Antigenic preparations

FIELD: immunotherapeutic agents.

SUBSTANCE: antigenic preparations are obtained from keratinophilic fungi Trichophiton or Microsporum species or yeast species Candida by alkali hydrolysis techniques. Thus obtained preparations can be, in particular used, as vaccines and for treating allergy and modulating immune response.

EFFECT: expanded immunotherapeutic possibilities.

17 cl, 5 dwg, 12 tbl, 20 ex

 

The present invention relates to antigenic preparations, including polysaccharides and/or glycopeptides derived from keratinophilic fungi, and yeast, methods of producing these antigenic preparations, their use as pharmaceuticals as well as for their use as vaccines, including, but not limited to, for the prevention and treatment of allergies, as well as to modulate the immune response.

Allergies in one form or another, affects more than 20% of the population, and threatening increase its spread, morbidity and mortality over the last decade has led to the fact that allergies were considered disease is the number one associated with the environment (Sutton and Gould, Nature 1993, 366, str-428). People and animals are affected by allergies to the same extent.

A key role in the development of allergies play an immunological reaction (Paul, William E. (ed), Fundamental immunology, Raven Press Books, Ltd., New York, 1984). In principle described two different types of allergic reactions. One of them is allergic reaction of immediate type (ARNT), for which the maximum allergic response to the allergen occurs minutes to hours. The second type is an allergic reaction of the delayed type (ARST). In the case of ARZT allergic response to the allergen reaches its maximum after 24 and 48 hours. Rather, everything about ARNT is mediated mainly through metabolism of IgE, at that time, as ARZT is more complex. It seems that in the development of ARZT involved other mediated cell responses (ie, b - and T-lymphocytes). For example, after the migration of lymphocytes and antibodies from donor animals with allergies to animals-recipients without allergies recipients developed ARZT (Askenase, P.W. (1973), J. exp. Med., 138, str-1155).

Due to the direct impact of antigens environment, fabrics, most allergies are epithelial tissue, especially the skin. For example, in the clinic of skin diseases acute allergic contact dermatitis and chronic allergic contact eczema up to 15% of all skin diseases. Allergic asthma is 20% of all asthma cases in people.

Allergic diseases, which can be classified as ARNT, are, for example, allergic eczema, allergic bronchial asthma, hay fever, rhinitis, conjunctivitis. These diseases can also develop into a chronic form, and should not be viewed as solely dependent IgE reactions. Examples of ARST are acute allergic dermatitis as a result of contact and chronic allergic eczema as a result of the contact, which can be further classified as ARZT (type IV) with epidermal lesions. Such a patient could reaestate sensitive when exposed to the allergen, and he has developed hypersensitivity. Renewed contact with the allergen causes an acute, subacute or chronic contact dermatitis.

An example of atopic dermatitis according to the veterinary clinic is summer eczema, also known as "sweet itch" or "itching after a bite of the Queen bee. Summer eczema is an allergic dermatitis of horses relating to the form of atopic allergic diseases (including reactions of type I and IV). Summer eczema is caused by the bite of a small two-winged insects of the families Culicidae and Ceratopgonidae and characterized by skin lesions with permanent erosion and exudates, mainly in the area of mane, tail and belly. In affected animals, there is a strong sensitivity of the skin against irritation, i.e. touch, rain, wind, etc., the deterioration of their General health and activity. As for other types of allergies, consider that the development of this disease is also influenced by factors related to nutrition. The symptoms of this disease are observed only from March to September, while induced by allergen skin sensitivity is observed throughout the year. Summer eczema is interesting common model system for studying allergies, and for the development of anti-allergic substances.

Depending on liricheskoy paintings were offered many options for treating allergies. For the treatment of acute allergic contact dermatitis, chronic allergic contact eczema and/or atopic eczema commonly used lipophilic creams, including corticosteroids, anti-infective agents, anti-inflammatory medications and/or calcium. To treat sweet itch various compounds were applied topically or parenterally, for example, steroid drugs, insecticides, various herbal dosage forms, salicylates, oil or peptides isolated from microorganisms. All the above treatment options are valid only on the symptoms and not the causes of allergies.

Weakened immune response or immunodeficient condition often plays an important role in the development of allergies. Therefore, for the treatment of eczema, atopic eczema, skin abscesses, and autoimmune diseases were used as immunotherapy methods, for example, the introduction of Immunostimulants as Bacille Calmette Guerin (BCG), levamisole and other stimulants (Aminha (editor), Kosha, ed. "Medicine", 1982, Moscow).

To ensure reddish-brown allergic dermatitis has been successfully applied introduction derived from an antibody peptides (British patent application No. 8913737). For the treatment of atopic eczema with relatively good results were also applied desensitization (Aminha(editor), Kosh, ed. "Medicine", 1982, Moscow).

Despite different approaches to the treatment of allergies, as far as we know, antigenic compounds derived from keratinophilic fungi or yeast, is not used to treat allergies.

In the context of the present invention, the term "soluble" or "insoluble" refers to solubility in aqueous solution. The term "antigenic preparation" refers to the substance of any composition capable of inducing an antigenic or immunogenic response. The term "modulation of the immune response" refers to the ability of the antigenic preparations of the present invention to stimulate or enhance the immune response, for example, as demonstrated by their ability to stimulate the proliferation of lymphocytes in cell culture (for a detailed overview can be found in Strube and others (1989) Vet. Med. Rev., 60, p.3-15, Buttner, M. (1993) List. Immun. Environ. Infect. Dis., 16, No. 1, p.1-10).

Suddenly it was recently revealed that antigenic preparations obtained from keratinophilic fungi or yeast, can be applied for the prevention and treatment of allergies, as well as to modulate the immune response, in particular, in mammals.

Developed methods for producing antigenic material from keratinophilic fungi, and yeast. Antigenic preparations prepared according to these methods include polysaccharides and/or glycopeptides. The antigen is s drugs can be used in the form of pharmaceutical compositions, as well as vaccines for the treatment of animals and people, especially for the treatment of allergies and to modulate the immune response. Note that the pharmaceutical compositions of this invention can be useful as immunological, and pharmacological.

Antigenic material according to this invention may also be derived from derived from keratinophilic fungi or yeast material, for example, from the cell walls of fungi or yeast.

To obtain antigenic preparations according to the present invention has been developed in three different ways. In accordance with these methods of keratinophilic fungi and yeast can be obtained three different antigenic fractions (ASMP, ANMP or EMR), which are further referred to as "faction". On the antigenic preparations containing more than one fraction, hereinafter referred to as "integrated product" or abbreviated as "complex".

Method 1: Fraction obtained according to this method, consists of soluble antigenic material comprising a polysaccharide and/or glycopeptides (ASMP). Briefly, this method, which is illustrated in detail with example 1 is as follows.

Keratinophilic fungi or yeast cultivated on tablets with agar, for example as described in European patent 0564620. The preferred medium is, for example, agar is an extract of malt company Oxoid. Can also be used in other environments, providing growth keratinophilic fungi or yeast. The resulting fungal biomass is recovered and treated with an aqueous alkali solution. Preferred aqueous alkaline solutions are solutions of caustic soda or caustic potash in a preferred concentrations of 0.1-5% (V/o). The alkaline treatment is carried out preferably at a temperature of from 20 to 150°during the time up to 30 hours. After processing in aqueous alkaline conditions separate solid and liquid phases of the drug, for example, by centrifugation, filtration or sedimentation. Preferably the separation is performed by centrifugation, which provides good separation of residues fungal cells, for example, when the accelerations of about 3500 g. Processing in aqueous alkaline conditions, and phase separation can be repeated several times.

After alkaline treatment of the resulting supernatant liquid is treated in an acidic aqueous conditions, for example, 0.2-1.5 M organic acid or 0.05-1 M mineral acid. For example, there can be used hydrochloric acid or acetic acid, preferably at pH values between 2.5 and 4.5. Preferably the processing in aqueous acidic conditions hold for 2 to 4 hours at a temperature of from 4 to 8°C, followed by separation of the solid and liquid layers. Processing in aqueous acidic conditions, and phase separation can be repeated several times, preferably under the conditions listed above. Then from the supernatant liquid from the stage of separation of the precipitated desired material. Preferably the deposition is carried out with the addition of a suitable organic solvent, such as alcohol, as, for example, low alkanol to the supernatant liquid, examples of such alcohols are methanol or ethanol. With respect to 1 volume of supernatant to 2-5 volumes of alcohol reaches the deposition of antigenic material. Can also be applied to other methods of deposition without the use of alcohol, known to specialists in this field, for example, precipitation with ammonium sulfate or other salts may also result in the deposition of antigenic material. The solid phase is then subjected to a further stage of separation, it is preferable in the above-described conditions. The resulting solid phase is isolated and, if desired, dissolved in an aqueous solution, preferably in distilled water, typically use 25 to 100 ml of water. Finally, the drug ASMP can be liofilizovane, and then can be stored for an extended period of time in anhydrous conditions.

Method 2: Fraction obtained according to this method, consists of antigenic not rastvorimogo material, comprising a polysaccharide and/or glycopeptides (ANMP). Briefly, this method, which is illustrated in detail by the example 2 is as follows.

Keratinophilic fungi or yeast cultivated on tablets with agar, for example as described in European patent 0564620. The preferred medium is, for example, the extract agar malt company Oxoid. Can also be used in other environments that provide growth keratinophilic fungi or yeast. The resulting fungal biomass is recovered and treated with an aqueous alkali solution. Preferred aqueous alkaline solutions are solutions of caustic soda and caustic potash in a preferred concentrations of 0.1-5% (V/o). Alkaline treatment is carried out preferably at 20-150°during the time up to 30 hours. After processing in aqueous alkaline conditions separate solid and liquid phases of the drug, for example, by centrifugation, filtration or sedimentation. Preferably the separation is carried out by centrifugation, which provides good separation of the residual cells of fungi, for example, when the accelerations of about 3500 g. Processing in aqueous alkaline conditions, as phase separation, can be repeated several times. After alkaline treatment, the solid phase is treated with mineral or organic acids. Preferably the solid f the se adds 0.2 to 1.5 M acetic acid or 0.05-1 M hydrochloric acid for from 0.5 hour to 3 hours at a temperature of from 70 to 100° C. After acid treatment of the solid phase is washed with an aqueous solution, preferably distilled water. It is useful to repeat the wash about five times. In conclusion, the solid phase is suspended in distilled water.

Method 3: Fraction obtained according to this method, consists of exogenous antigenic material comprising a polysaccharide and/or glycopeptides (AEMR). Briefly this method, which is illustrated in detail by the example 3 is as follows.

Keratinophilic fungi or yeast are incubated in an aqueous solution or cultivated in liquid medium for a certain period of time, up to 240 hours (volume of solution or culture is denoted here as the initial volume /ON/). You can use distrurbance water (see example 3.1.) as well as the environment described in the European patent 0564620. After incubating or culturing cells of fungi are separated, for example, by centrifugation, filtration or sedimentation, preferably by centrifugation under the conditions described above. The resulting supernatant is then subjected to lyophilization and then the substance after drying is dissolved in water.

Preferably the amount of water ranges from 0.1 to 0.2 volume from. The resulting solution is subjected to the deposition process. Preferably the deposition carry out way of the addition of distilled liquid of a suitable organic solvent, for example, alcohol such as low alkanol, for example, methanol or ethanol. The ratio of one volume of distilled liquid to 2-5 volumes of alcohol leads to good precipitation of antigenic material. Specialists know that can also be applied to other methods of deposition without the use of alcohol, for example, precipitation with ammonium sulfate or other salt may also lead to the separation of antigenic material. The resulting residue is separated and, if desired, dissolved in an aqueous solvent, preferably in distilled water. Preferably dissolved from 0.5 to 50 mg of the precipitate in 1 ml of aqueous solvent. Finally, the solution EMR can be liofilizovane and stored for extended periods in dry conditions, particularly at a temperature of from 2 to 10°C.

Preferred genera of fungi, which are indicated above fractions are genera Trichophyton, Microsporum or Candida. Preferred species are:

Trichophyton equinum,

Trichophyton mentagrophytes,

Trichophyton sarkisovii,

Trichophyton verrucosum,

Microsporum canis,

Microsporum gypseum or

Candida albicans.

The preferred strains of the aforementioned types are:

Trichophyton equinum "German Collection of Microorganisms and Cell Cultures GmbH (HKM) No. 7276,

Trichophyton mentagrophytes HKM No. 7279,

Trichophyton sarkisovii HKM No. 7278,

Trichophyton verrucosum HKM No. 7277,/p>

Microsporum canis HKM No. 7281,

Microsporum canis var. obesum HKM No. 7280,

Microsporum canis var. distortum HKM No. 7275,

Microsporum gypseum HKM No. 7274 or

Candida albicans HKM No. 9656.

All of the above strains have been deposited by the applicant in NYC (Mascheroder Weg 1B, D-38124 Braunschweig, Germany), according to the provisions of the Budapest Treaty on the Deposit of microorganisms. All strains, except for Candida albicans HKM No. 9656, have been described previously in the patent USSR No. 5006861 filed 21.10.1991, and related applications, i.e. in the published application for the European patent 0564620 filed 17.10.1992.

Depending on species, of which can be obtained fractions are designated in accordance with the following:

Fractions obtained from:

(i) Trichophyton equinum, referred to as ASMP-TE, ANMP-TE or EMR-THOSE

(ii) Trichophyton mentagrophytes, referred to as ASMP-TM, ANMP-TM or EMR-TM,

(iii) Trichophyton sarkisovii, referred to as ASMP-TS, ANMP-TS or AEMP-TS -

(iv) Trichophyton verrucosum, referred to as ASMP-TV, ANMP-TV or AEMP-TV,

(v) Microsporum canis, are referred to as ASMP-MC, ANMP-MC or EMR-MS,

(vi) Microsporum gypseum, referred to as ASMP-MG, ANMP-MG or AEMP-MG or

(vii) Candida albicans referred to as ASMP-CA, ANMP-CA or EMR-SA.

Where provides information concerning the specific strain, followed by the abbreviation for the strains are non special Deposit HKM, for example, EMR-SA refers to the fraction AEMR derived from a strain of Candida albicans HKM No. 9656.

Faction, Chemie, as indicated in any of the methods mentioned above (1-3), include at least one simple antigen, prepared from at least one fungus, referenced above. The antigenic preparations of the present invention include at least one of the above fractions or combinations thereof.

Antigenic preparations (ASMP and AEMR), as described in examples 1 and 3:

1) include monosaccharides, amino acids and nucleotides, which largely incorporate in the polymer structure and, to a lesser extent, are present in the form of free monomers.

2) mainly consist of individual monosaccharide elements: mannose, galactose, glucose and xylose, and others in different relative amounts.

3) contain a mixture of polymeric structures formed a significant number of these monosaccharides. The molecular weight of a substantial part of these polymer structures more than 20,000 kDa.

4) contain small amounts of free or bound amino acids.

5) contain a small number of DNA molecules, which, as shown, are subjected to enzymatic hydrolysis by desoksiribonukleaza I.

When NMR spectroscopy antigenic preparations ASMP and AEMR receive NMR spectrogram presented in figure 1-4.

Chemical shifts and multiplicity of the signals (summarized in table 12) are consistent with the literature and data for carbohydrates and amino acids.

For fractions AEMR and ASMP, for example, MG 7274, TM 7279 and SA 9656, the proton signals of the carbohydrates are in the area of 3.2 ppm to 5.5 ppm, the signals of the protons of the amino acids in the region from 0.75 ppm up to 3.45 ppm (without α-protons).

For ASMP also see the typical signals of 1.92 ppm for the protons of the methyl group of acetate.

For fractions AEMR also see the typical signals for protons of disaccharides and amino acids. For example, in the spectrum of TM 7279 see the signals of the protons of aromatic amino acids like phenylalanine, tyrosine and tryptophan, in the field 7,15 to 7.9 ppm

In terms of individual fractions ASMP or EMR, the preferred concentration is from 0.1 to 50 mg/ml In the case of individual fractions ANMP preferred concentration is from 0.1 to 5% (o/o).

In preferred variants of the invention, the antigenic preparations of the present invention include, for example, the following combinations of fractions (complexes):

Complex 1 consists of ASMP-TM and ASMP-MG, and ASMP-CA. Preferably, the concentration of each fraction is from 0.1 to 50 mg/ml Vysokoproizvoditelnykh of the embodiment of the invention in respect of complex 1 is a combination of ASMP-TM 7279, ASMP-MG7274 and ASMP-CA9656.

Complex 1.1 includes ASMP-MG and ASMP-CA. Preferably, the concentration of each fraction is from 0.1 to 50 mg/ml Vysokoproizvoditelnykh of the embodiment of the invention in relation to complex 1.1 one is by a combination of ASMP-MG7274 and ASMP-CA9656.

Complex 2 includes ANMP-TM and ANMP-MG, and ANMP-CA. Preferably, the concentration of each fraction is from 0.1 to 5% (o/o). Vysokoproizvoditelnykh of the embodiment of the invention in respect of complex 2 is a combination of ANMP-TM7279, ANMP-MG7274 and ANMP-CA9656.

Complex 3 includes EMR-TM and AEMP-MG, and EMR-SA. Preferably, the concentration of each fraction is from 0.1 to 50 mg/ml Vysokoproizvoditelnykh of the embodiment of the invention in relation to complex 3 is the combination EMR-TM, AEMP-MG7274 and EMR-SA.

Complex 4 includes ANMP and AEMR. Preferred are the following combinations of fractions: (1) ANMP-CA and EMR-TM or (2) ANMP-MG, ANMP-TM and EMR-TM. Preferably the concentration of the ANMP is from 0.1 to 5% (o/o), and the concentration EMR from 0.1 to 50 mg/ml Vysokoproizvoditelnykh variant implementation of the invention in relation to complex 4 are the following combinations:

4.1 ANMP-CA9656 and EMR-TM;

4.2 ANMP-MG7274 and ANMP-TM7279, and EMR-TM.

Complex 5 includes ANMP and ASMP. Preferred is a combination of ANMP-MG and ANMP-TM, and ASMP-CA. Preferably the concentration of the individual fractions ANMP is from 0.1 to 5% (o/o), and the concentration of individual fractions ASMP is from 0.1 to 50 mg/ml Vysokoproizvoitelnoj is a combination of ANMP-MG7274 and ANMP-TM7279, and ASMP-CA9656.

Other preferred antigenic complexes according to the present invention include, for example, ASMP and the Mrs or ASMP and EMR, and ANMP in concentrations for ASMP and EMR from 0.1 to 50 mg/ ml, and for ANMP from 0.1 to 5% (o/o).

The antigenic preparations of the present invention can be applied together with suitable physiologically acceptable carriers, which do not cause undesirable physiological side effects, and include buffers, solvents or adjuvants, for example, salt solutions, solutions of lactate or ringer's solution. The preferred carriers are, for example: media: aqueous solution comprising a 0.85% (V/o) sodium chloride; the carrier of B: an aqueous solution comprising 5% (V/o) glucose, and 0.3% (V/o) meat extract "lab-lemco" (Oxoid) and 0.1% (V/o) yeast extract (Oxoid); media: RPMI medium 1640 (acquired by the company Serva, catalogue No. 12-702).

The antigenic preparations of the present invention can be applied in pure form by themselves or in the form of solutions for injection, creams, sprayable solutions, aerosols, pills, or other forms known to specialists in this field. The antigenic preparations of the present invention can be further used to obtain highly effective vaccines.

The antigenic preparations of the present invention is capable of stimulating proliferation of cells of the immune system and, thus, is able to modulate the immune response. The antigenic preparations of the present invention is further capable of inhibiting proliferate the human keratinocytes.

The antigenic preparations of the present invention can give a high degree of resistance against allergic reactions, in particular, epithelial tissues and especially the skin. They are of interest for the prevention and treatment of allergies and, as shown by our experience, no adverse side effects, as demonstrated in vivo in laboratory animals (i.e. Guinea pigs and white mice) and horses (i.e. the first generation from crosses of different breeds and Icelandic horses).

In particular, acute allergic dermatitis and skin lesions can be effectively treated without side effects by introducing antigenic preparation of the present invention, i.e. by vaccination. After intramuscular injection (injection) antigenic preparations of the present invention can eliminate the symptoms of allergic skin inflammation, itching and skin sensitivity patients affected with atopic dermatitis. Full exemption from all allergic symptoms was achieved within 2-8 weeks after the last injection, disappeared caused by allergen skin sensitivity to stimuli. Further, within 1-6 weeks after the last injection itching could be terminated.

In a preferred variant of the invention, the antigenic preparations according to the present invention provide C the shield and treatment from the so-called summer eczema horses, especially Icelandic horses. After 1-3 intramuscular or intradermal injection of antigenic preparations of the present invention horses affected summer eczema can be cured or protected from summer eczema, preferred are complexes 1 and 1.1.

In another preferred variant of the invention, the antigenic preparations of the present invention provide protection and treatment for alopecia in mammals. After 1-3 intramuscular or intradermal injection of antigenic preparations according to this invention, mammals are affected by alopecia, can be cured or protected from it, the preferred complexes 1 or 1.1.

In another preferred variant of the invention, the antigenic preparations of this invention to improve the condition of hair and seasonal change cover mammals. After 1-3 intramuscular or intradermal injection can be significantly improved cover, and individuals with incomplete change of cover can be a total change of cover to normal, appropriate to the season, preferred are complexes 1 or 1.1.

According to another preferred variant of the invention, the antigenic preparations of the present invention provide protection from eczema and eczema. After 1-3 intradermal or in utilising injection of antigenic preparations according to this invention or after local treatment of these antigenic preparations mammals, i.e. the people affected by eczema can be cured or protected from eczema, are preferred faction ASMP-MG, ASMP-CA and ASMP-TM, i.e, ASMP-MG7274, ASMP-CA9656 and ASMP-TM7279 or complexes 1 and 1.1.

Further, in a preferred variant of the invention, the antigenic preparations of the present invention provide for the prevention and treatment of atopic dermatitis. After topical treatment antigenic preparations of the present invention mammals, i.e. people affected by atopic dermatitis, can be cured or protected from it, are preferred faction ASMP-MG, ASMP-CA and ASMP-TM, i.e, ASMP-MG7274, ASMP-CA9656 and ASMP-TM7279 or complexes 1 and 1.1.

The antigenic preparations of the presented invention can be used for the treatment for many indications, such as described in the book "Clinical immunology, Peter, H.H. (editor), published in 1991, Urban &Schwarzenberg, Munich, Germany, for example:

1. allergic diseases of the respiratory tract

1.1. allergic rhinitis and conjunctivitis

1.1.1. seasonal Rhino-conjunctivitis

1.1.2. perennial rhinitis

1.2. bronchial asthma

1.3. asthmatic condition

1.4. asthma in children

1.4.1. obstructive pulmonary disease after infection bronchiolitis

1.4.2. weak episodic or weak perennial bronchial asthma

1.4.3 strong perennial bronchial asthma

2. allerg the ical bronchopulmonary aspergillosis

3. food allergies

3.1. oposredstvovaniya IgE food Allergy

3.1.1. oposredstvovaniya IgE food Allergy infant

3.1.2. oposredstvovaniya IgE food Allergy adolescents and adults

3.2. oposredstvovaniya IgG and T-cell food Allergy

3.3. intolerance to cow's milk

3.4. syndrome Heiner

3.5. eosinophilic gastroenteropathy

3.6. abdominal disease

4. allergies with burning sensation in the result of an insect bite

5. hives all kinds

5.1. hives from contact

5.2. urticaria associated with allergic reactions

5.3. urticaria associated with intolerance of impurities and inhibitors of prostaglandin synthesis (pseudoallergy)

5.4. somatic urticaria

5.4.1. dermographism (urticaria artificial)

5.4.2. cholinergic and adrenergic urticaria

5.4.3. hives caused by cold

5.4.4. urticaria from light

5.4.5. urticaria from pressure

5.4.6. other rare forms of physical urticaria

5.5. related to urticaria vasculitis

5.6. labrocyte and related to urticaria pigmentation

5.7. urticaria associated with infectious diseases

5.8. urticaria associated with immunotropic

5.9. urticaria and amyloidosis

6. angioedema

6.1. hereditary angioneurotic edema (HANE)

6.2. prio is acquired angioedema

7. atopic dermatitis, atopic eczema

8. drug Allergy

Table 1

Properties and characteristics of Candida albicans HKM No. 9656
Properties and characteristics of the strainHKM No. 9656Epidemic strain No. 008
Description culture10-day colony on agar Saburo is a homogeneous creamy, pasty, bright and towering, with a Central depression, the edge of the colony is normal, with a diameter of 18-22 mm10-day colony on agar Saburo is a homogeneous creamy, paste-like and shiny with the folded segments, the edge of the colony of irregular shape, with a diameter of 15-18 mm
Morphological characteristicsspherical oval of blastospore 3.5-5×5-8 microns, pseudogap width of 5-8 microns, hyphae width of 2-3 microns. Chlamydospores on rice agar size 13-16 microns in diameterspherical oval of blastospore 3.5-5×5-8 microns, pseudogap width of 5-8 microns, hyphae width of 2-3 microns. Chlamydospores on rice agar size 13-16 microns in diameter
Pathogenic characteristics30 days after intraperitoneal injection of 10-100 million fungal cells, white mice, 80% of the animals had abdomina the performance communications granuloma, lethal effects were not observed30 days after intraperitoneal injection of 10 to 100 million fungal cells, white mice, 80% of the animals had abdominal granuloma, 40% of the animals died

The present invention further refers to a strain of Candida albicans HKM No. 9656, which was obtained by directed selection, based on the stabilization of cultural morphological characteristics and attenuation epidemic strain No. 008, which has been isolated in humans in 1990

Biological properties of strains of Candida albicans HKM No. 9656 are described in table 1.

Strain Candida albicans HKM No. 9656 differs further from the epidemic strain stability of populations, morphological characteristics during the long passage through the medium and lower virulence. Follow the instructions to obtain the antigenic preparations of the presented invention, this strain can be made highly effective and safe antigenic preparations.

The person skilled in the art will appreciate that the present invention is well suited for certain tasks and achieving results, and appreciate these and inherent in this invention the advantages. These compounds, methods and technique in this case represent the preferred embodiments of izobreteny is, they are given as examples and are not limiting in certain limits.

After describing in General terms the presented invention will be more clear if you refer to the following examples which illustrate the present invention but do not restrict it within certain limits.

Examples

In all examples, the centrifugation was carried out at the acceleration between 3000 g and 3500 g for 30-50 minutes. Environment were purchased from the firm Oxoid (Unipac GmbH, Am Lippeglacis 6-8, 46483 Oars, Germany) or Serva (Serva of Finmekkanika GmbH & Co. KG, rl-Benz-Str., 7, 69115 Heidelberg, Germany). Unless otherwise specified, the mushrooms were cultivated as described in the catalog Oxoid "5. Modern German edition" or in the European patent 0564620. The strains used for the preparation of antigenic preparations according to the present invention, obtained by breeding and attenuate strains of fungi, as described in the book Nowmaximize, 1981, "Spontaneous variation and cariology imperfect fungi", published by the publishing house "Nauka", Moscow, and Ivanova, G., 1992, "Systematics, morphological characteristics, biological properties of causative agents of dermatophytosis, common to animals and man", Moscow, Library of Moscow state University. The main methods of culturing mammalian cell cultures can easily knight is in the book Doyle, Griffiths and Newell (EDS), Cell & cell Culture tissue: Laboratory techniques, John Wiley & Sons (1995). For experiments with keratinocytes used cell Nasat (Boukamp and others (1988), J. Cell Biol., 106, pp. 761-771, and Ryle and others (1989), Differentiation, 40, p.42-54), can also be used isolated keratinocytes or other cell lines of keratinocytes. Lymphocytes horses were isolated and cultured as described in the book Friemel, N., " Immunological methods research", published by VEB Gustav Fischer Verlag, Jena, 1984; or in the book, Paul E., "Fundamental immunology", published by Raven Press, new York, 1984. Autoradiographically studies were mainly carried out as described in the article Boehncke and others, 1994, Scand. J. Immunol. 39, str-332, references are given here. Caustic soda, sodium hydroxide, hydrochloric acid and acetic acid were prepared in the form of aqueous solutions. Unless otherwise specified, the term soluble refers to solubility in aqueous solution. Examples of physiologically acceptable carriers used in the following experiments, are: media: aqueous solution comprising a 0.85% (V/o) sodium chloride; the carrier of B: an aqueous solution comprising 5% (V/o) glucose, and 0.3% (V/o) meat extract "lab-lemco" (Oxoid) and 0.1% (V/ o) yeast extract (Oxoid); media: RPMI medium 1640 (Serva).

Example 1

Antigenic soluble material comprising a polysaccharide and/or Glyco is eptide (ASMP), was obtained from:

Trichophyton mentagrophytes (ASMP-TM), Microsporum gypseum (ASMP-MG) or Candida albicans (ASMP-CA) according to the following methods:

Fungi were cultured on plates with agar, as described in European patent 0564620. Fungal biomass was removed and to receive:

I. ASMP-TM:

(i) biomass Trichophyton mentagrophytes were treated with 4.5% (V/o) with sodium hydroxide solution at a temperature of approximately 140°C for 1 hour, then centrifuged for 45 minutes. To the supernatant was added a 4 M solution of acetic acid to achieve a final pH of 3.5. After 2 hours sludge was separated by centrifugation and added to 3 volumes of ethanol to 1 volume of supernatant. The sediment deposited in the deposition of alcohol, were subjected to sedimentation by centrifugation and dissolved in distilled water. In the end the individual drugs ASMP was subjected to lyophilization.

(ii) biomass Trichophyton mentagrophytes were treated with 0.2% (V/o) a solution of sodium hydroxide at a temperature of approximately 140°C for 1 hour and then centrifuged. The supernatant was treated with 1 M solution of hydrochloric acid to a final pH of 3.5 to 4 hours at 4-10°C. the Precipitate was then separated by centrifugation and added 2 volumes of ethanol to 1 volume of supernatant. The precipitate resulting from the addition of alcohol, besieged at centrifuging the years and was dissolved in distilled water. Then individual drugs ASMP was subjected to lyophilization.

II. ASMP-MG:

(i) biomass Microsporum gypseum were treated with 0.2% (V/o) with sodium hydroxide solution at a temperature of approximately 140°C for 2 hours and then centrifuged. The residue was again treated with 0.2% (V/o) with sodium hydroxide solution at a temperature of approximately 140°C for 2 hours, then centrifuged and the method was repeated for the third time. The final supernatant was then treated with 8 M solution of acetic acid to a final pH of 3.5 for 3 hours at 18-20°C. the Precipitate was then separated by centrifugation and added to 3 volumes of ethanol to 1 volume of supernatant. The precipitate which was formed in the result of the addition of alcohol, besieged by centrifugation and dissolved in distilled water. At the end of the individual drugs ASMP was subjected to lyophilization.

(ii) biomass Microsporum gypseum was treated with 3% (V/o) a solution of sodium hydroxide at a temperature of about 75°C for 6 hours and then centrifuged. The residue was again treated with 3% (V/o) with sodium hydroxide solution at a temperature of about 75°C for 6 hours and then centrifuged. The final supernatant was then treated with 0.5 M solution of hydrochloric acid to a final pH of 3.5 to 4 hours at 4-10°C. the Precipitate was then separated by centrifugation and added to 3 volumes of methyl JV the mouth to 1 volume of supernatant. The sediment deposited in the result of addition of the alcohol was separated by centrifugation and dissolved in distilled water. At the end of the individual drugs ASMP was subjected to lyophilization.

III. ASMP-CA:

(i) the biomass of Candida albicans was treated with a 3.0% (V/o) with sodium hydroxide solution at a temperature of 75°C for 6 hours and then centrifuged. The residue was again treated to 3.0% (V/o) with sodium hydroxide solution at 75°C for 6 hours followed by centrifugation. The final supernatant was then treated with 12 M solution of acetic acid to a final pH 3.5 for 2 hours at 4-10°C. the Precipitate was then separated by centrifugation and added 2 volumes of methyl alcohol to 1 volume of supernatant. The sediment deposited in the deposition alcohol was separated by centrifugation and dissolved in distilled water. Then individual drugs ASMP was subjected to lyophilization.

(ii) the biomass of Candida albicans were treated with 4.5% (V/o) a solution of sodium hydroxide at a temperature of about 35°C for 3 hours and then centrifuged. The residue was again treated with 4.5% (V/o) with sodium hydroxide solution at a temperature of about 35°C for 3 hours, followed by centrifugation, the whole procedure was repeated for the third time. The final supernatant was then treated with 0.25 M solution of hydrochloric acid until the final rn,5 for 4 hours at 18-20° C. the Precipitate was then separated by centrifugation and 1 volume of supernatant was added to 2 volumes of ethanol. Fallen as a result of precipitation with alcohol the precipitate was separated by centrifugation and dissolved in distilled water. Then individual drugs ASMP was subjected to lyophilization.

Example 2

Antigenic insoluble material comprising a polysaccharide and/or glycopeptides (ANMP), was obtained from: Trichophyton mentagrophytes (ANMP-TM), Microsporum gypseum (ANMP-MG) or Candida albicans (ANMP-CA) in accordance with the following methods:

Fungi were cultured on plates with agar, as described in European patent 0564620. Fungal biomass was removed for preparation:

I. ANMP-TM:

(i) biomass Trichophyton mentagrophytes were treated with 0.2% (V/o) with sodium hydroxide solution at a temperature of about 35°within 24 hours, and then centrifuged. The residue was treated with 0.3 M acetic acid for 3 hours at a temperature of about 60°C and washed 5 times with distilled water. After each stage of leaching was performed centrifugation. The final precipitate is re-suspended in a 0.85% (V/o) aqueous solution of sodium chloride (medium) to a final concentration of ANMP-TM 0,5% (o/o). The drug ANMP-TM kept in suspension at 2-10°C.

(ii) biomass Trichophyton mentagrophytes were treated with 0.2% (V/o) a solution of sodium hydroxide at a temperature of about 35°within 24 hours, and the ATEM centrifuged. The residue was treated with 0.1 M hydrochloric acid for 30 minutes at 70°C and washed 5 times with distilled water. After each washing, centrifugation was performed.

The final precipitate is re-suspended in RPMI medium 1640 (medium) to a final concentration of ANMP-TM 1,5% (o/o). The drug ANMP-TM kept in suspension at 2-10°C.

II. ANMP-MG:

(i) biomass Microsporum gypseum was treated with a 3.0% (V/o) with sodium hydroxide solution at a temperature of about 75°C for 6 hours, then centrifuged. Separated resulting residue was again treated to 3.0% (V/o) with sodium hydroxide solution at a temperature of about 75°C for 6 hours, then centrifuged. Separated resulting residue was treated with 0.7 M acetic acid for about 4 hours at 60°C and washed 5 times with distilled water. Each washing accompanied by centrifugation. The final precipitate is re-suspended in an aqueous solution comprising 5% (V/o) glucose, 0.1 percent (V/o) yeast extract company Oxoid and 0.3% (V/o) meat extract "lab-lemco" firm Oxoid (carrier B), to a final concentration of ANMP-MG 2,5% (on/about). The drug ANMG-MG kept in suspension at 2-10°C.

(ii) biomass Microsporum gypseum was treated with 3% (V/o) a solution of sodium hydroxide at a temperature of about 35°C for 3 hours, then centrifuged. The residue was again treated with 3% (V/o) a solution of sodium hydroxide at a temperature of about 35� C for 3 hours, then centrifuged, this procedure was repeated for the third time. Separated resulting residue was treated with 0.5 M hydrochloric acid for 30 minutes at 80°C and washed five times with distilled water. After each wash was followed by centrifugation. The final precipitate is re-suspended in RPMI medium 1640 (medium) to a final concentration of ANMP-MG 2,0% (o/o). The drug ANMP-MG kept in suspension at 2-10°C.

III. ANMP-CA:

(i) the biomass of Candida albicans were treated with 4.5% (V/o) with sodium hydroxide solution at a temperature of approximately 140°C for 2 hours, then centrifuged. The residue was again treated with 4.5% (V/o) with sodium hydroxide solution at a temperature of approximately 140°C for 2 hours, centrifuged, the procedure was repeated a third time. Separated resulting residue was treated with 1 M acetic acid for 1 hour at 60°C and washed five times with distilled water. Each wash was followed by centrifugation. The final precipitate is re-suspended in a 0.85% (V/o) aqueous solution of sodium chloride (medium) to a final concentration of ANMP-CA 1.5% (o/o). The drug ANMP-CA kept in suspension at 2-10°C.

(ii) the biomass of Candida albicans were treated with 4.5% (V/o) a solution of sodium hydroxide at a temperature of approximately 140°C for 2 hours, then centrifuged. The residue was again treated with 4.5% (V/o) with sodium hydroxide solution when the temperature around 140° C for 2 hours, the resulting precipitate was treated with 0.1 M hydrochloric acid for 30 minutes at 100°C and washed five times with distilled water.

After each wash was followed by centrifugation. The final precipitate is re-suspended in RPMI medium 1640 (medium) to a final concentration of ANMP-CA 2,5% (on/about). The drug ANMP-CA kept in suspension at 2-10°C.

Example 3

Exogenous antigenic material comprising a polysaccharide and/or glycopeptides (AEMR), were obtained from liquid cultures: Trichophyton mentagrophytes (AEMP-TM), Microsporum gypseum (AEMP-MG) or Candida albicans (AEMP-CA). Liquid cultures were cultivated mainly in the conditions described in European patent 0564620. Individual drugs EMR received according to the following methods.

I. AEMP-TM: Trichophyton mentagrophytes incubated for 240 hours at 26°in 1000 ml of distilled water. Then the culture containing about 1.2×108cells per ml, was centrifuged. The supernatant was subjected to lyophilization, and then was dissolved in 100 ml of distilled water was added to 3 volumes of methyl alcohol and the residue was dissolved in aqueous solution. The supernatant was subjected to lyophilization, the received AEMP-TM.

II. AEMP-MG: Microsporura gypseum were cultured for 50 hours at 28°With 200 ml of media (RPMI medium 1640 company Serva). Culture, containing about 3×107cells per ml were subjected centres is Pirovano. The supernatant was subjected to lyophilization, and then was dissolved in 20 ml of distilled water, was added 2 volumes of methyl alcohol and the residue was dissolved in aqueous solution. The supernatant was subjected to lyophilization, resulting in AEMR-TM.

III. EMR-CA: Candida albicans was cultured for 30 hours in 800 ml of medium B (1% (V/o) meat extract "lab-lemco" firm Oxoid, 0,1% (V/o) yeast extract company Oxoid and 5% (V/o) dextrose) at 37°C. the Culture containing about 108cells per ml, was centrifuged. The supernatant was subjected to lyophilization, and then dissolved in a small amount of distilled water, was added 2 volumes of methyl alcohol and the residue was dissolved in aqueous solution. The supernatant was subjected to lyophilization, resulting in AEMR-TM.

Example 4

Determined the effect of different antigenic preparations on the growth of cell cultures of keratinocytes (cell culture Nast).

I. Antigenic fractions ASMP-TM, ANMP-TM and EMR-TM obtained from Trichophyton mentagrophytes HKM No. 7279, ASMP-MG, ANMP-MG and AEMP-MG obtained from Microsporum gypseum HKM No. 7274, and ASMP-CA, ANMP-CA and EMR-SA obtained from Candida albicans HKM No. 9656, was used in various concentrations. Faction ANMP obtained in accordance with example 2, was subjected to lyophilization and re-suspended in SFR (phosphate buffered saline solution with a concentration of 6.7 mm phosphate when Phi is biologicheskii the pH value of about 7.2; acquired in the company Serva, catalog No. 17-516).

For cultivation used a 12-cell tablets for tissue culture company Falcon (flat-bottomed cell, the surface area of 9.6 cm2). In each cell was added 0.15 ml of cell suspension of keratinocytes (cells Nasat) from about 1 million cells in 1 ml of culture medium (RPMI 1640, supplemented with 10 % (V/o) fetal calf serum), 2 ml of culture medium and 0.02-0.1 ml antigenic fractions dissolved in SFR. In the control cells did not add material antigenic fractions. Cultivation was performed in an incubator with 5% (V/o) carbon dioxide at a temperature of 37°C for 48 hours prior to the development in the control cells confluent cell monolayer.

Inhibition of cell growth was determined by comparison of the size of the area of cellular layers, processed antigenic fractions, and control, not processed antigenic fractions (control = 100%). The results are presented in tables 2 and 3.

Inhibition of cell growth was observed when the concentration of the ASMP-MG 0,1 mg/ml, the concentration of ASMP-TM 0.3 mg/ ml and the concentration of ASMP-CA 1 mg/ml For ANMP (MG, TM and CA) inhibition was observed at a concentration of 1 mg/ml For AEMP-MG inhibition of cell growth was observed at a concentration of 0.3 mg/ml, and for AEMR-TM and EMR-SA at a concentration of 1 mg/ml

Example 5

Determined the effect of different antigenic coat the s on cell proliferation of lymphocytes horses.

Used antigenic fractions ASMP and AEMR fungal strains of T. mentagrophytes HKM No. 7279, M. gypseum HKM No. 7274 and C. albicans HKM No. 9656. Preparing a suspension of approximately 40,000 lymphocytes (from Icelandic horses) in 1 ml of culture medium. Culture medium RPMI 1640 supplemented with 10% (V/o) fetal calf serum. Cultivation of lymphocytes was carried out in 96-cell tablets for tissue culture with a U-shaped bottom (firm Falcon, No. 3077). In each cell was placed 200 μl of cell suspension was added 20 μl of antigenic fractions dissolved in SFR. In the control experiments did not add material antigenic fractions.

Tablets with tissue culture were incubated at 37°C with 5% (V/o) carbon dioxide within 72 hours. Then the growth medium was replaced and was added to a solution containing [3H]-thymidine (1 ál of the cell). Conducted the second stage of cultivation within 12 hours, the culture was washed SFR. Cell proliferation was determined by measuring the radioactivity, as described in Boehncke and others, 1994, Scand. J. Immunol. 39, page 327 332. Measurement of cell proliferation was performed by comparison of the study with control cultures that were not exposed to material antigenic fractions. Reference values were defined as 100%. The result is presented in table 4. Separate antigenic extracts had either inhibitory or stimulatory effect on cleoc the th lymphocyte proliferation.

Example 6

This example illustrates a typical complex drugs. The complexes (1 to 5)described in this example were obtained from Trichophyton mentagrophytes HKM No. 7279, Microsporum gypseum HKM No. 7274 or Candida albicans HKM No. 9656.

I. Complex 1 includes ASMP-TM, ASMP-MG and ASMP-CA in a suitable carrier, example

 Concentration (mg/ ml)
Complex 1AndBIn
ASMP-TM727951030
ASMP-MG727451030
ASMP-CA965651030
 in the mediain the mediain the media
 A or B or CA or B or CA or B or C

Complex 1.1 includes ASMP-MG and ASMP-CA in a suitable carrier, example

 Concentration (mg/ ml)
Complex 1AndBIn
ASMP-MG727451030
ASMP-CA965651030
  in the mediain the mediain the media
 A or B or CA or B or CA or B or C

II. Complex 2 includes ANMP-TM, ANMP-MG and ANMP-CA in a suitable carrier, example

Concentration [% (o/o)]
Complex 2AndBInG
ANMP-TM72790,51,01,52,5
ANMP-MG72740,51,01,52,5
ANMP-CA96560,51,01,52,5
 suspensionsuspensionsuspensionsuspension
 in the mediain the mediain the mediain the media
 A or B or CA or B or CA or B or CA or B or C

III. Complex 3 includes EMR-TM, EMR-MG and EMR-SA in a suitable carrier, example

 Concentration (mg/ ml)
Complex 3AndBIn
EMR-TM51030
AEMP-MG727451030
EMR-SA51030
 in the mediain the mediain the media
 A or B or CA or B or CA or B, or

IV. Complex 4 includes ANMP and EMR in a suitable carrier, example

(i) Complex

4.1 ANMP-CA9656 of 2.5% (V/o)

EMR-TM 7,1 mg/ml

in medium a or B, or

(ii)

 Concentration
Complex 4.2AndB
ANMP-MG7274a 2.5% (V/o)3,0% (o/o)
ANMP-TM7279a 2.5% (V/o)3,0% (o/o)
EMR-TM10.5 mg/ml18,5 mg/ml
 in the mediain the media
 A or B or CA or B or C

V. Complex 5 includes ASMP and ANMP in a suitable carrier, example

Concentration
Complex 5AndB
ANMP-MG7274of 1.75% (o/o)3% (o/o)
ANMP-TM7279of 1.75% (o/o)3% (o/o)
ASMP-CA965615.6 mg/ ml15.6 mg/ ml
 in the mediain the media
 A or B or CA or B or C

Example 7

Security different antigenic preparations were studied in experiments on vaccination on model systems of animals (white mice, Guinea pigs and horses).

Antigenic fractions were obtained as described in examples 1 to 3 and 6, Trichophyton mentagrophytes HKM No. 7279, Microsporum gypseum HKM No. 7274 or Candida albicans HKM No. 9656.

Daily for 5 days after each vaccination was carried out following clinical observations regarding the status of the vaccinated animals:

1. General condition

appetite

the effect on locomotion

2. Local reaction

swelling and inflammation at the injection site

the temperature change at the injection site

the development of pain at the injection site

the necessity of processing the site of injection drugs

I. Antigenic preparations were injected with one or two times with an interval of 10 days intraabdominal white the mice and intraabdominal and subcutaneous Guinea pigs. Antigenic preparations, their concentrations and the results are presented in tables 5 and 6 (a and B). Subcutaneous or intra-abdominal injection of antigens of fungi in the form of single or multiple drugs in most cases had no adverse effect on the General condition of the animals, and the injection did not observe any local reaction.

II. Combination preparations of antigens from fungi, as described in example 6 (complexes 4.1, 4.2 and 5), were injected every once intramuscularly the same horse in different places (left and right side of the neck and in one of the pectoral muscles). Were vaccinated with three different horses: (i) one pregnant Mare, (ii) one foal, the age of 7-8 months, and (iii) one stallion-producer at the age of 6 years. Antigenic preparations, their concentrations and the results are presented in table 7.

Intramuscular injection of antigens of fungi in the form of a comprehensive drug had no effect on the General condition of the horses, there wasn't any local reactions at the injection site. These studies demonstrate the safety of antigenic preparations according to the present invention.

Example 8

We also studied the effect of different antigenic preparations on the skin and hair in white mice.

Antigenic preparations were obtained as described in examples 1-3 and 6, Trichophyton mentagrophytes HKM is the 7279, Microsporum gypseum HKM No. 7274 or Candida albicans HKM No. 9656.

Antigenic preparations were injected with two times with an interval of 10 days intraabdominal white mice. Monitoring the condition of the skin and hair lasted for five days. Antigenic preparations, their concentrations and the results of the studies are shown in table 8. Injections of antigenic preparations improved the condition of the skin and hair of white mice compared to control animals affected by dermatitis.

Example 9

The effectiveness of three different antigenic preparations were examined with vaccination Icelandic horses affected summer eczema, experience during the inspection using a placebo.

Antigenic preparations were obtained as described in examples 1-3 and 6, T. mentagrophytes HKM No. 7279, M. gypseum HKM No. 7274 and C. albicans HKM No. 9656. The volume of 1 ml of media containing individual antigenic preparations were injected with three times intramuscularly. The interval between each injection was five days. The injection was made alternately in the right and left side of the chest muscles. Antigenic preparations, their concentrations and the results of the studies are shown in tables 9 and 10.

The introduction of an antigenic preparation comprising ASMP-MG7274, ASMP-TM and ASMP-CA9656, resulted in complete recovery of all vaccinated horses (3) four weeks after the third injection. In horses from the control group (in the under media And without antigen) was not observed any signs of recovery.

Example 10

The safety of three different antigenic preparations were examined with vaccination Icelandic horses affected summer eczema, experience during the inspection using a placebo.

Antigenic preparations were obtained as described in examples 1-3 and 6, T. mentagrophytes HKM No. 7279, M. gypseum HKM No. 7274 and C. albicans HKM No. 9656. The volume of 1 ml of media containing separate antigenic preparations were injected with three times intramuscularly. The interval between each injection was five days. The injection was made alternately in the right and left side of the chest muscles. Watched the emergence of side effects in animals within three days after each injection. Antigenic preparations, their concentrations and the results of the studies are shown in table 11. The usual side effects, such as fever or loss of appetite, was not observed. Only one antigenic preparations caused swelling at the injection site. This minor side effect was observed only have one horse. Signs of pain were observed.

Example 11

In laboratory animal models have been studied anti-allergic efficacy of individual fractions ASMP-TM7279, ASMP-MG7274 and ASMP-CA9656 and complex 1, including ASMP-TM7279, ASMP-MG7274 and ASMP-CA9656.

Individual fractions were obtained in accordance with example 1. Complex 1 was obtained in accordance with examples 1 and 6.

Mouse CF-1 were sensitized following the model and the instructions of the test on the swelling of the ear mouse (Gad SC, Dimm BK, Dobbs, DW, Reilly C, Walsh RD: the Development and affirmation of alternative skin test sensitization: test ear swelling mouse (MEST). Toxicology and Applied Pharmacology, 84, 93 114, 1986. It is a well known, proven and accepted by the Organization for Economic Cooperation and Development test for the study of Allergy-causing substances. To prove anti-allergic efficacy of the complex or its components separate factions in laboratory animal must be prevented swelling of the ear caused by the allergen. Was conducted blind experience on mice with placebo and two different allergens.

MEST was conducted on mice CF-1, which is most sensitive to allergens. Mice CF-1 age 6-10 weeks preparing, vibrava the skin in the abdomen, inhazinue 0.05 ml adjuvant-blockers and making a local application from zero until the fourth day from 100 µl serum, 1-chloro-2,4-dinitrobenzene (DNCB), in one experiment and mite allergen in another experience on the shaved skin of the abdomen. After 7 days was made tapicerki 20 μl of allergen to the studied CCS, and the solvent was applied to the ear in the control experiment. After 24 hours and 48 hours was measured thickness of the ear. The same procedure was performed with the control group, which were treated with placebo instead of the complex and, accordingly, factions complex.

The introduction of separate fractions ASM-TM7279, ASMP-MG7274 and ASMP-S and complex 1, including ASMP-TM7279, ASMP-MG7274 and ASMP-CA9656, resulted in 90% reduction in ear swelling after sensitization of mite allergen and 87,5% reduction in ear swelling after sensitization TNHB 48 hours after repeated provocative samples compared to control groups.

Example 12

The effectiveness of the integrated product comprising antigenic preparations ASMP-MG7274 and ASMP-CA9656, obtained as described in example 1, was demonstrated by vaccination Icelandic horses affected summer eczema.

Intradermal injection of 0.4 ml of media containing 0.2 mg of MG and 0.2 mg, CA, three times, with an interval of five days between each injection, led to wean vaccinated horses three weeks after the last injection, as evidenced by a significant decrease in clinical symptoms. Side effects were not observed.

Example 13

The effectiveness of antigenic preparation obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, was indicated for vaccination of persons aged 41 years, suffering from eczema with inflammation, itching and erosion of the skin between the fourth and fifth toe.

Intradermally were injected with only one time 0.1 ml of medium containing 0.4 mg ASMP-MG7274. The skin has returned to normal in 4-5 days after treatment.

Itching stopped after 24 hours the donkey injection. No serious side effects were observed.

Example 14

The effectiveness of antigenic preparation obtained as described in example 1 (ASMP), Candida albicans HKM No. 9656, was demonstrated in the treatment of atopic dermatitis.

ASMP-CA9656 was mixed into the cream using cream "Camille, hand cream and nail", purchased from the company, Procter and gamble, to a final concentration of 60 mg AMR-SA/ml of cream. The drug was applied tapicerki three year old girl suffering from atopic dermatitis with yellow scabs on the skin around both ears. The cream was applied tapicerki on the damaged part of the skin once a day for 30 days. After such treatment, the skin returned to normal. Side effects were not observed.

Example 15

The effectiveness of antigenic preparation obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, was demonstrated in the treatment of eczema.

ASMP-MG7274 was mixed into the cream using cream "Camille, hand cream and nail", purchased from the company, Procter and gamble, to a final concentration of 60 mg ASMP-CA9656/ml of cream. A man aged 30 years, suffering from eczema with inflammation, erosions and itching on the ring finger, was treated by topical application of the cream on the affected areas of skin once a day for 30 days. This led to full recovery after treatment. Itching disappeared a few days after the start of treatment. The adverse is known not watched.

Example 16

The effectiveness of antigenic preparations obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, Trichophyton mentagrophytes HKM No. 7279 and Candida albicans HKM No. 9656 was tested by inoculation of five horses, which has not changed winter hair until June. Were injected with intramuscularly three times with an interval of 5 days, 1 ml of media containing 15 mg of each of the antigenic preparations ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656 (final concentration 45 mg ASMP/ml), this led to a complete replacement of the normal seasonal hair in 15 days. No side effects were observed.

Example 17

The efficiency of complex antigenic preparation obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, Trichophyton mentagrophytes HKM No. 7279 and Candida albicans HKM No. 9656, demonstrated in the treatment of alopecia.

Two horses at the age of 7 years, suffering from alopecia throughout the body, one in 3-5 and another 7-10 different places, was treated with vaccine containing 10 mg of each of the antigenic preparations ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656 in 1 ml of medium (final concentration 30 mg/ml). The vaccine was injected with intramuscularly three times at intervals of 5 days, which resulted in a full return cover both horses to normal within 10 days after the last injection. Side effects were not observed.

Example 18

The efficiency of complex antigenic preparation derived, campisano in example 1 (ASMP), from Microsporum gypseum HKM No. 7274, Trichophyton mentagrophytes HKM No. 7279 and Candida albicans HKM No. 9656, demonstrated in the treatment of alopecia horses.

The horse at the age of 10 years, suffering alopecia in 10-12 different places throughout the body, treated with vaccine containing 15 mg of each of the antigenic preparations ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656 in 1 ml of medium (final concentration of 45 mg/ml). The vaccine was injected with intramuscularly three times at intervals of 5 days, which resulted in a full return of the cover to the normal 15 days after the last injection. Side effects were not observed.

Example 19

The efficiency of complex antigenic preparation obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, Trichophyton mentagrophytes HKM No. 7279 and Candida albicans HKM No. 9656, demonstrated in the treatment of alopecia in dogs.

Three female dogs suffering alopecia in 2-3 different places throughout the body, treated with vaccine containing 10 mg of each of the antigenic preparations ASMP-MG7274, ASMP-TM7279 and ASMP-S in 1 ml of medium (final concentration 30 mg/ml). The vaccine was injected with intramuscularly three times with an interval of five days, which resulted in a full return of the cover to the normal 15 days after the last injection. Side effects were not observed.

Example 20

The efficiency of complex antigenic preparation obtained as described in example 1 (ASMP), Microsporum gypseum HKM No. 7274, Trichophyton mentagrophytes HKM is 7279 and Candida albicans HKM No. 9656, demonstrated in the treatment of alopecia in dogs.

Two male dogs, five and eight years of age, suffering from alopecia in 2-4 different sites throughout the body, treated with vaccine containing 15 mg of each of the antigenic preparations ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656 in 1 ml of medium (final concentration of 45 mg/ml). The vaccine was injected with intramuscularly three times at intervals of 5 days, which resulted in a full return of the cover to the normal 30 days after the last injection. Side effects were not observed.

ASMP-MG7274
Table 2

The effect of the concentration of different antigenic fractions on the growth of cell cultures of keratinocytes (cells Nasat % compared to the control non-exposed antigenic fractions, fused cell monolayer control experiments = 100%)
antigenic fractionthe concentration of the antigenic fraction (mg/ml)
 0,0030,0070,010,0150,030,10,30,450,61,01,251,51,752
 the area covered by the cells, in % compared with the control experiments
10010010010010075502525250000
ASMP-TM10010010010010010075502500000
ASMP-SA100100100100100100100100100755025250
ANMP-MG7274100100100100100100100100100757550250
ANMP-TM100100100100100100100100100755025250
ANMP-SA100100100100100 100100100100757550250
EMR-MG7274100100100100100100757550250000
EMR-TM100100100100100100100100100757550250
EMR-SA10010010010010010010010010075502500

Table 3

The concentration of different antigenic fractions, resulting in 50% increase inhibition of cell growth of keratinocytes (cells Nasat)
StrainThe concentration of the antigenic fraction (mg/ml)
 ASMPANMPAEMP
MG7274 0,31,50,6
TM0,451,251,5
SA1,251,51,25
Table 4

The influence of different antigenic fractions on cell proliferation of lymphocytes horses
The concentration of the antigenic fractions (mg/ml)Proliferation of lymphocytes horses in % (in control experiments without affecting antigenic fractions = 100%)
 ASMP-

MG7274
ASMP-TMASMP-SAEMR-

MG7274
EMR-

TM
EMR-SA
50081,536,2of 37.819,47,01,08
50117106,9109,876,110,246,9
5181172,8119,8129,7 47,3138,3
0,598,693,8134,1147,8133,3138,3
0,05271,394,3181,5143,594,8149,8
0,005146,7207,6144,7104,7109,9146,3

Table 5A

The reaction of the studied animals (white mice, weighing 12-14 g) after the first injection" separate antigenic fractions
antigenic fraction

concentration (mg/ml) or [% (/)]

the volume of injection (ml)

the number of vaccinated animals

the number of animals which have been observed
    local painlocal reactionthe increase in local temperatureloss of appetitethe deterioration of locomotionlethal reactions
ASMP-MG727410.5 mg/ml 0,510000000
ASMP-TM12.5 mg/ml0.510000000
ASMP-SA15,5 mg/ml0,59000000
ANMP-MG72743,3%0,58010000
ANMP-TM3,3%0,510020000
ANMP-SA3,3%0,510010000
EMR-

MG7274
15,5 mg/ml0,510010000
EMR-TM11,3 mg/ml0,510010000
And the Mr-SA 13.5 mg/ml0,510010000

Table 5B

The reaction of the investigated animals after the first injection" complex products
complexNo.the concentration of mg/ml or

[%(0/0)]
the volume of injection (ml)the number of vaccinated animalsthe number of animals which have been observed
     local painlocal reactionthe increase in local temperatureloss of appetitethe deterioration of locomotionlethal reactions
white mice (body weight 12-14 g)
ANMP-MG7274 ANMP-TM7279 EMR-TM4.22,5%

2,5%

10.5 mg/ml
0,510000000
ANMP-CA9656 EMR-TM4.12,5%

7,1 mg/ml
0,51000000 0
ANMP-MG7274 ANMP-TM7279 ASMP-CA965651,75%

1,75%

15.6 mg/ml
0,510000000
Guinea pigs (body weight 150-200 g)
ANMP-MG7274 ANMP-TM7279 EMR-TM4.22,5%

2,5%

10.5 mg/ml
0,55000000
ANMP-CA9656 EMR-TM4.12,5%

7,1 mg/ml
0.55000000
ANMP-MG7274 ANMP-TM7279 ASMP-CA965651,75%

1,75%

15.6 mg/ml
0,55000000

Table 6A

The reaction of the studied animals (white mice, weighing 12-14 g) after the second injection" separate antigenic fractions
antigenic fraction

concentration (mg/ml) or [% (/)]

the volume of injection (ml)

the number of vaccines the bathrooms animals

the number of animals which have been observed
    local painlocal reactionthe increase in local temperatureloss of appetitethe deterioration of locomotionlethal reactions
ASMP-MG727410.5 mg/ml1,010000000
ASMP-TM12.5 mg/ml1,010000000
ASMP-SA15,5 mg/ml1,09000000
ANMP-MG72743,3%0,58010000
ANMP-TM3,3%0,510020000
ANMP-SA3,3%1.010010 000
EMR-MG727415,5 mg/ml0,510010000
EMR-TM11,3 mg/ml1,010010000
EMR-SA13.5 mg/ml0,510010000

Table 6B

The reaction of the investigated animals after the second injection" complex products
complexNo.concentration (mg/ml) or [%(o/o)]the volume of injection (ml)the number of vaccinated animalsthe number of animals which have been observed
     local painlocal reactionthe increase in local temperatureloss of appetitethe deterioration of locomotionlethal reactions
white mice (body weight 12-14 g)
ANMP-MG7274 ANMP-TM7279 EMR-TM4.22,5%

2,5%

10.5 mg/ml
0,510000000
ANMP-CA9656 EMR-TM4.12,5%

7,1 mg/ml
0,510000000
ANMP-MG7274 ANMP-TM7279 ASMP-CA965651,75%

1,75%

15.6 mg/ml
0.510000000
Guinea pigs (body weight 150-200 g)
ANMP-MG7274 ANMP-TM7279 EMR-TM4.2the 2.5% 2.5% 10.5 mg/ml1,05000000
ANMP-CA9656 EMR-TM4.1the 2.5% and 7.1 mg/ml1,05000000
ANMP-MG7274 ANMP-TM7279 ASMP-CA965651,75% 1,75% 15.6 mg/ml1,05000000

Table 7

The reaction of the horses after injection (single injection) complex products (each horse entered complexes No. 4.1, 4.2 and 5 at the same time by separate injection in separate places)complexNo.concentration (mg/ml) or

[%(0/0)]the volume of injection (ml)the number of vaccinated horsesthe number of horses with local reactionsthe number of horses with the General reactions:     painswelling/inflammationthe temperature riseloss of appetitethe deterioration of locomotionthe death of animalsANMP-MG7274 ANMP-TM7279 EMR-TM4.23,0%

3,0%

18,5 mg/ml0,5 



300 



0 



0 



0 



0ANMP-CA9656 EMR-TM4.13,0%

15.6 mg/ml0,5 00     ANMP-MG7274 ANMP-TM7279 ASMP-CA965653,0%

3,0%

15.6 mg/ml0,5 00    

Table 8

The condition of the skin and hair after injection of the complex antigenic preparations of white mice (body weight 12-14 g)
complexNo.concentration (mg/ml) or [(on/o)]injection (ml)the number of animalsthe number of animals who have noted the following skin condition after vaccination:the number of animals who have noted the following condition hair after vaccination:
   firstsecond scalysmoothsmooth and shinymatted and dull
ANMP-MG7274 ANMP-TM7279 EMR-TM4.22,5%

2,5%

10.5 mg/ml
0,51,020220
ANMP-C9656

EMR-TM
4.12,5%

7,1 mg/ml
0,51,030330
ANMP-MG7274 ANMP-TM7279 ASMP-CA965651,75%

1,75%

15.6 mg/ml
0,51,010110
unvaccinated---33021

Table 9

The efficacy of vaccination in different combination products, some Icelandic horses are affected summer eczema
complexNo.concentration (mg/ml) or

[%(0/0)]
the volume of injection (ml)the number of injectionsthe number of vaccinated horsesfour weeks after the third vaccination:

the number of horses
      curedthe uncured
    &x000A0;  from summer eczema
ASMP-MG7274110 mg/ml13330
ASMP-TM7279 10 mg/ ml     
ASMP-CA9656 10 mg/ ml     
ANMP-MG727421%13303
ANMP-TM7279 1%     
ANMP-CA9656 1%     
AEMP-MG7274310 mg/ ml13312
EMR-TM 10 mg/ml     
EMR-SA 10 mg/ ml      

1
Table 10

The efficacy of vaccination in different combination products, some Icelandic horses are affected summer eczema
complexNo.the concentrations of individual fractions (mg/ml) or [%(o/o)]the volume of injection (ml)the number of injectionsthe number of vaccinated horsesfour weeks after the third vaccination:

the number of horses, cured
      itchingeczema
ASMP-MG7274

ASMP-TM7279

ASMP-CA9656
110 mg/ml

10 mg/ml

10 mg/ml
13333
ANMP-MG7274

ANMP-TM7279

ANMP-CA9656
21%

1%

1%
13310
AEMP-MG7274

EMR-TM

EMR-SA
310 mg/ml

10 mg/ml

10 mg/ml
1332
without antigen (only storage media)--

(control)
13300

Table 11

Safety of vaccination with different antigenic preparations, some Icelandic horses are affected summer eczema
complexNo.concentration (mg/ml) or[%(o/o)]the volume of injection (ml)the number of injectionsthe number of vaccinated horsesthe number of horses with local side effects observed from 1 to 3 days after 1-3 injectionsthe number of horses with common side effects observed from 1 to 3 days after 1-3 injections
      swellingpaintemperatureloss of appetite
ASMP-MG7274110 mg/ml1330000
ASMP-TM7279 10 mg/ml        
ASMP-CA9656 10 mg/ml       
ANMP-MG727421%1330000
ANMP-TM7279 1%       
ANMP-CA9656 1%       
AEMP-MG7274310 mg/ml1331000
EMR-TM 10 mg/ml       
EMR-SA 10 mg/ml      
without antigen--1330000
(only media) (control)       

Table 12

NMR spectra
antigenic preparationsacetate (s)doublet amino acidsthe triplet of amino acidsmultiplet carbohydratesisolated rooms. CH2amino acidsend-acyl-SN amino acidsaromatic. the proton of amino acids
ASMP       
MG7274/9-18-1 (Fig. 4)1,92 ppmCH3(d, 6.8 Hz)

of 1.33 ppm
CH3(t, 7,1 Hz)

1,18 ppm
3,2-4,3 ppm1,7-to 3.45 ppmof 0.95 ppm 
  CH3(d, 7.5 Hz)

to 1.48 ppm
  the 4.9 to 5.4 ppm   
    CH2(Dia,16 Hz)

2,7 ppm/2,5 ppm
  
SA 9656/b008

(2)
1,92 ppmCH3(d, 7.0 Hz)

of 1.33 ppm
CH3(t, 7.0 Hz)

1,18 ppm
3,4-4,6 ppmof 3.28 ppmof 0.95 ppm 
  CH3(d, 7,3 Hz)

to 1.48 ppm
 4,9-of 5.24 ppm   
CM/32-m-1-5

(3)
1,92 ppmCH3(d, 7.0 Hz)

of 1.33 ppm
CH3(t, 7,1 Hz)

1,18 ppm
3,5-of 4.35 ppm2,1-3,3 ppm0,85-0,95 ppm 
  CH3(e, 7,1 Hz)

to 1.48 ppm
 a 5.0 to 5.25 ppm   
AEMP       
TM/R-5-1

(Figure 1, a-b)
 CH3(d, 6.5 Hz)

,33 ppm
 3,2-4,07 ppm1,6-3,12 ppm  
  CH3(d, 7.5 Hz)

to 1.48 ppm
   0,84-1,08 ppm7,15 to 7.9 ppm
    SN(d, 8.2 Hz) 4,65 ppm   
    SN (d, 4.0 Hz) 5,23 ppm   

Fig. 1 to 4:

Experiments with the use of NMR spectroscopy for analysis of fractions ASMP and EMR, as shown in figure 1-4, carried out in accordance with the following:

Spectra were taken in deuterated water at digital NMR spectrometer 250 MHz firm Bruker model DRX 400) with a frequency of1H 400, 13 MS. Scan width of 14.5 ppm, the ambient temperature is 300K. The assignment of the chemical shifts of conduct in relation to the solvent.

Standard one-dimensional1H-NMR spectra were obtained using a suitable pulse program of the company Brooker.

1. The method of obtaining antigenic material having the properties of anti-allergic drugs, and modulation of the immune response and is ecene alopecia, characterized in that the cells of fungi, which belong to the group keratinophilic fungi, or yeast, or the material of which is treated with about 0.1-5% (V/o) a solution of caustic potash or caustic soda at a temperature of about 20-150°C in a period of time up to 30 h, centrifuged, obtained after centrifugation the supernatant is treated with 0.2 to 1.5 M organic acid or 0.05-1M mineral acid, obtained after further centrifugation the supernatant is treated with a suitable organic solvent, e.g. alcohol, such as lower alkanol, or salt, for example, ammonium sulfate, and the resulting precipitate distinguish antigenic material.

2. The method according to claim 1, characterized in that keratinophilic the fungus belongs to at least one of the genera Trichophyton and/or Microsporum, and these yeasts belong to the genus Candida.

3. The method according to claim 1 or 2, characterized in that the fungus belongs to any of the following species: Trichophyton equinum, Trichophyton mentagrophytes, Trichophyton sarkisovii, Trichophyton verrucosum, Microsporum canis, Microsporum gypseum, and yeast - Candida albicans.

4. The method according to one of claim 1 or 2, characterized in that the fungus belongs to any of the following strains: Trichophyton equinum DSM No. 7276, Trichophyton mentagrophytes DSM No. 7279, Trichophyton sarkisovii DSM No. 7278, Trichophyton verrucosum DSM No. 7277, Microsporum canis DSM No. 7281, Microsporum canis var. obesum DSM No. 7280, Microsporum canis var. distortum DSM No. 7275, Microsporum gypseum DSM No. 7274, and the yeast - to Candid albicans DSM No. 9656.

5. The method of obtaining antigenic material having the properties of anti-allergic drugs, and modulation of the immune response and treatment of alopecia, wherein the cells of fungi belonging to the group keratinophilic fungi, or yeast, or their material is treated with about 0.1-5% (V/o) a solution of caustic potash or caustic soda at a temperature of about 20-150°C in a period of time up to 30 h, centrifuged, after centrifugation the resulting solid phase is treated with 0.2 to 1.5 M organic acid or 0.05-1 M mineral acid, washed with aqueous solution, followed by centrifugation and sediment distinguish antigenic material.

6. The method according to claim 5, characterized in that keratinophilic the fungus belongs to at least one of the genera Trichophyton and/or Microsporum, and these yeasts belong to the genus Candida.

7. The method according to claim 5 or 6, characterized in that the fungus belongs to any of the following species: Trichophyton equinum, Trichophyton mentagrophytes, Trichophyton sarkisovii, Trichophyton verrucosum, Microsporum canis, Microsporum gypseum, and yeast - Candida albicans.

8. The method according to one of claim 5 or 6, characterized in that the fungus belongs to any of the following strains: Trichophyton equinum DSM No. 7276, Trichophyton mentagrophytes DSM No. 7279, Trichophyton sarkisovii DSM No. 7278, Trichophyton verrucosum DSM No. 7277, Microsporum canis DSM No. 7281, Microsporum canis var. obesum DSM No. 7280, Microsporum canis var. distortum DSM No. 7275, Microsporum gypseum DSM No. 7274, and yeast - Candida albicans DSM No. 956.

9. The method of obtaining antigenic material having the properties of anti-allergic drugs, and modulation of the immune response and treatment of alopecia, wherein the cells of fungi belonging to the group keratinophilic fungi, or yeast, or material cultivated in liquid medium during the time up to 250 h, the share of solid and liquid phases of the final product, after separation to the supernatant add alcohol and sediment distinguish antigenic material.

10. The method according to claim 9, characterized in that before adding the alcohol supernatant is subjected to lyophilization, followed by dissolving in an aqueous solution, after deposition of about 1 to 5 volumes of alcohol is dissolved in an aqueous solution, the resulting solution is subjected to lyophilization.

11. The method according to claim 9 or 10, characterized in that keratinophilic the fungus belongs to at least one of the genera Trichophyton and/or Microsporum, and these yeasts belong to the genus Candida.

12. The method according to one of claim 9 or 10, characterized in that the fungus belongs to any of the following species: Trichophyton equinum, Trichophyton mentagrophytes, Trichophyton sarkisovii, Trichophyton verrucosum, Microsporum canis, Microsporum gypseum, and yeast - Candida albicans.

13. The method according to one of claim 9 or 10, characterized in that the fungus belongs to any of the following strains: Trichophyton equinum DSM No. 7276, Trichophyton mentagrophytes DSM No. 7279, Trichophyton sarkisovi DSM No. 7278, Trichophyton verrucosum DSM No. 7277, Microsporum canis DSM No. 7281, Microsporum canis var. obesum DSM No. 7280, Microsporum canis var. distortum DSM No. 7275, Microsporum gypseum DSM No. 7274, and yeast - Candida albicans DSM No. 9656.

14. Antigenic material, having the properties of anti-allergic drugs and money modulation imanage response and treatment of alopecia, obtained by treating the cells of fungi, which belong to the group keratinophilic fungi, or yeast, or material from them, about 0.1-5% (V/o) a solution of caustic potash or caustic soda at a temperature of about 20-150°C in a period of time up to 30 h, centrifugation, processing obtained after centrifugation of the supernatant liquid of 0.2-1.5 M organic acid or 0.05-1 M mineral acid treatment received after further centrifugation of the supernatant liquid with a suitable organic solvent for example, alcohol such as low alkanol, or salt, e.g. ammonium sulfate, or by treating the cells of fungi belonging to the group keratinophilic fungi, or yeast, or material, about 0.1-5% (V/o) a solution of caustic potash or caustic soda at a temperature of about 20-150°C in a period of time up to 30 h, centrifugation, processing obtained after centrifugation of the solid phase of 0.2-1.5 M organic acid or 0.05-1 M mineral acid, washing with an aqueous solution, followed by centrifugation, or to what litivinovka fungi cells, belonging to the group keratinophilic fungi, or yeast, or their material in a liquid medium during the time up to 250 h, the separation of the solid and liquid phases of the final product, if necessary, by lyophilization obtained after separation of the supernatant liquid, followed by dissolving in an aqueous solution, and adding alcohol in an amount of about 1 to 5 volumes, followed by separation of the resulting precipitate the final product.

15. Antigenic material 14, characterized in that it contains material from strains of T. mentagrophytes DSM No. 7279, from a strain of M. gypseum DSM No. 7274, or from a strain of C. albicans DSM No. 9656.

16. Yeast strain Candida albicans DSM No. 9656 designed to receive an antigenic material according to claims 1, 5 or 9.



 

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EFFECT: improved intensifying method.

2 tbl

FIELD: biotechnology, microbiology, veterinary science.

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9 cl, 8 tbl, 2 ex

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2 dwg, 12 ex

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

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