The use of an exopolysaccharide fraction produced by Lactobacillus rhamnosus

The subject of the present invention is a use of exopolysaccharides produced by Lactobacillus rhamnosus in the manufacturing of drugs for the prophylaxis and treatment of autoimmune diseases.

Application PL344117 reveals a strain of Lactobaccilus ramnosus, PLl, deposited as PCM 2572, which is capable of producing an extracellular substance whose composition consists of 42.4% glucose and 57.6% galactose. Aside from this, no other data regarding the properties of the revealed exopolysaccharide were published.

Patent PL195110 reveals the properties of several probiotic microorganisms, including two strains of L. rhamnosus. No mention was made of other uses of said strains nor of their products, which would be appropriate for such uses.

Patent PL195657 reveals a strain of Lactobacillus rhamnosus, 573L/1, deposited under the accession B/00004 and a pharmaceutical composition containing said strain. According to the description, this strain of possesses unique properties and exhibits a particular affinity for human cells. It also has very good probiotic properties. Patent PL195656 claims a strain of Lactobacillus rhamnosus, 573L/3, deposited under the accession number B/00006 as well as a pharamceutical composition containing said strain. According to the description, the claimed strain of Lactobacillus rhamnosus, 573L/3, is active against microaerophyllic bacteria and cures chronic inflammation. The described strain is capable of inhibiting disease-causing bacteria of the gastrointestinal tract, of binding to the epithelium of the human gastrointestinal tract as well as being resistant to the activity of gastric juices.

Patent PL195510 claims a strain of Lactobacillus rhamnosus, 573L/2, deposited under the accession number B/00005 as well as a pharamceutical composition containing said strain. According to the description, the claimed strain of Lactobacillus rhamnosus, 573L/2, is active against microaerophyllic bacteria and cures chronic inflammation. The revealed strain is singularly effective against bacteria of the genus Clostridium as well as possessing medicinal properties against post-antibiotic diarrhoea, and possesses excellent probiotic properties.

In none of the above-cited documents were other uses shown for the revealed strains of Lactobacillus rhamnosus not to mention any substances derived therefrom, which could be appropriate to such uses.

The publication T. Lipiήski et al., Carbohydrate research 338 (2003) 605-609 reveals the chemical structure of an exopolysaccharide produced by Lactobaccilus ramnosus KL37C. The publication indicates no uses of the isolated substance, nor any fractions containing it. The goal of the present invention is to deliver a substance which could be used to produce a drug for the treatment of autoimmune diseases. Unexpectedly, this goal was achieved by the present invention. The subject of the present invention is a use of a fraction containing exopolysaccharide encompassing repeated pentasaccharide units of the formula:

→ 3)-α-D-Glςp-Cl → 2)-β-D-Ga1/-(l → 6)-α-D-GaIp-(l → 6)-α-D-Glςp-(l → 3)-β-D-Gal/-{l → isolated from Lactobacillus rhamnosus in the production of a drug for the prophylaxis and treatment of autoimmune diseases. Preferentially, the strain used is Lactobacillus rhamnosus KL37C deposited at the PCM under the accession number B/00022 on May 13, 2008.

The subject of the present invention is also a method of obtaining the active exopolysaccharide fraction from the KL37C strain of Lactobacillus rhamnosus. A preferential embodiment of this method is shown in Example 1. Detailed description of the present invention

The present invention relates to the use of a bacterial exopolysaccharide (EPS), or its fraction, in the prophylaxis and treatment of autoimmune diseases as well as chronic inflammatory diseases, in whose pathogenesis bacterial infection has been implicated (acuteness, reappearance of clinical symptoms such as repeated inflammations like excema and arteriosclerosis).

The classical use of probiotics is based on the oral administration of live probiotic bacteria, whose activity is dependent on their colonisation of the gastrointestinal mucosa. The therapeutic effect based on the immunomodulatory activity of probiotics can be obtained through the administration of biologically active cell wall structures instead of whole bacterial cells. In the case of bacteria of the genus Lactobacillus, attempts have been made to use teichoic acids and peptidoglycan, as well as initial attempts to use exopolysaccharides. Unexpectedly, the EPS fraction isolated from L. rhamnosus KL37C, containing a polysaccharide whose structure is known from the publication by T. Lipiήski et al. entitled "Structural analysis of the Lactobacillus rhamnosus strain of KL37C exopolysaccharide",

Carbohydrate Research 2003, 338, 605-609 showed strong immunostimulatory activity against immune titre cells in vitro.

Instead of using whole bacteria (classical use of probiotics, orally administered live bacteria) the present invention reveals the use of biologically active bacterial structures, of a defined structure and defined immunomodulatory properties. After analyzing research performed to date, the prime candidate is an exopolysaccharide isolated from Lactobacillus rhamnosus EPS37, or its fraction (i.e. the pentasaccharide being the base structural unit of the EPS). The choice of EPS is further supported by initial results indicating the different immunoregulatory properties of the EPS and the whole bacteria from which it has been isolated. If, indeed, even non-specific infective agents acting upon persons with a particular genetic predisposition can initiate systemic diseases such as chronic intestinal inflammation, excema or rheumatoid arthritis, then an attempt to "oversteer" (block) Toll-like receptors or other locations on cellular membranes responsible for the initiation of autoimmune inflammation may stop the process at a very early juncture. Assuming that the exopolysaccharide or its active fragment will have a blocking effect on the activity of LPS or another bacterial product, then its role may be deemed as that of an "anti-adjuvant". Such a concept is entirely novel and may be effective, if said "anti-adjuvant" can be administered such that it reaches apprpriate sites of activity. One may, however, also consider administering the exopolysaccharide alongside an antibiotic. The latter is also an entirely new concept. One of the proposed innovative medicinal compositions is an EPS preparation given orally with taurine during bowel inflammation (EPS+Taurine+HOCl produced by inflammatory neutrophiles = antiinflammatory activity+antioxidant activity [taurochloramine]+immunosuppresive activity [EPS]). Instead of taurine, one may administer a preparation of Taurolidine, which releases taurine in vivo. Taurolidine (Taurolin) is a drug used against bacterial infections (Watson, R.W, Redmond, H.P., Mc Carthy, J., Bouchier-Hayes, D., 1995, Taurolidine, an antilipopolysaccharide agent, has immunoregulatory properties that are mediated by the amino acid taurine. J Leukoc Biol. 58:299.). A similar treatment strategy has been used against IBD by administering a conjugate of 5-amino salicylic acid and taurine. The presence of taurine augments the therapeutic activity of the preparation due to the formation of taurochloramine. The proposition of this composition of EPS+Taurine has not been studied empirically. It is based on research indicating the antiinflammatory and antioxidant activity of taurine derivatives, taurochloramine and taurobromamine: (Marcinkiewicz J, et al. (2006) "Anti- inflammatory effects of taurine derivatives (taurochloramine, taurobromamine, and taurolidine) are mediated by different mechanisms", Adv Exp. Med Biol 583: 481-492)

It seems, however, that the administration of EPS during the acute phase of an inflammation (intensification of chronic inflammations or autoimmune disease) is not effective due to the absence of an inhibitory effect of EPS on the production of inflammation titre mediators (i.e. cytokin ThI - TNF) Example 1. Production of the active exopolysaccharide fraction.

Bacteria of the Lactobacillus rhamnosus strain KL37C were cultured on MRS (Rogosa) medium in anoxic conditions, using a BBL Anaero-Pac System (Becton Dickinson) for 48 hours at 37°C. Bacterial cells were centrifuged at 8000 RPM, for 30 min, at 4°C. The exopolysaccharide was isolated from the bacterial biomass suspended in water using sonification: 3x5 min in an icebath. The precipitate was centrifuged at 6000 RPM, for 30min, at 4°C. The supernatant was recentrifuged twice at 12000 RPM for 1 hour at 4°C and then precipitated with 5 volumes of ethanol and kept overnight at -20 ⁰ C. The precipitate was centrifuged at 12000 RPM for 20 min at 4°C, and then suspended in water, frozen at -70 ⁰ C and lyophilised. The preparative efficiency was 2% of dry bacterial mass. Using known methods (see T.Lipiήski et al. Carbohydrate research 338 (2003) 605-609), it was determined that the isolated fraction contains an exopolysaccharide encompassing repeated pentasaccharide units of the formula:

→ 3)-α-D-Glφ-(l -→ 2)-β-D-Ga1/-(l → 6)-α-D-Oal/Hl → 6)-α-D-Glςp-(l → 3)-β-D-Gal/-{l →

Example 2. Biological activity The fraction isolated according to Example 1 was subjected to biological testing.

The animal test results were based on the following animals : CBA mice - induction of humoral response against ovalbumine [OVA] ; DBA mice - induction of a humoral response against collagen type II as well as on in vitro studies performed on cells of an immune titre (peritoneal macrophages isolated from mice as well as J774 macrophages). We studied the immunomodulation of EPS vs. LPS: binding strength to the macrophage cell membrane, cytokine synthesis induction, modulation of the synthesis of IgG & OVA. The research was performed at the Faculty of Immunology of the Medical College of the Jagiellonian University as part of statutory research, No. 501/P/22/L.

In vitro assays: the research was performed on peritoneal macrophages of CBA mice. The cells were stimulated in vitro with killed Escherichia coli, Lactobacillus rhamnosus as well as components of their cell walls (LPS-lipopolysaccharide and EPS- exopolysaccharide). We examined their ability to induce cytokine secretion (ELISA measurement) (Fig. 1), as well as their affinity for macrophage surface receptors (using atomic force microscopy- AFM). AFM examination showed that macrophages preincubated with EPS augment their ability to bind LPS (Ref. 1). During studies on the ability of macrophages to produce ROI, it was

shown that Lactobacillus rhamnosus and EPS generate lower levels of ROI than E.coli and LPS do. Furthermore, it was shown that Lactobacillus (EPS) exhibit a lower induction potential for cytokine synthesis by macrophages than E.coli (LPS). We used standard techniques described in M. Targosz, A. Labuda, P. Czuba, R. Biedroή, M. Strus, A. Gamian, J. Marcinkiewicz, M. Szymoήski. "Influence of macrophage activation on their capacity to bind bacterial antigens studied with atomic force microscopy." Nanomedicine. 2006 Iun;2(2):82-8.

The results obtained are shown in the Figures. Fig. 4 shows cytokine synthesis by macrophages stimulated in vitro by EPS37 and LPS, depending on the dose of substance administered. The adjuvant properties of EPS vs. LPS were examined in CBA mice with ovalbumine mixed with LPS (OVA+LPS) or EPS (OVA+LPS). The control consisted of mice immunized with OVA alone. LPS exhibited strong adjuvant properties, inducing a twofold increase in IgG&OVA synthesis. The level of IgG&OVA was significantly lower in the OVA+EPS group. The most interesting result was the blocking by EPS of the adjuvant properties of LPS.

We also examined the effect of EPS on CIA (collagen-induced arthritis) (the study was performed at the Faculty of Immunology of Medical College of the Jagiellonian University in collaboration with the Department of Rheumatology). Animal tests: the studies on the effect of EPS on arthritis were performed on DBA mice, using collage induced arthritis (CIA), an animal in vivo experimental model corresponding to rheumatoid arthritis in humans.

In order to elicit CIA, the mice were injected intraperitonneally with type II collagen (CII) administered jointly with LPS (positive control). The experimental groups were: mice given collagen+EPS or collagen+LPS+EPS. We evaluated the development of arthritis (frequency /arthritic index). Furthermore, during the experiment we measured the limb diameter of the animals. We observed limb distension only in the group treated with colagen+LPS (CIA+ >50% cases; 8/14); in LPS+EPS (CIA+ -6%; 1/15); in the EPS group there were no cases of arthritis (frequency - Fig.l; arthritic index - Fig. 2). The clinical observations correlated directly with the MPO level (neutrophile enzyme) in the studied tissue. Because anti-CII antibodies are key in the development of arthritis, we also evaluated the serum anti-CII IgG levels individually in each animal. We observed an anti-CII IgG level 80% lower in animals given collagen+EPS than when collagen+LPS was given. Whereas the administration of collagen+LPS+EPS lowered the antibody levely by 60% in relation to the CII+LPS group [Fig. 3].

The above results make it possible to conclude that the presence of EPS protects against the occurrence of CIA induced by the administration of collagen with bacterial adjuvants (Gram(-) or LPS-producing bacteria). The research was also performed on a "classical" CIA model, the induction of arthritis through a twofold administration of collagen with Freund's adjuvant (Thl-mediated response stimulator, as opposed to LPS, which stimulates the Th2-mediated response), and we observed no effect of EPS on CIA. Furthermore, we also examined the effect of EPS on CIA progression induced by the passive intravenous transfer of commercial anti-collagen IgG - without any EPS effect. These results show that EPS inhibits the development of inflammation dependent on auto-antibodies (antiCII IgG - arthritis induction) through the inhibition of antibody production. However, EPS is unable to quench the inflammation once the antibodies (see passive CIA transfer) are already produced.

The results of animal testing are shown in the attached figures. Fig. 1 shows the frequency of the occurrence of CIA in DBA mice immunised with type II collagen in the presence of LPS, LPS+EPS or EPS. (n=15). Fig. 2 shows confirmed augmentation of arthritis (points - Arthritic index). Fig. 3 shows the observed humoral response to type II collagen in CIA. The level of specific serum IgG in mice was determined using ELISA. The results are shown in relative units [U] (IU = 1/100 of IgG titre).