WO2010023178A1

WO2010023178A1 - Pharmaceuticals comprising a bacterial polysaccharide

Info

Publication number: WO2010023178A1
Application number: PCT/EP2009/060872
Authority: WO
Inventors: Jens Kildsgaard; Thomas Janzen; Benedicte Flambard
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2008-08-28
Filing date: 2009-08-24
Publication date: 2010-03-04
Anticipated expiration: 2011-02-28

Abstract

The present invention relates to a bacterial polysaccharide from a bacterium belonging to a species of the group consisting of Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium bifidum, Streptococcus thermophilus and Lactobacillus reuteri. The use of said polysaccharide for treatment of diseases, in particular inflammatory bowel diseases and pharmaceutical composition comprising such a polysaccharide.

Description

PHARMACEUTICALS COMPRISING A BACTERIAL POLYSACCHARIDE

FIELD OF INVENTION

The present invention relates to a bacterial polysaccharide, its use for treatment of diseases, and pharmaceutical composition comprising such a polysaccharide. SUMMARY OF INVENTION

The present inventors have shown that polysaccharide (CPS and/or EPS) producing lactic acid bacteria strains are able to inhibit a pro-inflammatory stimulation of cells in culture. Based on this surprising finding, it is contemplated that EPS may reduce gastric mucosal inflammation in humans and have a general beneficial effect on the human immune system. This inhibition was not observed by an isogenic strain that is unable to produce EPS. As the EPS seems to "shield-off" the cells in culture, it is contemplated that EPS are beneficial for patients suffering from a break-down in their intestinal barrier function, e.g. patients suffering from Crohns disease. The data suggests that EPS might be helpful for maintaining a healthy gastrointestinal function, i.e. improving intestinal mucosal barrier function, for subjects suffering from irritable bowel syndrome as well as for normal, healthy subjects. Further, the data suggests that EPS is able to influence the immune system (immune modulation), that EPS decreases or inhibit inflammatory responses (EPS has anti-inflammatory properties), and that EPS is able to maintain gut health by enhancing the intestinal mucosal barrier function. The beneficial effect of EPS will be applicable in a number of settings: o For healthy people wishing to improve intestinal health o For people suffering from intestinal complications, e.g. irritable bowel syndrome (IDS),

Crohn's disease, or colitis. o The CPS/EPS concept may be able to play a role during early life development

DETAILED DISCLOSURE In a first aspect, the present invention relates to a polysaccharide isolated from a bacterium belonging to a species of the group consisting of:

Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium bifidum, Streptococcus thermophilus, and Lactobacillus reuteri.

In a second aspect, the present invention relates to a polysaccharide isolated from the growth medium (e.g. supernatant) of a bacterium belonging to a species of the group consisting of: Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium bifidum, Streptococcus thermophilus, and Lactobacillus reuteri. In an interesting embodiment the growth medium may comprise milk from a mammal. It is presently preferred that the bacterium belongs to a strain selected from the group consisting of: BB-12 ® free (CHCC8902), BB-12 ® (CHCC5445), ST143 (CHCC2389), RC-14 ® (CHCC5151), Bb. Longum sp. (CHCC8879), ST6008 (CHCC6008), ST9204 (CHCC9204), ST4239 (CHCC4239), BB-02 (CHCC2228), and a mutant/variant of any of these.

It is contemplated that the polysaccharide is an extracellular polysaccharide (EPS) or a capsular polysaccharide (CPS).

In a further aspect, the present invention relates to a pharmaceutical composition comprising the polysaccharide of the invention, and to a pharmaceutical composition comprising a bacterium belonging to a species of the group consisting of:

Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium bifidum, Streptococcus thermophilus, and Lactobacillus reuteri, especially a pharmaceutical composition comprising a bacterium belonging to a strain selected from the group consisting of: BB-12 ® free (CHCC8902), BB-12 ® (CHCC5445), ST143 (CHCC2389), RC-14 ® (CHCC5151), Bb. Longum sp. (CHCC8879), ST6008 (CHCC6008), ST9204 (CHCC9204), ST4239 (CHCC4239), BB-02 (CHCC2228), and a mutant/variant of any of these. The pharmaceutical compositions which comprise living cells may further comprises a prebiotic or a growth medium, e.g. a growth medium which comprises milk from a mammal, either as a mixture or as a kit of parts, wherein the bacterial cells are packed separately from the prebiotic or growth medium.

In a still further aspect, the present invention relates to the use of a polysaccharide of the invention, or the pharmaceutical composition of the invention, for treatment of inflammatory disease. In an embodiment, the disease is inflammatory bowel disease, colitis, or Crohn's disease.

In another aspect, the present invention relates to a method of treatment of an inflammatory disease by administering a polysaccharide or a pharmaceutical composition of the invention to the person in need of treatment. The disease may be inflammatory bowel disease, colitis, or Crohn's disease.

DEFINITIONS

In the present context, the term "mutant" should be understood as a strain derived from a strain of the invention by means of e.g. genetic engineering, radiation and/or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, polysaccharide producing properties as the mother strain. Such a mutant is a part of the present invention. Especially, the term "mutant" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant.

In the present context, the term "variant" should be understood as strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same polysaccharide producing properties. Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

EXPERIMENTAL

Purification of polysaccharides The polysaccharides are purified from the bacteria according to the method described in International Dairy Journal 9 (1999) : Separation, purification and characterisation of extracellular polysaccharides produced by slime-forming Lactococcus lactis ssp cremoris strains.

Analysis of the in vitro anti-inflammatory potential of the purified CPS/EPS.

The anti-inflammatory potential of the purified CPS/EPS are analysed in a murine dendritic cell assay and/or an assay involving a human intestinal epithelial cell line (HT-29). o The selected strains and the purified CPS/EPS are screened for immuno-modulatory and anti-inflammatory potential in murine bone marrow derived dendritic cells • The selected strains are screened for immuno-modulatory and anti-inflammatory potential in human intestinal epithelial cell cultures (Caco-2 and/or HT-29). o Markers of particular relevance are IL-8 (pro-inflammatory cytokine), and NF-KB¹ and PPAR-γ² (pro-inflammatory and anti-inflammatory transcription factors, respectively). These markers are measured in cell culture supernatants, and in cytoplasmic and nuclear extracts of the cells.

Analysis of the in vitro anti-inflammatory potential of whole strains producing CPS/EPS.

• The anti-inflammatory potential of whole strains producing CPS/EPS are analysed in a murine dendritic cell assay and/or an assay involving a human intestinal epithelial cell line (HT-29).

Results

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

¹ NF-κB is a family of transcription factors composed of homo- or heterodimers. Transcription factor of many genes in both innate and adaptive immoπe responses.

2 Nuclear hormone receptor. Activation of this transcription factor inhibits inflammatory gene expression (suppression of NF- B) REFERENCES

International Dairy Journal 9 (1999) : Separation, purification and characterisation of extracellular polysaccharides produced by slime-forming Lactococcus lactis ssp cremoris strains.

WO05021575A, WO07095958A, WO06119780A, WO05072641

All references cited in this patent document are hereby incorporated herein in their entirety by reference.

Claims

1. A polysaccharide isolated from a bacterium belonging to a species of the group consisting of:

2. A polysaccharide isolated from the growth medium (e.g. supernatant) of a bacterium belonging to a species of the group consisting of:

3. A polysaccharide of claim 2, wherein the growth medium comprises milk from a mammal.

4. The polysaccharide of any preceding claims, wherein the bacterium belongs to a strain selected from the group consisting of: BB-12 ® free (CHCC8902, DSM 17281), BB-12 ®

(CHCC5445, DSM 15954), ST143 (CHCC2389, ), RC-14 ® (CHCC5151), Bb. Longum sp. (CHCC8879), ST6008 (CHCC6008, DSM18111), ST9204 (CHCC9204), ST4239 (CHCC4239), BB-02 (CHCC2228), and a mutant/variant of any of these.

5. The polysaccharide of any preceding claim, which is selected from the group consisting of: an extracellular polysaccharide (EPS) or a capsular polysaccharide (CPS).

6. Pharmaceutical composition comprising the polysaccharide of any preceding claims.

7. Pharmaceutical composition comprising a bacterium belonging to a species of the group consisting of:

8. Pharmaceutical composition comprising a bacterium belonging to a strain selected from the group consisting of: BB-12 ® free (CHCC8902), BB-12 ® (CHCC5445), ST143 (CHCC2389), RC-14 ® (CHCC5151), Bb. Longum sp. (CHCC8879), ST6008 (CHCC6008), ST9204 (CHCC9204), ST4239 (CHCC4239), BB-02 (CHCC2228), and a mutant/variant of any of these.

9. Pharmaceutical composition of claim 7 or 8, which further comprises a prebiotic or a growth medium, e.g. a growth medium which comprises milk from a mammal.

10. Use of a polysaccharide of any preceding claims for treatment of inflammatory disease.

11. Use of a pharmaceutical composition of any preceding claims for treatment of inflammatory disease.

12. Use according to any preceding claim, where the disease is inflammatory bowel disease, colitis, or Crohn's disease.

13. Method of treatment of an inflammatory disease by administering a polysaccharide or a pharmaceutical composition of any preceding claims to the person in need of treatment.

14. Method according to the preceding claim, where the disease is inflammatory bowel disease, colitis, or Crohn's disease.