Polysaccharide from bifidobacterium infantis strain and use thereof to treat or prevent inflammatory disorders

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology and medicine. Disclosed is a polysaccharide which is isolated from the Bifidobacterium infantis NCIMB 41003 strain and has the structure [-β(1,3)-D-GalpNAc-β(1,4)-D-Glcp-]n, where said disaccharide unit repeats n times, which yields a polysaccharide with molecular weight greater than 100000 Da. The polysaccharide exhibits immunomodulating activity and is used in preparing medicinal agents for treating or preventing undesirable inflammatory activity, undesirable gastrointestinal inflammatory activity, rheumatoid arthritis and autoimmune disorders.

EFFECT: pharmaceutical composition for treating and preventing inflammatory disorders and a food product containing the isolated polysaccharide are disclosed.

9 cl, 6 dwg, 3 ex

 

The scope of the invention

The present invention relates to the exopolysaccharide and to its use in the treatment and prevention of inflammatory disorders.

Prior art

The gastrointestinal tract provides a protective interface between the internal environment and the constant threat of antigens derived from food and microorganisms from the external environment (Sanderson et al., 1993). According to estimates, the complex ecosystem of the intestinal microflora of adults contains 500 different bacterial species. Some of these species are considered to be potentially harmful due to the production of toxins, invasion through the mucous membrane or activation of carcinogens and inflammatory reactions (Salminen, 1998). However, were identified bacterial strains with strengthening health activities.

Probiotics are beneficial bacteria that exist in the healthy microflora of the intestine and were defined as a group of living organisms that are beneficial to the animal host by improving its intestinal microbial balance. They consist of "friendly bacteria"that are cultivated in the laboratory and then used to restore the balance of microflora, which has become unbalanced, for example, due to stress, illness or antibiotic use. It is important, as it was the shows the ingestion of probiotic bacteria may stabilize immunological barrier in the intestinal mucosa by reducing the formation of local proinflammatory cytokines (Isolauri, 1993; Majamaa, 1997). It is shown that the change of properties of local flora by probiotic therapy reverses some immunologic disorders characteristic of Crohn's disease (Malin, 1996), food allergies (Majamaa, 1997) and atopic eczema (Isolauri, 2000).

One of the predominant bacterial species present in the intestinal microflora is Bifidobacterium. Intestinal Bifidobacterium fermented sugar with obtaining lactic acid. Genomic codes of many proteins Bifidobacterium longum specialized in relation to the catabolism of oligosaccharides that allows the bacteria to use the so-called "neoslavery" vegetable polymers or glycoproteins and glycoconjugates of the owner. It is believed that the ability of Bifidobacterium to compete with other intestinal bacteria and make up a large percentage of the bacterial flora of the gastrointestinal Department may be partially due to the large variety of molecules, which they can use for energy.

While B.infantis, B.breve and S.longum are major bacterial group in the intestine of infants, Bifidobacteria are considered to be only the 3rd or 4th is about the size of a group of bacteria in adults (and are only 3-6% of the faecal flora adult). The number of these bacteria in the human body with age usually decreases. Babies who breastfeed breast, Bifidobacteria constitute about 90% of their intestinal bacteria; however, this number is lower in infants fed artificially. When the power breastfed infants change to cow's milk and solid food, Bifidobacteria are combined with the increasing number of other bacteria found in the human body, such as Bacteroides Streptococci and lactobacilli.

It was shown that bifidobacteria play a role in modulating the immune system. Believe that B.breve release metabolites, providing anti-NF effect, able to penetrate through the intestinal barrier. It was reported that the inflammation of the mucosa in mice with absence of interleukin-10 (IL-10) is reduced when feeding test animals preparation of lactic acid bacteria (Madsen, et al., 1997; O'mahony et al., 2001; McCarthy et al., 2004). In W0 00/41168 discovered a strain of Bifidobacterium infantis isolated from resected and washed the gastrointestinal tract of man, which is significantly immunomodulatory oral use.

Scientific research indicates an increasing incidence of diseases that can be caused by incomplete or disturbed microflora (natural microbial resident population of the digestive system is), such as infections of the gastrointestinal tract (GIT), constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, food allergies, diarrhea caused by antibiotics, cardiovascular disease and some cancers, such as colorectal cancer. The data indicate that after treatment with a single strain of Bifidobacterium infantis severity of IBS symptom decreases (Whorwell et al., 2006). Efficiency is associated with modulation of systemic immune responses, which indicates that the mechanism of action, in part, is immuno-mediated (O'mahony et al., 2005). In the present invention are described compound isolated from Bifidobacterium infantis, which simulates together with immunomodulating activity of Bifidobacterium infantis in vitro.

Summary of the invention

In the present invention proposed a polysaccharide produced by Bifidobacterium infantis, which demonstrates immunomodulatory properties. The polysaccharide may be secretively (exopolysaccharide) or secretively.

According to one aspect of the invention proposed by the selected polysaccharide containing structure: [-β(1,3)-D-GalpNAc-β(1,4)-D-Glcp-]n.

The polysaccharide can be isolated from the bacterial strain Bifidobacterium. The strain may represent such a strain, as NCIMB 41003. According to another aspect of the invention proposed the use of polysaccharide p the invention as a medicine.

According to another aspect of the invention the application of the polysaccharide according to the invention in getting medicines for the treatment or prevention of undesirable inflammatory activity.

According to another aspect of the invention the application of the polysaccharide according to the invention in getting medicines for the treatment or prevention of undesirable gastrointestinal inflammatory activity.

In one embodiment of gastrointestinal inflammatory activity represents Crohn's disease, ulcerative colitis, irritable bowel syndrome, pauci, post-infectious colitis, diarrhea associated with Clostridium difficile, diarrhoea associated with rotavirus, or post-infectious diarrhea.

According to another aspect of the invention the application of the polysaccharide according to the invention in obtaining drugs for treating or preventing rheumatoid arthritis.

According to another aspect of the invention the application of the polysaccharide according to the invention in obtaining drugs for treating or preventing autoimmune disorders.

According to another aspect of the invention proposed pharmaceutical composition comprising a polysaccharide according to the invention and a pharmaceutically acceptable carrier.

In an additional embodiment of the of Britania also offered food, containing the selected polysaccharide. For example, the food product may constitute one or more selected from the group including yoghurts, cereals, drinks and the like.

Detailed description of the invention

Below various preferred features and embodiments of the present invention are described by non-limiting example.

In the implementation in practice of the present invention are used, unless otherwise indicated, conventional technological methods of chemistry, Microbiology, and immunology, which are within the skill of a person skilled in the art. Such methods are described in the literature.

The authors of the invention was identified Bifidobacterium, secreting exopolysaccharide, which has immunomodulating properties.

The exopolysaccharide

The present invention relates to an exopolysaccharide, biointensive with Bifidobacterium infantis. Polysaccharides are synthesized on a broad range of microorganisms and are usually repeating units of sugar that remains or associated with the cell surface or are secreted, or both. They play a role in both cellular stress reactions or may contribute to the virulence of the pathogen. Recently it was demonstrated immunomodulatory role of polysaccharide of Bacteroides fragilis (Mazmanian et al., 2005).

You should note that all references in this description of the invention for treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly preferably. As the treatment of a human or veterinary treatment are included in the scope of the present invention.

Inflammation

Inflammation is a local response to cellular injury that is characterized by capillary expansion, infiltration of leukocytes, redness, heat, pain, swelling, and often loss of function. The control of the inflammatory response is carried out on a number of levels (for an overview, see Henderson B. and Wilson, M. 1998.) Controlling factors include cytokines, hormones (eg, hydrocortisone), prostaglandins, reactive intermediate compounds and leukotrienes.

Cytokines are low-molecular biologically active proteins that are involved in education and the control of immune and inflammatory responses, and regulate development, tissue repair and hematomas. They provide connections between leukocytes and other cell types. Most cytokines are pleiotropic and demonstrate biologically diverse overlapping activity.

Cytokine cascades and network control inflammatory response, and not the action of specifically what about cytokine specific type of cells (Arai Kl, et al., 1990). Attenuation of the inflammatory response leads to lower concentrations of the respective trigger signals and other inflammatory mediators, which leads to the suspension of the inflammatory response. The tumor necrosis factor alpha (TNFα) is a major proinflammatory cytokine, as it initiates a cascade of cytokines and biological effects that lead to the inflammatory state. Thus, agents that inhibit TNFα, such as infliximab, are currently used for the treatment of inflammatory diseases.

It is believed that proinflammatory cytokines play an important role in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease (IBD). Modern therapy for the treatment of IBD is aimed at reducing the levels of these proinflammatory cytokines. The exopolysaccharide of the present invention may have potential application in the treatment of inflammatory disorders. This can be achieved, for example, by increasing the concentration of non-inflammatory cytokines, such as IL-10, but not limited to them, and/or reduce the concentration of inflammatory cytokines.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the intestine. IBD is divided into Fe is otype Crohn's disease and ulcerative colitis. Crohn's disease can cover any part of the gastrointestinal tract, but most commonly the terminal ileum and the colon. Approximately 10% of cases, limited to the rectum and colon, definitive classification of Crohn's disease or ulcerative colitis cannot be made, and they are called "non-deterministic colitis". Both extraintestinal disease include inflammation of the skin, eyes or joints.

Crohn's disease and ulcerative colitis are usually classified as autoimmune diseases, as both are marked by an abnormal response of the immune system, leading to chronic inflammation in the lining of the intestine. The prevalence of inflammatory bowel disease is increased in subjects with other autoimmune diseases, including ankylosing spondylitis, psoriasis, sclerosing cholangitis, and multiple sclerosis.

Crohn's disease

Crohn's disease is a chronic disorder that causes inflammation of the digestive or gastrointestinal Department, in which the immune system attacks the intestines.

Although Crohn's disease most commonly affects the end of the ileum and the beginning of the large intestine, it can cover any part of the gastrointestinal tract. Inflammation of the intestine is discontinuous transmural; it may include granuloma or be associated with colonic or perianal hiss. Suppose that the gene CARD 15 gene and allele ASV associated with susceptibility to Crohn's disease.

Ulcerative colitis

Ulcerative colitis is a disease that causes inflammation and ulcers in the lining of the colon. It is a nonspecific chronic inflammatory disease that affects the intestines. Ulcers are formed and bleed in places where inflammation has destroyed the cellular lining. Unlike Crohn's disease, the inflammation is continuous and is limited to the layers of the mucosa of the rectum and colon; fistula and granulomas were not observed.

Both genetic and environmental factors are important in its etiology. Fuss et al. studied T-cell's own records of patients with ulcerative colitis and found that they produced significantly large number of IL13 and IL5 than control or cells with Crohn's disease, and few of γ-interferon. It was concluded that ulcerative colitis is associated with an atypical Th2 response mediated non-classical NKT cells (non-specific killer T-cells)that produce IL13 and have cytotoxic potential against epithelial cells.

Pouchet

Chronic and/or acute inflammation of the ileal reservoir, the so-called "pouch the t", is often observed long-term complication of anastamose ileoanal pouch procedure of the pocket. In patients with ulcerative colitis distribution of paucity varies from less than 10% to over 40%. The definition of "pouchet includes clinical signs, macroscopic inflammatory tissue lesions by endoscopy and histological evidence of intense acute inflammation of the tank mucous membrane.

Diarrhea associated with Clostridium difficile

Clostridium difficile is an anaerobic, gram-positive asporogenous Bacillus, first isolated in 1935 from the fecal flora of healthy newborns. Only in 1978 established its relationship with pseudomembranous colitis (PMC), induced by antibiotics. Almost all antibiotics have been associated with C.difficile diarrhoea and colitis, including vancomycin and metronidazole (used to treat them) and anticancer chemotherapy. The frequency relationship refers to the frequency of use, method of administration and effect of antibiotics on the microflora of the large intestine.

Irritable bowel syndrome

Irritable bowel syndrome (IBS) is a chronic disorder that affects the normal functions of the large intestine (colon). It is characterized by a group of symptoms - spastic abdominal pain, bloating, constipation and diarrhea.

IBS is one is responsible for a large part of the discomforts and ailments, but he does not cause permanent harm to the intestines and does not lead to intestinal bleeding, or any serious disease, such as cancer. The signs and symptoms of IBS vary widely from one patient to another and often occur in many other diseases.

Other active ingredients

It should be understood that the exopolysaccharide of the present invention may be inserted prophylactically or as a treatment either by itself or with other probiotic and/or prebiotic substances. In addition, bacteria can be used as part of a prophylactic or therapeutic schemes using other active substances, such as used for treatment of inflammation or other disorders, especially disorders of the gastrointestinal tract. Such combinations can be entered in a single preparation or in the form of individual drugs administered simultaneously or at different times and using the same or different routes of administration.

The pharmaceutical composition

The pharmaceutical composition is a composition that contains a therapeutically effective amount of pharmaceutically active agent or consists of it. It preferably contains a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof). Acceptable carriers or diluents for terapevticheskii use are well known in the pharmaceutical field and are described, for example, in Remington''s Pharmaceutical Sciences. The choice pharmaceutical carrier, excipient or diluent can be carried out with regard to the intended route of administration and generally accepted pharmaceutical practice. The pharmaceutical compositions may contain as carrier, excipient or diluent, or in addition thereto, any(s) right(s) bind(s) substance(s), lubricant(s) substance(s), suspendresume(s) agent(s)that cover(s) substance(s), solubilizer(s).

Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, petroleum ether (petroethral) esters of fatty acids, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

If necessary, the pharmaceutical compositions can be entered using any one or more of the following ways: inhalation, in the form of a suppository or pessary, locally in the form of a lotion, solution, cream, ointment or powder, by use of a skin patch, orally in the form of tablets containing excipients, such as krahmaly lactose, or in capsules or Beulah, either individually or in a mixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents, or they can be injected parenterally, for example, intracavernous, intravenously, intramuscularly or subcutaneously. For parenteral compositions can best be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to get the solution isotonic with blood. For buccal or sublingual injection composition can be introduced in the form of tablets or lozenges that can be prepared in the form of the drug accepted way.

Might have different requirements for composition/drug depending on the different delivery systems. For example, the pharmaceutical composition of the present invention can be prepared in the form of drug delivery using minipump, or mucosal route, for example in the form of nasal spray or aerosol for inhalation or solution taken orally, or parenterally, when the composition is prepared in the form of an injectable form, for delivery, for example, intravenous, intramuscular or subcutaneous route. Alternatively, the composition may be intended for delivery using a single still other ways.

Additional preferred features and embodiments of the present invention will be further described by non-limiting example and with reference to the accompanying figures.

Figure 1 shows the scheme of purification of exopolysaccharide (PS1) from the conditioned medium produced by Bifidobacterium 35624 (NCIMB 41003).

Figure 2 shows the structure of the exopolysaccharide (PS1)produced by Bifidobacterium 35624 (NCIMB 41003).

Figure 3 shows that the purified exopolysaccharide (PS1) from Bifidobacterium 35624 [NCIMB 41003] shows together with immunomodulating activity in the joint incubation in vitro with mononuclear cells of peripheral blood.

Figure 4 shows that PS1 restricts the release of proinflammatory cytokines in response to stimulation of Toll-like receptor 4 (TLR-4) in vitro.

Figure 5 shows that PS1 restricts the release of proinflammatory cytokines in response to stimulation of Toll-like receptor 4 (TLR-4) in vivo.

Figure 6 shows that PS1 restricts the release of proinflammatory cytokines in response to repeated stimulation of Toll-like receptor 4 (TLR-4) in vitro.

Strain Bifidobacterium 35624 (NCIMB 41003) described in WO 00/42168, the full contents of which are incorporated in this description by reference. The strain was deposited at the NCIMB (National collections of industrial and marine bacteria) 13 January 1999.

Example 1 Purification and opredelenieslova.ru of exopolysaccharide (PS1) of the air-conditioned environment, produced by Bifidobacterium 35624.

The cleaning. 100 ml of sterile medium MRS (SM MRS Broth, Oxoid Ltd., Basingstoke, Hampshire, England) with the addition of 0.05% (wt./about.) cysteine, were placed in a sterile 250 ml Erlenmeyer flask, inoculated with Bifidobacterium 35624. Inoculated medium incubated under anaerobic conditions (10-01 Pack-Anaero, Mitsubishi Gas Chemical Company America, New York, NY) at 37°C without shaking. Sample pinacolborane environment MRS was used as the control environment and processed identically inoculated sample in the methods set forth below.

After 48 h of growth the culture of Bifidobacterium 35624 reached stationary phase and showed OD optical density at 600 nm of approximately 3 (2-3×109colony forming units/ml). The culture was transferred to a polycarbonate centrifuge tubes and centrifuged at 40000xg for 30 min (rotor JA-20 centrifuge, Beckman J2-21, Beckman Coulter, Inc., Fullerton, CA), which resulted in the receipt of a transparent supernatant and dense cellular precipitate. The supernatant (conditioned medium) was carefully removed and used in the purification of exopolysaccharide (EPS). The cell precipitate was discarded.

The purification scheme EPS from conditioned medium produced by Bifidobacterium 35624, shown in figure 1. If not stated otherwise, all steps were performed on ice or at 4°C. 80 ml of culture supernatant was loaded into the ultrafiltration device with a threshold molecular weight MW) 100000 Yes (hub VC1042 Vivacell 100 ml, Vivascience, Hannover, Germany). The samples were concentrated by centrifugation at 2000xg in the centrifuge for clinics. After reaching a volume of approximately 0.5 ml UltraConnect (high molecular weight fraction HiMW) once subjected diafiltration using phosphate-saline buffer solution (PBS), and again concentrated to approximately 0.5 ml of Ultraconnected was transferred into a standard 15 ml centrifuge tube. The ultrafiltration device was washed with 4 ml PBS and the wash liquid were combined with ultracetultracet obtaining HiMW fraction ultraconnected.

A solution of 100% trichloroacetic acid (TCA) was added to the fraction UltraConnect HiMW to a final concentration of 20% (vol./about.) Tja. Samples were incubated for 2 h on ice and then centrifuged at 8000xg for 20 min (rotor JA-20). The supernatant, containing EPS, was transferred to a 30 ml test tube from corex methods. The precipitate, containing the proteins was discarded. The supernatant, containing EPS, was treated with 3 volumes of ice-cold 95%ethanol, and incubated overnight at -20°C. Then the tubes were centrifuged at 8000xg for 20 min (rotor JA-20). The supernatant was discarded. The precipitate, containing EPS, resuspendable in 5 ml of PBS and then laid siege to 3 volumes of ethanol, as described above. The precipitate was air-dried on ice for 60 min and then resuspendable in 9 ml of 10 mm MgCl2, 50 mm Tris-HCl (pH 7,).

Removal of nucleic acids each of deoxyribonuclease I (LS006331, Worthington Biochemical Corporation, Lakewood, NJ) and ribonuclease A (R5250, Sigma-Aldrich Corporation, St.Louis, MO) was added to a final concentration of 0.1 mg/ml and incubated for 2 h at 37°C. Residual protein was removed by adding proteinase K (R, Sigma-Aldrich) to a final concentration of 0.02 mg/ml and then incubating the mixture for 2 h at 37°C. Proteinase K iactiveaware by incubation for 15 min at 70°C, with the subsequent addition of phenylmethylsulfonyl (R, Sigma-Aldrich) to a final concentration of 0.2 mm for 15 min at room temperature. The purified EPS was besieged from the solution by adding 3 volumes of ethanol, as described above. Sediment resuspendable in 9 ml of phosphate-saline buffer solution. Resuspending the sample was loaded into dialysis tubing SnakeSkin (68035, Pierce 10 Biotechnology, Rockford, IL)with a threshold molecular weight of 3500 and then were dialyzed against 7 liters of water (2 cycle) for 48 h at 4°C.

A small aliquot detalizirovannoi sample was collected to determine the amount of polysaccharide present, using the standard phenol/sulfuric acid method of Dubois et al. (Anal. Chem. 28, 350-356 (1956)). After you have determined the concentration of EPS was taking appropriate aliquots were frozen on dry ice and liofilizirovanny dry. Liofilizirovannoe substance was stored at -0°C.

NMR analyses. Analysis by proton nuclear magnetic resonance (1H-NMR) was performed on the spectrometer Varian Inova 600 MHz (Varian Medical Systems, Palo Alto, CA). Lyophilized samples were dissolved in D20 and after providing extensive exchange of deuterium received spectrum at 25°C. Chemical shifts were referred to internal TSP. Data1H1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), spectroscopy nuclear Overhauser effect (NOESY) and proton-carbon heteronuclear odnoslotovoy correlation spectroscopy (HSQC) were collected in phase-sensitive mode using the quadrature of Stats-Abercorn-ruby (States-Haberkorn-Rubin). All sequences of pulses supplied by the supplier of the spectrometer and were used without modification. Pre-saturation of low power was applied to the residual HDO pulse. Typically, the data sets were collected with 512 x 2048 complex data points and 16-32 scans/increment. Pulse program TOCSY contained 60-MS MLEV-17 mixed sequence, and the mixing time in NOESY was 150 MS.

Analysis of carbohydrates. Analyses helicoiling songs performed by combined gas chromatography/mass spectrometry (GC/MS) per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methylglycosides obtained from purified EPS Bifidobactrium 35624 using acidic methanolysis (York et al., (1986); 5 Merkle, R.K. and Poppe, I. 1994). For analysis picatinney communication sample EPS has perimetrically, depolimerization, restored and azetilirovanie. The resulting partially methylated alliterate (PMAAs) were analyzed using GC-MS as described previously (York et al., (1986); Merkle and Poppe (1994)).

Structure determination of EPS.

Purified EPS were analyzed using1H-NMR. The spectra showed that the substance is composed exclusively of carbohydrate; not indicated the presence of nucleic acid, protein, lipid, or small organic impurities. In the analysis using two-dimensional NMR, using experiments known in the art and described above, found that a large part of the present carbohydrate contained a linear polysaccharide, which consists of disaccharide glycosides of repetitions. In combination with the data analyses of the structure and relations of the structure of this polysaccharide (named PS1) is a [-β(1,3)-attached-D-N-acetyl-galactosamine-pyranosyl-β(1,4)-attached-D-glucose-pyranosyl-]nwhere n indicates that this disaccharide glycosides unit is repeated n times, which gives the polysaccharide with a molecular weight of greater than 100,000 Da. The structure of PS1 can be abbreviated to designate as [-β(1,3)-D-GalpNAc-β(1,4)-D-Glcp-]nand it is shown in figure 2. Note that PS1 was not detected in the sample environment control.

Example 2 - Ecopolitical is d (PS1) B.infantis 35624 has immunomodulatory activity when combined incubation with cells of the human immune system in vitro.

The EPS fraction was analyzed using analysis of induction of cytokines RVMS (mononuclear cells in peripheral blood). In this analysis RVMS separated from blood by dividing the density gradient and incubated for 72 hours at 37°C (in the presence of penicillin and streptomycin) with control medium or with increasing concentrations of purified PS1 of the Century infantis 35624. The supernatant was analyzed for levels of IL-1β, IL-6, IL-8, IL-10, IL-12, TNF-α and IFN-y, using sets for mesoscale studies (MSD), and analyzed using a tablet reader for MSD.

Figure 3 shows the results of this analysis. PS1 stimulated the secretion of all tested cytokines upon stimulation RVMS 1-5 µg/ml PS1. The activity of stimulating cytokines was reduced to background levels of many cytokines using 10 µg/ml PS1.

In addition to the test resting RVMS, to activate RUMS in the presence or in the absence of PS1 stimulation used TLR-4-ligand lipopolysaccharide (LPS). Since it was found that 5 μg/ml PS1 represents the optimal dose in preliminary experiments, this dose was used in the following analyses. These results are illustrated in figure 4 in the form of average values for cytokines to cells stimulated by LPS+PS1, minus values for cytokines to cells, the incentive is only new LPS. When combined incubation with PS1, RVMS, LPS-stimulated, secrete significantly less IL-6 and significantly less IL-8, TNF-α and IFN-y.

Example 3 - Exopolysaccharide (PS1) from B.infantis 35624 has anti-inflammatory activity when injected into a mouse model of sepsis.

PS1 were injected with I.P. (IPR) healthy mice and these mice were observed for 24 hours. There were obvious signs of discomfort, which gave the possibility to assume that this polysaccharide was well tolerated by the animals, and PS1 did not cause sepsis or inflammatory response. After a 24-hour period of observation, the animals were administered intraperitoneally injected with lipopolysaccharide (LPS)to induce sepsis-like response. All animals were penalized after 2 hours and the secretion of cytokines in splenocytes was measured in vitro. Splenocytes isolated from mice treated PS1+LPS, released significantly less TNF-α compared to mice that received only LPS (Figure 5). In addition, splenocytes from these animals re-stimulated with LPS in vitro, and again observed blunted response of proinflammatory cytokines in splenocytes obtained from animals previously exposed to PS1 (6).

Taken together, these data show that EPS1 derived from Bifidobacterium infantis 35624, has immunomodulatory activity and protects from Pospolita the different reactions, mediated LPS or TLR-4.

Although the above experiment describes the indirect exopolysaccharide (PS1), it is assumed that secretively form of exopolysaccharide, such as affiliated/associated with the cell or with membrane or capsule form exopolysaccharide, will behave similarly Sekretareva EPS. The experiment described in this description of the invention, was designed to study secreted EPS, but the specialist in the art it is obvious that part of the secreted EPS may be associated with a cell (e.g., associated with the cell or capsule) in the process of making EPS and before transport or release EPS of cells.

The invention is not limited to the embodiments described in this description of the invention above, which can vary in the details.

Links

Arai Kl, et al., Annu Rev Biochem 59: 783-836, 1990.

Fuss et al., "Nonclassical CD Id-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis." Inflamm Bowel Dis. 2005 Jan; ll(l):74-5.

Henderson B., and M. Wilson ln "Bacteria-Cytokine interactions in health and disease".

Isolauri et al., Clin Exp Allergy 30:1605-10, 2000.

Isolauri et al. Pediatr Res. 33:548-53, 1993.

Madsen et al., Gastroenterology 112:A1030, 1997.

Majamaa et al., J Allergy Clin Immunol 99:179-86, 1997.

Malin et al., Br J Rheumatol 35:689-94, 1996.

McCarthy et al., Gut 53:694-700, 2004.

Mazmanian et al., Cell 122:107-118, 2005.

Merkle and Poppe Methods Enzymol. 230:1-15, 1994.

O'mahony et al., Aliment oil displayed pure Pharmacol Ther 15:1219-25, 2001.

O'mahony et al., Gastroenterology 128:541-51, 200 Portland Press, 79-130, 1998.

Remington's Pharmaceutical Sciences, Mack Publishing Co. 1985 (Editor: A.R. Gennaro).

Salminen, Int J Food Environ 20:93-106, 1998.

Sanderson et al., Gastroenterology 104:622-39, 1993.

Whorwell et al., Am J Gastroenterol 101:1581-90, 2006.

York et al., Methods Enzymol. 118:3-40, 1986.

1. Polysaccharide isolated from a strain ofBifidobacterium infantisNCIMB 41003 and having the structure:

where n indicates that this disaccharide glycosides unit is repeated n times, which gives the polysaccharide with a molecular mass of more than 100 000 Da.

2. The use of polysaccharide according to claim 1 in the manufacture of a medicinal product for the treatment or prevention of undesirable inflammatory activity.

3. The use of polysaccharide according to claim 1 in the manufacture of a medicinal product for the treatment or prevention of undesirable gastrointestinal inflammatory activity.

4. The use according to claim 3, where gastrointestinal inflammatory activity represents Crohn's disease, ulcerative colitis, irritable bowel syndrome, pauci, post-infectious colitis, diarrhea, associated withClostridium difficile, diarrhoea associated with rotavirus, or post-infectious diarrhea.

5. The use of polysaccharide according to claim 1 in the manufacture of a medicinal product for the treatment or prevention of rheumatoid arthritis.

6. The use of polysaccharide according to claim 1 in the manufacture of a medicinal product for treating or preventing autoimmune R is straist.

7. Pharmaceutical composition for the treatment and prevention of inflammatory disorders, containing the polysaccharide according to claim 1 and a pharmaceutically acceptable carrier.

8. Food product containing polysaccharide according to claim 1.

9. The food product of claim 8, which represents one or more than one product selected from the group consisting of yoghurt, cereals, drinks.



 

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FIELD: chemistry.

SUBSTANCE: invention relates to production of acidic polysaccharides and can be used in producing cosmetics. The acidic polysaccharides are selected from hyaluronic acid, chondroitin sulphate, dermatan sulphate, heparan sulphate and keratan sulphate and are characterised by simultaneous presence of alcohol groups that are esterified with butyric acid and formic acid. The method of producing said acidic polysaccharides involves dissolving an acidic polysaccharide in form of a salt with sodium or other alkali metals in formamide while heating. Butyric anhydride is then added to the obtained solution at room temperature in the presence of an organic base. The obtained homogeneous viscous reaction mixture is then diluted with aqueous NaCl solution and neutralised to pH 6-7.5. Further, the diluted reaction mixture is purified by dialysis or cross-flow filtration and the purified polysaccharide solution is frozen. The product is recovered by freeze-drying or spray-drying. In the disclosed compounds, presence of butyric acid and formic ester substitutes of a modified polymer protect from enzymatic degradation by hyaluronidases presence if tissue.

EFFECT: invention enables to obtain polysaccharides which can be used in cosmetics and medical drugs for local application as moisturising, smoothing and tonic components or as adjuvants when treating skin damages.

16 cl, 1 dwg, 1 tbl, 9 ex

Glucan compositions // 2490279

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biochemistry. Disclosed is a composition, having immune-stimulating properties, which contains unmodified, soluble β-glucan with average molecular weight from about 120000 Da to about 205000 Da. The composition also contains soluble β-glucan with molecular weight greater than 380000 Da in amount of less than or equal to 10% and β-glucan with molecular weight of less than 25000 Da in amount of less than or equal to 17%. The unmodified, soluble β-glucan can be used in single doses of up to about 6 mg/kg. Also disclosed is a method of producing soluble β-glucan. The method involves applying pressure of up 35 lb/in2 (24607.4 kg/m2) to a suspension of powdered β-glucan and an acid. The suspension is then heated to 135°C for a period of time which is sufficient to form soluble β-glucan. Also disclosed are compositions (versions), having immune-stimulating properties, which contain a pharmaceutically effective amount of unmodified, soluble β-glucan obtained using said method.

EFFECT: invention enables to obtain improved β-glucan compositions, which can be administered in a higher concentration than the maximum dose with fewer undesirable reactions.

28 cl, 4 tbl, 2 dwg

FIELD: food industry.

SUBSTANCE: method for production of an inulin-containing solution from girasol includes tubers washing and cutting into chip-shaped slices, the latter drying and grinding and production of a suspension by way of mixing the produced flour and water. Fructosans are extracted from the suspension with the solution purified after extraction. The purified solution is cooked till the dry substances content is equal to 42-45%. Additionally proposed is an inulin-production method according whereto the purified and boiled out inulin-containing solution is passed through nanofilters with attenuation threshold equal to first 5000 Da and then - 6000 Da with separation of a solution containing inulin with molecular weight equal to 5000 - 6000 Da. The produced inulin solution undergoes crystallisation. Sedimented inulin crystals are mixed with the initial inulin-containing solution and undergo drying. Additionally one proposes fructooligosaccharides production method.

EFFECT: inventions group allows to enhance the ready products quality and yield.

4 cl, 3 dwg, 3 ex

FIELD: wood working industry.

SUBSTANCE: method provides for production of a wood material and its treatment by means of hydrothermic treatment with water for production of wood hydrolysate and wood remains. Used wood remains are suitable for use when making a fibrous mass. The specified hydrolysate contains oligo- and polysaccharides as the main component of the dry substance. At the same time hydrothermic treatment is carried out at the temperature in the range from 100°C to 190°C fo 10 - 360 minutes. Further the wood hydrolysate is divided into at least first and second fractions due to different molecular mass. The first fraction has a higher molecular mass compared to the second fraction. The specified first fraction is used to produce a polymer product. The produced product contains at least 80% oligo- and polysaccharides, 5-15% lignin, 0-5% monosaccharides and less than 0.1% of ash in terms of the dry substance. The specified oligo- and polysaccharides are characterised by the extent of substitution of acetyl groups (DsAC) from 0.05 to 2.0.

EFFECT: invention makes it possible to ecologically process wood material in the form of wood chips and produce a polymer product without use of chemical reagents.

43 cl, 1 dwg, 5 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a crossed-linked chitosan composition. The composition contains chitosan with a degree of deacetylation 30-75% and a cross-linking agent. Chitosan is randomly deacetylated. The molar ratio of the cross-linking agent to chitosan makes 0.2:1 or less at a number of functional groups in the cross-linking agent and a number of deacetylated amino groups in chitosan. The invention also represents chitosan hydrogel made of the specified composition, a method for preparing it and the versions of using it.

EFFECT: invention provides higher bioavailability of the active ingredient and ensures that hydrogel has storage-stability of the visco-elastic properties of a plastic solid.

15 cl, 1 dwg, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a sizing composition for glass fibres, particularly meant for reinforcing organic and/or inorganic matrices, the obtained glass fibres as well as composite materials containing said glass fibres. The sizing composition for glass fibres in form of a physical gel contains the following, wt %: 0.1-5 of at least one texturising agent selected from xanthanes, guar gum and succinoglycans; 2-8 of at least one film-forming agent; 0.1-8 of at least one compound selected from a group consisting of plasticisers, surfactants and dispersants; 0.1-4 of at least one binder; 0-6 of at least one additive. The subject of the invention is also glass fibres coated with said sizing composition and composite materials containing an organic or inorganic material which is reinforced with said glass fibres.

EFFECT: reduced migration of the sizing agent when drying coils.

23 cl, 1 dwg, 6 tbl, 34 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound CL168 of general structural formula I where R is oxygen. The invention also relates to a method of producing a compound of formula I and use of the compound of formula I to produce a medicinal agent for preventing or treating tumorous and immunological diseases.

EFFECT: compound of formula I for producing a medicinal agent for preventing or treating tumorous and immunological diseases.

4 cl, 11 tbl, 19 ex

Iimmunomodulator // 2504371

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an immunomodulator. An immunomodulator for the immunocorrection accompanying the integrated treatment of chronic non-specific pulmonary diseases, chronic obstructive pulmonary disease, bronchial obstruction syndrome, chronic bronchial pneumonia, pulmonary fibrosis, tracheobronchitis, chronic laryngitis, pulmonary, tracheal and pharyngeal cancer; the immunomodulator is prepared by mixing a water infusion of rose bay leaves and a water infusion of yellow melilot taken in equal proportions, with a cattle lung and larynx powder, settling the prepared mixture, keeping on a boiling water bath, cooling; further, the mixture is filtered; the prepared solution is added with cattle blood serum containing leukaemia oncovirus antibodies, hemlock infusion, ascorbic and sorbic acids until all the ingredients fully dissolved; the prepared solution is placed in the water bath, cooled, filtered, sterilised under certain conditions.

EFFECT: above preparation provides higher effectiveness and reduces the length of treating the above diseases, and ensures the higher immunobiological properties of the human body.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an extract of one or more bacterial strains Lactobacillus. The extract of one or more bacterial strains Lactobacillus representing a soluble extract, wherein the extract contains chemically modified bacterial molecules prepared by the action of an alkaline medium on one or more bacterial strains Lactobacillus; the extract is effective in treating diseases associated with the anti-inflammatory cytokine production imbalance. A method for preparing the extract of one or more bacterial strains Lactobacillus. A pharmaceutical composition effective for reducing at least one symptom associated with at least one condition specified in a respiratory disorder, an allergic condition, an urinary disorder and a gastric disorder, containing the extract. A nutritional composition. A pharmacological composition effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance, containing the extract. A method of relieving the above symptoms. The extract prepared by the above method.

EFFECT: extract is effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance.

21 cl, 7 dwg, 23 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to using at least one immunomodulatory compound of general formula (1) or a pharmaceutically acceptable, solvate or isomer thereof for preparing a pharmaceutical composition for treating a disease or disorder specified in asthma, atopic dermatitis, allergic rhinitis, inflammatory intestinal disease, diabetes or rheumatoid arthritis in homoiothermal animal, including a human. What is also presented is using (5S,11R)-1-amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetradecanoylamino]dodecan-12-ol-12-dihydrophosphate (OM-294-BA-MP (S,R)) or a pharmaceutically acceptable salt, solvate or isomer thereof for preparing the pharmaceutical composition.

EFFECT: group of inventions provides treating the above diseases by modulating the TH1/TH2 cytokine balance by reducing TH2-cytokine release and enhancing TH2-cytokine production.

11 cl, 16 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to preparing interferonogenic antiviral agents of yeast RNA, and may be used in medicine. The IFN-γ inducer is presented by a soapy amphiphilic complex of high-polymer Saccharorayces cerevisiae RNA and oleic acid penetrating easily through biological membranes in the following proportions: high-polymer RNA - 80-90%, oleic acid - 10-20%.

EFFECT: invention enables inducing interferon γ (IFN-γ) production effectively with using available and low-cost ingredients.

2 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a method for preparing an agent possessing immunomodulatory activity. The method for preparing an agent possessing immunomodulatory activity characterized by the fact that herbal composition containing common motherwort herb, knotgrass herb, pot marigold blossom, licorice rhizome and root, Alexandrian laurel rhizome and root, rosehip, schizandra fruit, linseeds, sequentially twice extracted in 60-70% ethanol, twice in 40-50% ethanol, and once in purified water under specific conditions; then the aqueous-alcoholic extracts are concentrated in vacuum; the aqueous still residues are combined with the aqueous extract, then filtered, boiled out, purified by separation, boiled out additionally, dried in a vacuum dryer and further milled.

EFFECT: method enables preparing the agent possessing manifested immunomodulatory activity with the high content of active substances.

13 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pyridine derivatives of formula (I) wherein A, R1, R2, R3, R4, R5, R6 and R7 are presented in the description, preparing and using them as pharmaceutically active compounds possessing SP1/EDG1 receptor agonist activity.

EFFECT: using the declared compounds or pharmaceutically acceptable salts thereof for preparing a pharmaceutical composition for preventing or treating the diseases or disorders associated with the activated immune system.

13 cl, 76 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: hemorrhagic pancreonecrosis is preliminarily modelled on animals. After that, tamerit in dose 2 mg/kg is introduced intramuscularly. After that, histological analysis of pancreas tissue after the first, third and seventh day is performed and its results are used to make conclusion about sufficiency and efficiency of performed treatment.

EFFECT: method ensures correction of the course of acute phase of systemic inflammatory reaction syndrome, which leads to stimulation of migration of immunocompetent cells into necrosis foci, acceleration of formation of granulation tissue with proliferation of cells of fibroblast line without phenomena of purulent inflammation, stimulation of proliferation of pancreas duct structures.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and is intended for treatment of vaginal infections. Method includes complex therapy with application of immunocorrector. As immunocorrector solution of medicine Betaleukin, introduced intravaginally during 40-90 minutes daily at least during 5 days, is applied.

EFFECT: method is highly efficient in complex therapy of vaginal infections.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to method of obtaining immunotropic preparation. Method of obtaining immunotropic preparation by extraction of air-dry leaves of Lophanthus anisatus Benth with aqueous ethyl alcohol, heating, cooling, filtering, solvent evaporation, residue dissolving and extraction with chloroform, filtering and drying of sediment, under specified conditions.

EFFECT: method makes it possible to obtain efficient immunotropic medication.

3 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to using mixed metal compounds for preparing a drug preparation for neutralisation of gastric acid or buffer action thereon, as well as for treatment of a condition or a disease related to high gastric levels of acid. The mixed metal compound is a compound of formula (I): MII1-aMIIIaObAn-c.zH2O (I), wherein MII and MIII mean a two-valence and three-valence metal respectively, An- means an n-valence anion, 0.2≤a≤0.4, 0.2≤b≤1.5, 2+a is equal to 2b+Σcn, Σcn<0.9a, and z is equal to 2 or less. According to the invention, the mixed metal compound is presented in the form of a granulated material, wherein a diametre of 50 wt % of the granules makes 106 to 1180 mcm with the material comprising 50 wt % of the mixed metal compound, 3 to 12 wt % of noncovalent water and no more than 47 wt % of an excipient on granulated material weight basis. The mixed metal compound in the form of the granulated material represents a compound of formula (I) or a compound of formula (III): MII1-xMIIIx(OH)2An-y·mH2O (III) ; wherein MII and MIII mean a two-valence and three-valence metal respectively, An- means an n-valence anion, x=Σyn, 0<x≤0.4, 0<y≤1, and 0≤m≤10.

EFFECT: invention provides the buffer action on gastric acid, causing no effect of rebound acid hypersecretion.

46 cl, 3 tbl, 1 dwg

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