HK1061644B - Solid composition containing bacillus-type non-pathogenic bacterial spores - Google Patents

Description

The present invention relates to a solid composition containing non-pathogenic bacterial spores, for use in pharmaceutical, veterinary or nutritional applications, including a composition containing bacterial spores of the genus Bacillus.

The formulation of spores for administration to humans or animals is difficult and problematic since, in the case of living material, the risk of loss of a considerable part of the active substance is high if inappropriate processes are used.

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One of the main disadvantages of the earlier technology is the low stability of the resulting compositions, a critical factor which does not allow storage for reasonably long periods and which makes it necessary to store and preserve the composition before use at low temperatures in order to keep the properties of the spores as intact as possible.

Another disadvantage of earlier art compositions is the low concentration of active ingredient, thus of spores, which can be introduced into the composition itself.

Liquid spore-based formulations are available on the market, for example the bacillus spore suspension, which has been marketed in Italy for a long time under the brand name Enterogermina®. Although it is effective and has long been appreciated by consumers, this suspension cannot contain a very high concentration of the active substance (more than 2 billion spores/5 ml).

The purpose of the present invention is to provide a solid composition from non-pathogenic bacterial spores, hereinafter referred to simply as spores , which is simple to produce, easy to store and contains a high amount of active substance and stable over time.

It has now been found that by adsorbing the spores onto a suitable matrix using the fluidized air bed technique, a solid form of spores is obtained at very high concentration, which is easy to process industrially and very stable.

In particular, it was found that this solid form, hereinafter referred to as composition , allows a significant amount of spores to be attached to the matrix by creating a high-concentration solid composition which, due to its granularity and specific surface area, is particularly suitable for the in vivo release of spores throughout the gastrointestinal tract.

In addition, it was found that the composition has excellent fluidity properties and can therefore be industrially processed for formulation into single or multi-dose compositions without the need for further processing.

Thus, in one aspect, the invention concerns a composition of spores of nonpathogenic bacteria of the genus Bacillus adsorbed on a matrix consisting of at least one water-insoluble compound and a cellulose derivative, which may be obtained by the fluidised air bed technique.

In particular, the invention relates to a composition of spores of non-pathogenic bacteria of the genus Bacillus which can be obtained by a process which involves the treatment of a liquid suspension of these spores with a matrix of an adsorbent compound insoluble in water and a cellulose derivative using the fluidised air bed technique.

According to the present invention, the expression adsorbent compound means any chemical compound, or a mixture of chemical compounds, which can be ingested and has adsorbent properties; preferably, the adsorbent compound insoluble in water is chosen from the group consisting of clays, kaolin, calcium carbonate, colloidal silicates, magnesium silicate and aluminium, with kaolin being particularly advantageous.

According to the invention, the expression cellulose derivative refers to any cellulose derivative that can be ingested, such as, for example, microcrystalline cellulose, methylcellulose, hydroxypropylmethylcellulose, etc. of which a wide range is commercially available, with microcrystalline cellulose being particularly preferred.

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The spores can be retained on the matrix by any means of attachment, i.e. by any possible bonding (chemical, biological, physical, etc.) depending on the type of matrix used.

The relative amounts of the two components that make up the matrix can vary over a wide range, for example the weight ratio between the adsorbent compound and the cellulose derivative can be between 90:10 and 10:90, preferably between 70:30 and 30:70, even more preferably between 60:40 and 40:60, with the two components being advantageously present in the matrix in a weight ratio of about 50:50.

In any case, the matrix of the invention is a water-insoluble mixture, inert to spores, where by inert to spores it is meant that it does not interfere negatively with spores.

The spores that may be used in the present invention are preferably spores of non-pathogenic bacteria particularly useful in pharmaceutical, veterinary and/or nutritional applications. The composition of the present invention may contain spores of a single Bacillus or spores of several mixed Bacillus.

Depending on the preferred aspect, the composition of the invention includes Bacillus subtilis or Bacillus clausii spores.

Preferably, the composition of the invention includes spores of one or more strains of Bacillus clausii (formerly known as Bacillus subtilis), which were registered under the Budapest Treaty with the CNCM Institut Pasteur under serial numbers: I-273, I-274, I-275, I-276.

For the preparation of the composition, the technique of the fluidized air bed is used, which is well known to the professional.

This technique involves spraying an aqueous suspension containing the spores, hereinafter referred to as concentrated suspension, on a matrix obtained by mixing at least one adsorbent compound and a cellulose derivative, agitated continuously by an air stream throughout the process, in a tool designed for this purpose, such as a fluidised air bed granulating machine.

The concentrated suspension is aqueous and has a very high concentration of spores.

According to a particularly advantageous aspect of the invention, the concentrated suspension is obtained by inoculation of one or more strains of Bacillus in a culture medium (e.g. based on peptones and mineral salts), by incubating the mixture at an appropriate temperature for 48-72 hours under aerobic conditions, by centrifuging the cells from the depleted medium with distilled water and then by pasteurizing the resulting suspension.

The concentrated suspension thus prepared has a Bacillus spore concentration of more than 10 billion per gram, normally between about 15 and about 25 billion per gram or even more.

In general, the concentrated suspension is best preserved after freezing because of its high instability at room temperature; in this particular case, the concentrated suspension will be thawed just before use in the process of the present invention.

Alternatively, the concentrated suspension may be an extemporaneous suspension of spores preserved in freeze-dried form in water.

The airflow temperature used in the process of the present invention is between room temperature and the maximum temperature supported by the spores; according to a preferred aspect, the process is preferably carried out at a temperature between 40° and 90°C, preferably between 60° and 80°C.

Once the spraying and adsorption of the concentrated matrix is completed, the resulting composition is then kept in suspension by means of the heated air stream until it reaches the desired residual humidity, preferably until the humidity is less than 3%, preferably less than or equal to 2%.

The process time is defined by the amount of concentrated suspension to be sprayed, the spraying speed and the air flow temperature.

The method of preparation of the composition is a further subject of the present invention.

As shown above, the composition of the invention can be obtained in a highly concentrated form and have a concentration of, for example, 3 to 30 billion spores per gram of final composition, for example, 5 to 20 billion spores per gram of final composition, preferably about 10 billion spores per gram, thus allowing the administration of substantial amounts of spores in small volumes. This important property makes the composition particularly easy to use and, for example, can be introduced into small cells or bags or incorporated into food or other compositions. If necessary, the composition can also be administered again after being suspended in water or other liquids.

The composition of the invention has been stable while maintaining its title unaltered for a long time, even at temperatures above room temperature (about 40°C).

The determination of the spore content of the invention can be carried out by any method, for example by counting on conventional culture plates.

As shown above, the composition of the invention has a large specific surface area, due to a very fine particle size, up to 90 per cent of the composition particles with particle size equal to or less than 130 micrometres, preferably with 60 per cent of the composition particles with particle size equal to or less than 60 micrometres.

In addition, the composition has no odor or taste and can thus be added to foods, beverages, or other compositions without altering their original flavor.

If desired, the composition may contain additives suitably chosen according to the final consumer, the mode of absorption or the type of further treatment to which it is intended to be subjected, provided that the additives are inert against the spores.

For example, lubricants, diluents, etc., or any other agent which may increase the flow or enhance other particular physical properties, may be added to facilitate further processing of the composition.

For example, if the composition is to be introduced into hard gelatine capsules, it may be useful to add magnesium stearate and/or microcrystalline cellulose.

Alternatively or in combination with the above additives, flavourings may be added which may give the composition particular fragrances or flavors.

Any subsequent components may be added to the matrix prior to the adsorption of the spores or simply to the final composition obtained by the process of the invention.

If necessary, the composition may be modified further; the composition may therefore be granulated according to the well-known techniques in case compression is desired, or treated to obtain controlled-release compositions according to the well-known techniques, in order to alter the time of its release into the intestine.

The composition may be administered in varying amounts depending on the purpose for which it is administered.In general, 10 billion spores/day or more can be expected for human administration, preferably 1 to 8 billion spores/day, for example 2, 4 or 6 billion spores/day, with administration possible as a single dose or repeatedly.

For the purpose of its administration, the composition of the invention may, if necessary, be formulated in dosage units; for example, due to its qualities, it can be easily formulated in gelatine capsules, such as capsules in the 0, 1 or 2 format, chosen by the expert in the field according to the dosage.

The dosage units, in the form of capsules or sachets, containing the composition of the invention represent a further object of the present invention.

For example, these dosage units may contain 1 to 10 billion of these spores, preferably 2 to 5 billion, 50 to 500 mg, for example 50 to 250 mg of kaolin and 50 to 600 mg, for example 50 to 300 mg of microcrystalline cellulose.

These dosage units may be administered once or several times a day, depending on the need and concentration of the dosage unit.

For example, dosage units containing 5-7 billion spores, preferably about 6 billion spores, can be prepared and administered once a day.

In the case of living material, it is obvious that the microbiological titer may vary; therefore, an excess of 10-20% of the intended dose of spores is preferably added to the preparation of the composition.

Some batches of the invention's compositions packaged in glass or polyethylene were subjected to stability studies to evaluate their behaviour at different temperatures (5°C to 40°C) and humidity levels (up to 75% relative humidity). Results after 24 months showed that the composition title was not significantly altered under any of the conditions tested. Antibiotic resistance and biochemical characteristics were also evaluated under the same conditions and were found to be consistent with the original properties of the product.

The composition according to the invention is useful in the pharmaceutical, veterinary and/or nutritional fields.

It has the same commercial applications as Enterogermina®, in particular the composition of the invention has a beneficial effect on the gut and the immune system and is particularly suitable for the treatment and prevention of intestinal dysmicrobiosis and endogenous dysvitaminosis as well as as adjuvant treatment in the recovery of the gut microbial flora affected by antibiotic therapy and chemotherapy, and is suitable for use, for example, in combination with antibiotics to combat Helicobacter pylori.

The invention also concerns a medicinal product containing the bacterial spore composition as defined above.

The following examples illustrate the invention without limiting it.

The Commission has decided to initiate the procedure laid down in Article 8 (2) of Regulation (EC) No 1049/2006.

Pre-fermentation of 600 ml of a suspension of four strains of Bacillus clausii I-273, I-274, I-275 and I-276 (in equal proportions) at a concentration of 500 million spores/ml in 30 litres of fermentation medium for 7 hours is carried out. The pre-fermented suspension is inoculated in 1000 litres of peptone and mineral salt fermentation medium; incubation at 37°C for 48-72 hours under aerobic conditions, centrifugation of the cells of the culture medium with distilled water until a final volume of 100 litres is obtained and the suspension is passed at 70°C for 30 minutes. A concentrated suspension containing a concentration of Bacillus spores of about 20 billion ions per gram is obtained.

The following paragraphs are added:

In a system for the fluidised airbed process, 15 kg of pharmaceutical grade kaolin and 15 kg of pharmaceutical grade microcrystalline cellulose are loaded and the mixture is heated for a few minutes by means of a 60 °C air stream. Under a laminar flow hood, 15 kg of an aqueous suspension of a mixture of Bacillus clausii spores I-273, I-274, I-275 and I-276 (concentrated suspension as prepared above) is loaded into a container with a peristaltic pump connected to 1.2 mm diameter mist nozzles and connected to the fluidised airbed installation at a concentration of 20 billion grams per spamme.The concentrated suspension of spores is then sprayed on the heated mixture at a pressure of 2 bar and a flow rate of 135 ml/minute, the mixture being kept in suspension with air at 60°C. After about 110 minutes the system is stopped, allowed to cool and the composition is recovered, thus obtaining a composition having the following characteristics: - titer determined by plate count: 10 billion spores/g; residual moisture ≤ 2 %; granulometry - determined by a Malvern® laser granulometer in demineralized water: 90% particles < 100 μm 60 per cent of particles < 50 μm specific surface: 3-5 m2/g.

The following paragraphs shall apply:

By operating as described in Example 1, but using only Bacillus clausii strain I-274, a product with the following characteristics is obtained: - titer determined by plate count: 10 billion spores/g; residual moisture ≤ 2 %; granulometry - determined by a Malvern® laser granulometer in demineralized water: 90% particles < 130 μm 60 per cent of particles < 60 μm specific surface area: 3,5 to 5 m2/g.

The Commission has

Operating as described in Example 1, but using only Bacillus clausii strain I-276, a product with the following characteristics is obtained: 12 billion spore/g; residual moisture ≤ 3 %; particle size - determined by a Malvern® laser particle size meter in demineralized water: 90% particles < 130 μm 60 per cent of particles < 60 μm specific surface: 3-5 m2/g.

The Commission shall adopt implementing acts.

To 240 g of a composition of Example 1 57 g of microcrystalline cellulose and 3 g of magnesium stearate are added. After mixing, the resulting composition is distributed in hard gelatine operculated capsules of size 1, each containing 300 mg of the following composition: - What? The following shall be added to the list of products: containing approximately 2 billion spores of - What?

Bacillus Clausii (I-273, I-274, I-275, I-276)	mg	240,00
Cellulose microcristalline	mg	57,00
Stéarate de magnésium	mg	3,00

The Commission shall adopt implementing acts. The following information is provided in the package leaflet: The following is the list of active substances:

Matrice (Kaolin + Cellulose microcristalline) contenant environ 2 milliards de spores de Bacillus Clausü (I-273, I-274, I-275, I-276)	mg	200,00*
Cellulose microcristalline	mg	72,25
Stéarate de magnésium d'origine végétale	mg	2,75

(* dans la formule de fabrication 220,00 mg correspondant à un surdosage de 10%)

The Commission has The following information is provided in the package leaflet: The following information is provided for the purpose of the analysis:

Matrice (Carbonate de calcium + Cellulose microcristalline) contenant environ 2 milliards de spores de Bacillus Clausü (I-273, I-274, I-275, I-276)	mg	200,00*
Cellulose microcristalline	mg	52,25
Stéarate de magnésium d'origine végétale	mg	2,75

(* dans la formule de fabrication 220,00 mg correspondant à un surdosage de 10%)

Claims (19)

1. Solid composition of spores of nonpathogenic bacteria of the Bacillus genus, adsorbed onto a matrix made up of:

   - at least one water-insoluble adsorbent compound chosen from the group made up of clays, kaolin, calcium carbonate, colloidal silicas and magnesium and aluminium silicate, and

   - a cellulose derivative,

   characterized in that:

   the ratio by weight of said adsorbent compound to said cellulose derivative is between 90:10 and 10:90, and

   the composition can be obtained by the fluidized air bed technique.
2. Composition according to Claim 1, characterized in that it can be obtained by a process which comprises treating, according to the fluidized air bed technique, a liquid suspension of said spores with a matrix made up of at least one water-insoluble adsorbent compound and a cellulose derivative.
3. Composition according to Claim 1 or 2, characterized in that it contains from 3 to 30 billion of said spores per gram of composition.
4. Composition according to Claim 3, characterized in that it contains from 5 to 20 billion of said spores per gram of composition.
5. Composition according to Claim 4, characterized in that it contains approximately 10 billion of said spores per gram of composition.
6. Composition according to one of Claims 1 to 5, characterized in that said adsorbent compound is kaolin.
7. Composition according to one of Claims 1 to 5, characterized in that said adsorbent compound is calcium carbonate.
8. Composition according to one of Claims 1 to 7, characterized in that said cellulose derivative is microcrystalline cellulose.
9. Composition according to one of Claims 1 to 8, characterized in that the ratio by weight of said adsorbent compound to said cellulose derivative is between 70:30 and 30:70.
10. Composition according to Claim 9, characterized in that said ratio is approximately 50:50.
11. Composition according to one of Claims 1 to 10, in the form of a dosage unit in gel capsules or sachets.
12. Composition according to Claim 11, containing for each dosage unit from 1 to 10 billion of said spores, from 50 to 500 mg of kaolin and from 50 to 600 mg of microcrystalline cellulose.
13. Composition according to Claim 12, containing for each dosage unit from 2 to 5 billion of said spores, from 50 to 250 mg of kaolin and from 50 to 300 mg of microcrystalline cellulose.
14. Composition according to Claim 11, containing for each dosage unit approximately 2 billion of said spores in gel capsules.
15. Composition according to Claim 11, containing for each dosage unit approximately 6 billion of said spores in gel capsules.
16. Composition according to one of Claims 1 to 15, characterized in that said spores are spores of Bacillus subtilis or of Bacillus clausii.
17. Composition according to Claim 16, characterized in that said spores originate from one or more strains chosen from Bacillus clausii I-273, I-274, I-275 and I-276.
18. Composition according to one of Claims 1 to 15, characterized in that said spores are a mixture of spores of different Bacilli.
19. Medicinal product comprising the composition according to one of Claims 1 to 18.