Bacillus spp. strains for use in the prevention or treatment of diseases caused by the der p1 allergen

Abstract

The invention relates to elimination of an allergen produced by Dermatophagoides pteronyssinus from contaminated surfaces and to the strains of Bacillus spp. and formulations containing said strains for use therein.

Description

[0001] Bacillus spp. strains for use in the prevention or treatment of diseases caused by the DER P1 allergen

[0002] The invention relates to elimination of an allergen produced by Dermatophagoides pteronyssinus from contaminated surfaces and to the strains of Bacillus spp. and formulations containing said strains for use therein.

[0003] House dust mites, in particular Dermatophagoides pteronyssinus, produce a number of inhaled allergens characterized by high sensitizing potential. The most common symptoms of sensitization to allergens originating from dust mites are runny nose, nasal congestion, itching, watery eyes, redness and swelling of the eyelids, sneezing, coughing, skin lesions, headache, fatigue or drowsiness. In some individuals, prolonged exposure to mite allergens may lead to other allergic diseases such as recurrent rhinosinusitis, chronic rhinitis, and even the development of atopic bronchial asthma.

[0004] The main allergen originating from Dermatophagoides pteronyssinus, DER P1 is a proteolytic enzyme present in mite cultures (including natural cultures such as those present in bedding and house dust) and mite faeces, leading to bothersome allergies in many patients.

[0005] The object of the invention is to provide an effective method suitable for use in the prevention or treatment of diseases caused by the allergens originating from Dermatophagoides pteronyssinus, resulting in effective inactivation of the DER P1 allergen. In particular, it is desirable to obtain a method for elimination of the active form of this allergen from surfaces inhabited by house dust mites, in particular Dermatophagoides pteronyssinus, particularly from furniture surfaces such as upholstered furniture surfaces, bed mattresses, and carpets. A particular objective of the invention consists in providing a method and means for implementation thereof facilitating elimination of at least 80% of active mite allergens, in particular the DER P1 allergen as produced by Dermatophagoides pteronyssinus.

[0006] Surprisingly, the aforementioned problem has been solved by the instant invention.

[0007] The invention relates to a composition containing bacterial strains of Bacillus spp. as deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506, for use in the prevention or treatment of diseases caused by the allergens originating from Dermatophagoides pteronyssinus, wherein said composition is intended for use in the form of a solution, preferably administered as an aerosol, wherein the concentration of bacteria in said solution is at least 50 cfu / mL and said composition is applied to the allergen-contaminated surface daily for at least 5 consecutive days, leading to the elimination of at least 80% of active allergen from the contaminated surface.

[0008] Preferably, the surface contaminated with the allergen is a furniture surface, preferably a bed surface.

[0009] Preferably, the disease is an allergy or respiratory asthma.

[0010] Preferably, the allergen is DER P1 .

[0011] The invention also relates to a method for the elimination of allergen originating from Dermatophagoides pteronyssinus from the contaminated surface characterized in that said contaminated surface is treated with a solution containing a Bacillus spp. strain selected from those deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505 and B / 00506, wherein said solution is applied to the surface contaminated with the allergen daily for at least 5 consecutive days.

[0012] Preferably, the surface contaminated with the allergen is a furniture surface, preferably a bed surface.

[0013] Preferably, the allergen is DER P1 .

[0014] Preferably, a solution containing all indicated strains of Bacillus spp. is used.

[0015] The invention also relates to a formulation for the elimination of allergen originating from Dermatophagoides pteronyssinus characterized in that said formulation contains a Bacillus spp. strain selected from those deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506.

[0016] Preferably, said formulation is in the form of an aqueous solution comprising water, Bacillus spp. strains in an amount of at least 50 cfu / mL, ethanol in an amount of not more than 5% w / w and optionally a fragrance composition in an amount of not more than 5% w / w.

[0017] Preferably, said formulation contains all strains of Bacillus spp. as listed in claim 1 .

[0018] Preferably, said formulation is provided in the form of nebulizable solution, particularly in the form of an aerosol. The invention also relates to a Bacillus spp. strain selected from those deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506.

[0019] For a better explanation of the inventive concept, the invention shall be hereinbelow described using its exemplary embodiments.

[0020] Example 1. Identification of Bacillus spp. strains suitable for use according to the invention.

[0021] Studies were carried out on various strains of probiotic bacteria or other gram-positive strains of Bacillus sp. The aim of these studies was to identify strains thatform spores and at the same time show high productivity in the formation of appropriate extracellular enzymes under specific conditions. The following selection criteria were used in the screening:

[0022] 1 ) limitation to Gram-positive Bacillus spp. strains showing no toxicity absence of toxigenic activity and ne very low antibiotic resistance, and at the same time were characterized by high survival in the formulation for administration in the aerosol form and showed proteolytic activity against DER P1 ;

[0023] 2) selection of strains producing maximum amounts of desirable extracellular enzymes within short time periods, wherein the enzymes considered desirable included lipases, ureases, amylases, proteases, and catalases.

[0024] As the result of the above-mentioned first stage of selection 25 different strains of Bacillus spp. were selected. Then, the quantity and quality of the extracellular enzymes secreted by them were examined.

[0025] The results of this selection phase are presented in the table below.

[0026] Table 1.

[0027] Key:

[0028] ++ very good productivity

[0029] + good productivity

[0030] + / - moderate productivity inactivity or trace productivity

[0031] Surprisingly, as the result of the search four strains of Bacillus spp. were identified to which simultaneously meet the requirements: of high efficiency in the production of specific enzymes, not producing toxins, upon to inhale, high susceptibility to antibiotics, and high effectiveness of degradation of DER P1 . No such characteristics were observed for other tested strains. Most often frequently, other strains as pre-selected in the first stage of the screening process were not able to simultaneously produce satisfactory quantities of extracellular enzymes with specific activities.

[0032] The identified preferred strains of Bacillus spp. were deposited in accordance with the Budapest Treaty within the Polish Collection of Microorganisms (PCM) held at the Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw on January 5 and February 22, 2024 under accession numbers as listed in Table 2.

[0033] Table 2. Deposited strains of Bacillus spp.

[0034] Example 2. Formulation containing Bacillus spp. strains and preparation thereof

[0035] Strains of probiotic bacteria meeting the requirements described in Example 1 are suitable for use according to the invention. Examples of such strains include the listed strains of Bacillus spp. deposited in connection with the present patent application.

[0036] In a preferred embodiment, said strains are used in the form of a liquid-suspension suitable for nebulization, particularly, aerosol.

[0037] It can be in the form of a liquid with the following composition: Preferably, strains of Bacillus spp. in the form of stabilized spores are used in the formulation.

[0038] A formulation having the above composition was obtained by growing separate cultures of each strain as listed in Table 2, followed by combining all cultures within an aqueous solution with the addition of a fragrance composition and bioethanol at appropriate pH. A unique BoV (Bag on Valve) container filled with compressed air without the use of any other gases was used. Formulation is not in contact with air, thereby being protected against contamination. A suitable spray head was used to administer the formulation at a rate of 6 mL / 10 s. The container used facilitates generation and delivery of aerosol at constant speeds regardless of the container position.

[0039] Example 3. Elimination of allergens originating from Dermatophagoides pteronyssinus

[0040] Studies were conducted to verify the suitability of the formulation of the invention for the elimination of house dust mite allergens from surfaces contaminated therewith.

[0041] The elimination of purified DER P1 allergen using the formulation described in Example 2 was tested in a cellulose filter disc assay by repeated addition of the formulation onto the surfaces of cellulose filter discs previously charged with allergen-containing samples. The test was performed in conditions mimicking the natural environment, i.e. moist bedding and liquid sweat. Sterile cellulose filter discs having the diameter of 0.5 cm, the thickness of 0.4 mm, and moisture content of 80-90%, were used in the test. Samples containing 5 micrograms of the allergen were treated with successive doses (15 pL) of the formulation of the invention under 80-90% RH and temperature of 20 °C or 37 °C for 5 days. Formulation was applied once a day for 5 consecutive days. The level of DER P1 elimination was determined using a quantitative Western blot SDS Page assay measuring the content of the non-degraded DER P1 in cellulose discs filtrates following centrifugation. The studies were carried out using products for the research on the Dermatophagoides pteronyssinus DER P1 protein as manufactured by CITEQ: primary rabbit polyclonal anti-DER P1 antibodies, recombinant DER P1 antigen (protein), excrement extract (lyophilisate), whole mite culture (lyophilisate).

[0042] The results of the experiment are provided in FIG. 1 . FIG. 1 A shows the results of the 12% SDS-PAGE assay: lane M corresponds to the PageRuler™ Plus Prestained Protein Ladder molecular weight marker, lanes K1-K5 correspond to control reactions involving DER P1 without the addition of the formulation of the invention, and lanes 1-5 (duplicates) correspond to reactions with DER P1 and the dosed formulation of the invention (15 pL administered at 1-day intervals). FIG. 1 B presents the results of a quantitative DER P1 elimination assay.

[0043] Unexpectedly, the formulation of the invention was found to eliminate at least 80% of the active DER P1 following once-daily dosing for at least 5 consecutive days.

[0044] An experiment was also conducted to investigate the possibility of DER P1 from being eliminated from various samples contaminated with this allergen following a single administration of the formulation obtained accordingto Example 2.

[0045] To this end, the degree of elimination of the pre-purified DERP1 allergen, DER P1 in mite excrements, and DER P1 in whole mite cultures by the formulation of the invention was measured was in a liquid phase assay. The results are shown in FIG. 2, with panel A showing samples (10 pg) following the treatment with the formulation at 20°C or 37°C for 12 h in a buffered neutral-pH solution. Lanes: M - PageRuler™ Plus Prestained Protein Ladder molecular weight marker; D20, D37, E20, E37, C20, C37 - DER P1 elimination reactions using the formulation of the invention at 20°C or 37°C on pre-purified DERP1 , excrement, and live mite culture, respectively. Panel B shows the results of the quantitative DER P1 elimination assay carried out as part of the same experiment.

[0046] The experiment confirmed that the main allergen, DER P1 , is effectively eliminated already following a single application of the formulation of the invention under conditions similar to the real environment inhabited by dust mites within the tested temperature range of 20 °C or 37°C, at 80-100% RH and neutral pH values.

[0047] BP / A / IW2 page 14

[0048] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURE

[0049] INTERNATIONAL FORM

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[0051] !Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority.

[0052] Form BP / 4 (sole page) BP / A / II / I2 page 14

[0053] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURE

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[0055] Form BPM (sole page) BP / A / II / 12 page 14

[0056] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSI T OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURE

[0057] INTERNATIONAL FORM 1. IDENT

[0058] Identifica DEPOSI iiTsci^

[0059] The micr

[0060] (Mark w IIL RECE

[0061] This Inter (date of t

[0062] IV. INTE

[0063] Address: Polish Ac ul. Weigi Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority w as acquired: where a deposit made outside the Budapest Treaty after the acquisition of the status of international deposit ary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was t -eceived by the international depositary' authority.

[0064] Form BP / 4 (sole page) BP / A / II / 12 page 14

[0065] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURE

[0066] INTERNATIONAL FORM

[0067] RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the

[0068] INTERNATIONAL DEPOSITARY AUTHORITY identified at the botom of this page " Where Rule 6 4 (d) applies, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest T reaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority.

[0069] Form BP / 4 (sole page)

Claims

Claims1. Composition containing bacterial strains of Bacillus spp. as deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506, for use in the prevention or treatment of diseases caused by the allergens originatingfrom Dermatophagoides pteronyssinus, wherein said composition is intended for use in the form of a solution, preferably administered as an aerosol, wherein the concentration of bacteria in said solution is at least 50 cfu / mL and said composition is applied to the allergen-contaminated surface daily for at least 5 consecutive days, leading to the elimination of at least 80% of active allergen from the contaminated surface.

2. Composition for use according to Claim 1 characterized in that the surface contaminated with the allergen is a furniture surface, preferably a bed surface.

3. Composition for use according to Claim 1 characterized in that the disease is allergy or respiratory asthma.

4. Composition for use according to Claim 1 characterized in that the allergen is DER P1.

5. Method for the elimination of allergen originating from Dermatophagoides pteronyssinus from the contaminated surface, characterized in that the contaminated surface is treated with a solution containing a Bacillus spp. strain selected from those deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505 and B / 00506, wherein said solution is applied to the surface contaminated with the allergen daily for at least 5 consecutive days.

6. Method according to Claim 5 characterized in that the surface contaminated with the allergen is a furniture surface, preferably a bed surface.

7. Method according to Claim 5 characterized in that the allergen is DER P1 .

8. Method according to Claim 5 characterized in that a solution containing all indicated strains of Bacillus spp. is used.

9. Formulation for the elimination of allergen originating from Dermatophagoides pteronyssinus characterized in that said formulation contains a Bacillus spp. strainselected from those deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506.

10. Formulation according to Claim 9 characterized in that said formulation is in the form of an aqueous solution comprising water, Bacillus spp. strains in an amount of at least 50 cfu / mL, ethanol in an amount of not more than 5% w / w and optionally a fragrance composition in an amount of not more than 5% w / w.11 . Formulation according to Claim 9 characterized in that said formulation contains all strains of Bacillus spp. as listed in claim 1 .

12. Formulation according to Claim 9 characterized in that said formulation is provided in the form of nebulizable solution, particularly in the form of an aerosol.

13. Bacillus spp. strain selected from among the strains deposited in the Polish Collection of Microorganisms (PCM) under accession numbers: B / 00503, B / 00504, B / 00505, and B / 00506.

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