production of specific antibodies against IgE, but also specific answers to IgG2 and IgG3. Kinetics and values rPhl p 5b - specific responses to IgGi were similar in groups I and II.

Example 2

Mice immunized with CBP-bound rPhl p 5b, demonstrate a strong cytokine response to the extract of pollen of Timothy grass meadow

Profile INFγ, IL-5 and IL-4, secreted by spleen cells of mice of the three groups of immunization, cultured in the presence of the extract natural pollen of Timothy grass meadow, was similar in all three groups. Figure 2 shows the production of cytokines in vitro culture of spleen cells. The levels of INF-γ, IL-5 and IL-4 were measured in supernatant antigen-stimulated cells in the spleen of mice, which is s were immunized with SVR-rPhl p 5b - group I (black bars), Alum-rPhl p 5b - group II (white bars) or with SVR+rPhl p 5b - group III (shaded bars). The bars indicate the average of 5 separate units, the magnitude of the error mean standard error mean. U-Mann-Whitney test, *p<0,05; ***p< 0,001.

Based on the data it can be concluded that cells in the spleen of mice that received SVR-conjugated rPhl p 5b demonstrate significantly higher production of cytokines (INF-γ, IL-5)than cells in the spleen of mice treated with Alum-adsorbed rPhl p 5b. The lowest level of release of cytokines was found in cultures of spleen cells of mice that had received a joint introduction rPhl p 5b and SVR.

Example 3

CBP-bound rPhl p 5b induces a weaker reaction granulomatous tissue than Alum-adsorbed allergen

For analysis of tissue reactions at the injection sites took skin sections from mice of group I and II and were processed for histological examination. Then researched representative skin sections of mice immunized with the SVR-rPhl p 5b and Alum-rPhl p 5b. The sites of injection in mice immunized with SVR-rPhl p 5b and Alum-rPhl p 5b, analyzed histopathologically.

In mice treated with Alum, it was possible to see the General, more pronounced inflammatory response and additional granulomatous reaction with preimuschestvennokonets cells in the outer shell and granular debris in the center of the granuloma. Inflammatory tissue reactions of mice immunized with the SVR-rPhl p 5b, tended to weaken and contain less granular fragments than in mice treated with Alum. Next analyzed the histograms of tissue sections of mice immunized with SVR and Alum-bound rPhl p 5b. They contained a mixed inflammatory cell infiltrate, including macrophages and lymphocytes with rare mast cells and eosinophils at sites of injection in the skin depth, partly with granulomatous changes.

Example 4

Mice immunized with CBP-bound rPhl p 5b, have cross-reactivity of IgG with natural extracts of pollen from grasses, containing the group 5 allergens

In order to study, induces whether immunization with CBP antibodies to IgG, which cross-react with group 5 allergens from the pollen of other species of herbs, experiments using ELISA was performed with preimmune serum pool and a serum pool containing serum from the second descent of blood. The results are shown in figure 3. Figure 3 shows the 30 cross-reactivity of antibodies to IgG from mice immunized with the SVR-rPhl p 5b, with the natural allergen group 5 of 9 species of herbs. The values of optical density (OD 405 nm), corresponding to the levels of serum antibodies to IgG for extracts of pollen from 9 species of herbs (Y-axis). Phleum pratense, Lolium perenne, Poa pratensis, Anthoxantum odoratum, Triticu sativum, Avena sativa, Cynodon dactylon, Zed mays, Phragmites antralis display the serum pool was collected before immunization (P) and during the second descent of blood (2), on the x-axis.

Specific allergen rPhl p 5b of IgG antibodies to react with a group of 5 natural allergen from Timothy grass meadow (Phleum pratense) and of the five grass species (Lolium perenne, Poa pratensis, Anthoxantum odoratum, Triticum sativum, Avena sativa). The highest levels were registered for Poa pratensis. No IgG reactivity to extracts of pollen from grasses (Cynodon dactylon, Zea mays, Phragmites antralis), missing allergens associated with a group of 5 were detected. Pridumannyi serum pool showed no significant IgG reactivity to any of the 9 herbal extracts of pollen.

Example 5

Sera of mice immunized with CBP-bound rPhl p 5b, inhibit the binding of IgE to the allergen in patients with allergic

Can serum of mice immunized with CBP-rPhl p 5b, or Alum-rPhl p 5b, to inhibit the binding of IgE patients allergic to grass pollen with allergen rPhl p 5b was investigated using competitive ELISA experiments. Associated with the tablet for micrometrology rPhl p 5b pre-incubated serum pool taken at day 63, obtained from mice immunized with CBP-rPhl p 5b or Alum-rPhl p 5b and for control purposes with the corresponding pool of sera pre-immunization, and then exposed them to the effects of serum IgE from PA is antov allergic to nine species of grass pollen.

The results are shown in figure 4. Figure 4 shows the inhibition of binding of IgE patients allergic to grass pollen with rPhl p 5b using mouse serum. Allergen rPhl p 5b associated with the tablet ELISA, pre-incubated with a serum pool of mice immunized with CBP-rPhI p 5b (group I) or Alum-rPhl p 5b (group II). The percentage inhibition of IgE specific for sera of patients allergic to nine species of grass pollen, shown on the y-axis. Rectangles and horizontal lines indicate the 50% values and not the abnormal range, respectively. Are average values, and open circles indicate anomalous values.

Sera of mice from group I, immunized with CBP-rPhl p 5b, inhibited the binding of IgE allergic patients (n=9) with rPhl p 5b from 37 to 80% (mean 59.1 per cent), whereas inhibition between 51 and 90% (average is 67.9 percent) was observed for sera from group II (Alum-rPhl p 5b). Although inhibition of IgE binding obtained with the sera of mice treated with Alum-related Phl p 5b, was slightly higher than that obtained with the sera of mice group I, no significant difference between group I and group II in relation to the blocking ability was observed (R=0,31, U-Mann-Whitney test).

1. The microparticle for parenteral administration, including

(a) sphere, essentially consisting of a three-dimensional cross-linked is on carbohydrate and

(b) the allergen that is covalently bound to the field and derived from plant pollen and specified carbohydrate sphere consists mainly of agarose.

2. The microparticle according to claim 1, where the allergen is derived from grass pollen.

3. The microparticle according to claim 2, where the allergen is derived from the pollen of Timothy grass meadow.

4. The microparticle according to claim 1, where the particle size is in the range of 0.1-10 microns.

5. The microparticle according to claim 4, where the particle size is in the range of 0.5-5 microns.

6. The microparticle according to any one of claims 1 to 5, wherein the microparticle is used for vaccination.

7. The microparticle according to any one of claims 1 to 5, wherein the microparticle is used to treat allergies.

8. Drug for the treatment of immune system, characterized in that it comprises microparticles according to any one of claims 1 to 7.

9. The drug of claim 8, characterized in that it is prepared for parenteral use.



 

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9 cl, 9 tbl, 24 ex

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Indanol derivatives // 2323937

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (I): (where R1 and R2 may be identical or different, and each is a 1,3-substituted aryl with substituents from group α; R3 stands for any of the following groups: -CO-R4, -CO-O-R4, -CO-NH-R4, -CO-CH2-N(Ra)Rb, -(CH2)m-CO-R5, -(CH2)m-R5, -CO-NH-CO-N(Ra)Rb, -CO-NH-SO2-N(Ra)Rb, -CO-NH-CO-(CH2)m-N(Ra)Rb, or -CO-NH2; R4 stands for a lower alkyl, cycloalkyl, cycloalkyl substituted with 1-3 substituent from group α, lower alkenyl, lower alkynyl, halogen-substituted lower alkyl, hydroxyl-substituted lower alkyl, lower alkoxyalkyl, lower aliphatic acyloxyalkyl or lower alkoxycarbonylalkyl; R5 stands for hydroxyl, -OR4 or -N(Ra)Rb; Rа and Rb may be identical or different, each of them stands for hydrogen, hydroxyl, lower alkoxy group, hydroxyl-substituted lower alkoxyl, hydroxyl-substituted lower alkoxyalkyl, lower alkoxy lower alkoxyalkyl, cyano lower alkyl, cyano lower alkoxyalkyl, carboxy lower alkyl, carboxy lower alkoxyalkyl, aliphatic lower alkoxycarbonyl lower alkoxyalkyl, carbamoyl lower alkyl group, carbamoyl lower alkoxyalkyl, lower aliphatic acylamino lower alkyl, lower aliphatic acylamino lower alkoxyalkyl, lower alkylsulphonylamino lower alkyl, lower alkylsulphanylamino lower alkoxyalkyl, (N-hydroxy-N-methylcarbamoyl) lower alkyl, (N-hydroxy-N-methylcarbamoyl) lower alkoxyalkyl, (N-lower alkoxy-N-methylcarbamoyl) lower alkyl, (N-lower alkoxy-14-methylcarbamoyl) lower alkoxyalkyl or R4, or both, including associated nitrogen, stand for nitrogen-containing heterocyclic group or nitrogen-containing 1-3 substituted heterocyclic group with substituents from group α; m is an integer from 1 to 6; А stands for carbonyl; В stands for straight bond; D stands for oxygen atom; Е stands for С14 alkylene; n is an integer from 1 to 3; and α group is a group of substituents, which consist of halogen atoms, lower alkyls, hydroxy lower alkyls, halogen lower alkyls, carboxy lower alkyls, lower alkoxyls, hydroxy lower alkoxyls, hydroxy lower alkoxyalkyls, lower alkoxycarbonyls, carboxyls, hydroxyls, lower aliphatic acyls, lower aliphatic acylamines, (N-hydroxy-N-methylcarbamoyl) lower alkyls, (N-lower alkoxy-N-methylcarbamoyl) lower alkyls, hydroxy lower aliphatic acylamines, amines, carbamoyls and cyano groups), or pharmacologically suitable salt thereof. Invention also relates to pharmaceutical composition and method for disease prevention and treatment.

EFFECT: preparation of novel biologically active compounds.

18 cl, 117 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula I or pharmaceutically suitable salt or solvate thereof, where dashed line stands for additional bond, а is a number from 0 to 2, b is a number from 0 to 2, n is 2, p is 2, r is 1, М1 stands for nitrogen, М2 stands for С(R3), X stands for either a bond or alkylene group with number of carbon atoms from 1 to 6, Y stands for -С(О)- group, Z stands for a bond, or alkylene group with number of carbon atoms from 1 to 6, or alkenylene group with number of carbon atoms from 1 to 6, or -С(O)-, -CH(CN)-, -SO2- or СН2С(O)NR4- group, R1 stands for groups, R2 stands for six-membered heteroaryl ring with one or two heteroatoms chosen independently of each other from either nitrogen atom or N-O group, other atoms of the cycle being carbon, five-membered heteroaryl ring with one, two, three or four heteroatoms chosen independently of each other from nitrogen, oxygen or sulphur, other atoms of the cycle being carbon, R32 stands for substituded quinoline group, R32 stands for substituted aryl group, heterocycloalkyl group, cycloalkyl group with number of carbon atoms from 3 to 6, alkyl group with number of carbon atoms from 1 to 6, group, where the said six-membered heteroaryl ring or the said five-membered heteroaryl ring may be R6-substituted, R12 independently of others is chosen from an alkyl group with number of carbon atoms from 1 to 6, hydroxyl group or fluorine atom, provided in case R12 stands for hydroxyl or fluorine the rest of R12 cannot be bonded to a nitrogen-bonded carbon atom, or two R12 substituents form an alkyl bridge with number of carbon atoms from 1 to 2, which bonds two non-adjaicent carbon atoms of the ring, R13 independently of the others is chosen from an alkyl group with number of carbon atoms from 1 to 6, hydroxyl group, alcoxy group with number of carbon atoms from 1 to 6, or fluorine atom, provided in case R13 stands for hydroxyl or fluorine the rest of R13 cannot be bonded to a nitrogen-bonded carbon atom, or two R13 substituents form an alkyl bridge with number of carbon atoms from 1 to 2, which bonds two non-adjacent carbon atoms of the ring. See description for meaning of the other structural elements. Invention relates also to pharmaceutical compositions, as well as to application of compounds of formula I.

EFFECT: preparation of novel biologically active substances and pharmaceutical compositions.

20 cl, 659 ex

FIELD: organic chemistry, medicine, neurology, pharmacy.

SUBSTANCE: invention relates to novel hydrogenated pyrrolo[4,3-b]indoles of the general formula (1): , their racemates, optical isomers, geometric isomers, pharmaceutically acceptable salts and/or hydrates that can be used, for example, in treatment and prophylaxis of different neurodegenerative diseases, such as Alzheimer's syndrome. In the general formula (1): a dotted line with accompanying unbroken line represents ordinary or double bond; R1 and R2 represent independently of one another substitutes of amino group chosen from hydrogen atom, possibly substituted (C1-C6)-alkyl substituted possibly with aryl, possibly substituted phenyl, possibly substituted carbonylamino or thiocarbonylamino group, substituted acyl, possibly substituted aryl sulfonate wherein substituted in indicated R1 and R2 are chosen from (C1-C6)-alkyl, halogen atoms, nitro, carboxy, alkoxy group, aryl; R1n represents one or some similar or different substituted of cyclic system chosen from hydrogen atom, alkyl, aryl, cyano group, halogen atom, 5-6-membered nitrogen-containing heteroaryl. Also, invention relates to methods for synthesis of these compounds, pharmaceutical compositions and their using, and to using compounds in libraries with their using.

EFFECT: valuable medicinal properties of compounds and pharmaceutical compositions, improved methods of synthesis.

20 cl, 2 tbl, 12 ex

FIELD: pharmaceutics.

SUBSTANCE: the present innovation deals with developing compositions for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria. Composition for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria containing two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen. Composition for preventing or treating pollinosis which should be applied for introduction for a pollinosis-suffering mammal, annually before pollinosis season that contains two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen as an efficient component. Method for obtaining a composition for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria containing two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen deals with the following stages: plant raw material should be mixed, extracted; the extract should be purified and concentrated and, if necessary, one should add the fillers followed by granulation. Method for obtaining a composition for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria containing two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen deals with mixing plant raw material, if necessary, with the fillers, the mixture obtained should be mixed with a plastifying solvent followed by granulation and drying. Method for preventing or treating pollinosis deals with the fact that a pollinosis-suffering patient should be annually introduced before pollinosis season with a composition that contains two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen. Dietetic food product or functional food product for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria obtained due to introducing two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen as an efficient component. Dietetic food product or functional food product for preventing pollinosis that contains two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen as an efficient component. Dietetic food product for animals or functional food product for animals for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria containing two types of medicinal raw material out of Cucurbita moschata and Carthamus tinctorius seeds and, at least, one medicinal raw material chosen out of Plantago asiatica, Lonicera japonica, Coix lachrymal-jobi var. ma-yuen as an efficient component. The above-mentioned compositions and dietetic products are highly efficient for preventing or treating pollinosis, allergic rhinitis, atopic dermatitis, asthma or urticaria.

EFFECT: higher efficiency of prophylaxis or therapy.

23 cl, 9 dwg, 15 ex, 2 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention elates to novel derivatives of imidazolidine of the formula (I): , wherein symbols B, E, W, Y, R, R2, R3, R30, e and h have values given in claim 1 of the invention. Compounds of the formula (I) represent pharmaceutically active substances. Compounds of the formula (I) relates to inhibitors of adhesion and migration of leukocytes and/or antagonists of adhesion receptor VLA-4 that relates to group of integrins. Proposed compounds can be used in treatment of diseases that are induced or associated with undesirable degree of leukocyte adhesion and/or migration of leukocytes, or wherein interaction between cells or between cells and matrix are very significant and important and these interactions are based on interaction of VLA-4 receptors and ligands. Except for, invention relates to methods for synthesis of compounds of the formula (I) and pharmaceutical compositions containing compounds of the formula (I).

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition, improved method of synthesis and preparing.

12 cl, 19 ex

FIELD: organic chemistry, chemical technology, medicine, pharmacy.

SUBSTANCE: invention relates to novel hydrogenated azepino[4,3-b]indoles of the general formula (1): , their racemates, optical isomers, geometric isomers and their pharmaceutically acceptable salts and/or hydrates. In the general formula (1) a dotted line with accompanying unbroken line represents a simple or double bond; R1 and R2 represent independently of one another substitutes of amino-group chosen from hydrogen atom, possibly substituted (C1-C8)-alkyl possibly substituted with aryl, 5-6-membered azaheterocyclyl, (C1-C8)-alkoxycarbonyl, possibly substituted phenyl, possibly substituted carbonylamino- or thiocarbonylamino-group, substituted acyl, (C1-C8)-alkylsulfonyl, possibly substituted arylsulfonyl and wherein substitutes in indicated R1 and R2 are chosen independently from (C1-C8)-alkyl, halogen atoms, nitro-, carboxy-, alkoxy-group, aryl; Rin represents one or some similar or different substitutes of cyclic system chosen from hydrogen atom, (C1-C8)-alkyl, (C6-C10)-aryl, halogen atom, 5-6-membered azaheterocyclyl. Also, invention relates to methods for synthesis of these compounds, their using and pharmaceutical composition and libraries of compounds. Synthesized compounds possess neuroprotective, cognitive-stimulating and anti-histaminic properties and can be used in treatment of different neurological disorders, allergic and autoimmune diseases, for example, for memory improvement.

EFFECT: valuable medicinal and biological properties of compounds and pharmaceutical composition, improved method of synthesis.

25 cl, 2 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention concerns methods of small intestinal water adsorption detection. Lithium carbonate solution is introduced to small intestine lumen in dosage 8 mg/kg. 60 minutes after blood serum is tested for its concentration. Water absorption is diagnosed as satisfactory, if lithium cation concentration in blood serum is 0.18 mmol/l and higher.

EFFECT: method provides analysis simplification and acceleration, decreased and increased accuracy of estimated water-absorbability function of small intestine.

1 tbl, 2 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention concerns application of perfluoralkyl-containing metal complex characterised by critically concentrated micelle formation less 10-3 moles/l, hydrodynamic micelle diameter (2Rh) more than 1 nm and proton relaxation in plasma (R1) more than 10 l/mmole-sec, as contrast medium applied to visualise intravascular thrombi during magnetic resonance imaging (MRI).

EFFECT: extended range of agents used as contrast medium for magnetic resonance imaging.

50 cl, 7 dwg, 1 tbl, 7 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention concerns application of perfluoralkyl-containing metal complex characterised by critically concentrated micelle formation less 10-3 moles/l, hydrodynamic micelle diameter (2Rh) more than 1 nm and proton relaxation in plasma (R1) more than 10 l/mmole-sec, as contrast medium applied to visualise intravascular thrombi during magnetic resonance imaging (MRI).

EFFECT: extended range of agents used as contrast medium for magnetic resonance imaging.

50 cl, 7 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: one performs the scintigraphy with marker T1 - chloride during incipient and tardive periods, following visual detection of the region of marker increased uptake, quantitative evaluation of its accumulation in abnormal focus during the incipient and tardive testing periods are ER and DR180 relatively, retention quotient definition RI180, equal to DR180/ER. At this time as a marker 199T1-chloride is used. Scintigraphy is performed in planar variant and when RI180<-0.182ER+l.197 and heterospecific phlogixtic process is detected, when RI180>-0.182ER+l.197 cacoethic neoplastic process is diagnosed.

EFFECT: high accuracy and informativity of the differentiating diagnostic of heterospecific phlogistic and cacoethic neoplastic processes.

4 dwg, 2 ex

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: method involves detecting duodenum wall integrity disorders in performing endoscopic examination of duodenal ulcer after removing necrotic detritus from ulcer bottom. Available wall defect is subjected to endoscopic probing with catheter introducing water-soluble radiopaque solution through the probe and followed with fistulographic examination. The radiopaque solution being observed outside of duodenum, duodenal fistula or perforated ulcer are diagnosed.

EFFECT: high reliability of early stage diagnosis.

3 dwg

FIELD: medicine.

SUBSTANCE: method involves adding 52-523 mg/l guanosine disodium triphosphate and 3.5-4.7 g/l of sodium chloride to radiopaque preparation before introducing it.

EFFECT: prevented cardiac function disorder; high safety of coronography without worsening radiopaque preparation quality.

1 tbl

FIELD: medicine, in particular treatment of pathological diseases mediated by activated macrophages.

SUBSTANCE: Disclosed is pharmaceutical composition containing conjugate or complex of general formula Ab-X and uses thereof in production of pharmaceutical composition, wherein Ab group contains ligand capable of binding to vitamin receptor, and X group contains immunogen, cytotoxin, or compound capable of macrophage function reducing. Pathological conditions mediated by activated macrophages are selected from group including Crone disease, inflammations, infections, organ transplant rejection, osteomyelitis, etc.

EFFECT: improved method for treatment and diagnosis of macrophage mediated diseases.

26 cl, 15 dwg, 19 ex

FIELD: medicine, surgery.

SUBSTANCE: one should insert a gastroscope into fistular foramen in the wall of a hollow organ to fulfill sanitation followed by cavitary nasodrainage. The innovation enables to decrease traumatism and drain distant small-sized sites of congestion.

EFFECT: higher efficiency.

1 ex

FIELD: medicine.

SUBSTANCE: it is important to fulfill roentgen computed tomography at retrograde introduction of roentgenocontrast mixture and gas into urinary bladder. Roentgenocontrast mixture contains roentgenocontrast substance diluted with physiological solution ranged 15-30% and Dimexid at 8-10 ml 100% Dimexid solution/100 ml roentgenocontrast mixture.

EFFECT: higher accuracy of diagnostics.

3 cl, 5 ex

FIELD: medicine; pharmacology.

SUBSTANCE: medicinal agent is mixed with preaerated and irradiated with accelerated electron current or pulse UV-laser radiation 5.0-50.0% polyethylene oxide solution of molecular weight 0.4-20 kilodaltons in presence of 0.01-0.1 M phosphate buffer, pH 6.0-8.0 and containing 0.05-0.3 M sodium chloride. Offered method is easy and multipurpose, and can be applied to increase enteral bioavailability of medicinal agents introduced mainly or solely parenterally due to low resorption by intestine walls.

EFFECT: increased enteral bioavailability of medicinal agents.

4 cl, 3 tbl, 10 ex, 1 dwg

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