D-fagomine for the control of inflammatory processes related to an overactivation of the humoral immune response

Abstract

The present invention belongs to the field of nutraceuticals or functional agents with immunostimulating capacity for triggering mechanisms of the innate immune response at a mucosal level. More specifically, it refers to the use of the compound D-fagomine as immunostimulating agent of the innate immune response in the mucosa and for the prevention or prophylaxis of inflammatory processes or diseases associated with an overactivation of the humoral immune response. The invention also relates to a composition comprising D-fagomine for the above referenced application.

Claims

1. A method of immunostimulating the innate immune system in a subject suffering from the onset of an autoimmune disease selected from the group consisting of Crohn's disease, ulcerative colitis, celiac disease, psoriasis, polymyalgia, and rheumatoid arthritis, the method comprising administering D-fagomine to the subject in an amount effective to stimulate the innate immune system of the subject's mucosa.

2. The method of claim 1 wherein the innate immune system is at the level of the oral mucosa, the esophageal mucosa, the gastric mucosa, enteric mucosa, the colonic mucosa, the nasal mucosa, the bronchial mucosa, the urethral mucosa or the uterine mucosa.

3. The method of claim 1 wherein the innate immune system is at the level of the colonic mucosa.

4. The method of claim 1 wherein the D-fagomine is administered in the form of a solid or aqueous composition.

5. The method of claim 1 wherein the D-fagomine is administered in the form of an aqueous composition and the amount of D-fagomine in the aqueous composition is less than 500 g/ml.

6. The method of claim 5 wherein the amount of D-fagomine in the aqueous composition is between 50 and 500 g/ml.

7. The method of claim 1 wherein the D-fagomine is administered in the form of an enriched extract from a natural source or a fine chemical.

8. The method of claim 7 wherein the natural source of D-fagomine is the seeds of Fagopyrum esculentum or Fagopyrum tataricum, leaves of Morus bombycis or Morus alba, roots of Lycium chinense, roots and leaves of Xanthoceris zambesiaca, fruits of Angylocalyx pinaertii, leaves of Baphia nitida, seeds of Castanospermum austral or roots of Lonchocarpus latifolius.

9. The method of claim 1 wherein the D-fagomine is administered in the form of a pharmaceutical, veterinary or food composition.

10. The method of claim 1 wherein the D-fagomine is administered in combination with at least one additive.

11. The method of claim 1 wherein the D-fagomine is administered in combination with rutin.

12. The method of claim 1 wherein the administering increases production of TNF-.

13. The method of claim 1 wherein the autoimmune disease is Crohn's disease.

14. The method of claim 1 wherein the autoimmune disease is ulcerative colitis.

15. The method of claim 1 wherein the autoimmune disease is celiac disease.

16. The method of claim 1 wherein the autoimmune disease is psoriasis.

17. The method of claim 1 wherein the autoimmune disease is polymyalgia.

18. The method of claim 1 wherein the autoimmune disease is rheumatoid arthritis.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: represents the recovery of E. coli 434 from the colon mucosa. A negative control without addition of E. coli to the preparation was used. As positive control a sample with E. coli but without D-fagomine was used. In addition, three different samples of E. coli with D-fagomine at different doses (50, 500 and 5000 g/ml) were tested. The results demonstrate that D-fagomine has antiadherent properties against E. coli in a dose dependent manner.

(2) FIG. 2: represents the TNF-alfa release by the cells from the innate immune system present in the colon mucosa. The TNF was measured from the supernatant. As in FIG. 1 negative control without addition of E. coli to the preparation and positive control with E. coli but without D-fagomine was used. Again three different samples of E. coli with D-fagomine at different doses (50, 500 and 5000 g/ml) were tested. The results demonstrate that D-fagomine has the capacity to stimulate the release of TNF-alfa and that the lower is the dose the more potent is such release. This is a direct indication that D-fagomine has an immunostimulating effect on the innate immune system helping the immune system detecting and attacking microorganisms at the mucosa level.

DETAILED DESCRIPTION OF THE INVENTION

(3) A first aspect of the invention refers to the compound D-fagomine or a pharmaceutical acceptable salt thereof, or a veterinary salt thereof or an edible salt thereof, for use as immunostimulating agent of innate immune system at a mucosal level and as an agent for the prevention of inflammatory processes related with an overeactivation of the humoral immune response.

(4) The immunostimulation of the innate immune system at the mucosal level has the advantage that it decreases the incidence of more potent and potentially damaging humoral immune responses. As such the D-fagomine helps to prevent the onset of autoimmune diseases such as diabetes, psoriasis metabolic syndrome Crohn's disease, celiac disease, polymyalgia or rheumatoid arthritis, and reduces the progression of diseases related to an abnormal humoral inflammatory response, as is metabolic syndrome and other diseases such as obesity, hypertension, type II diabetes, fatty liver, Alzheimer or cancer (e.g. breast, colon).

(5) Non-limiting D-fagomine salts which may be used within the context of the present invention are tartrate, hemitartrate, citrate, hemicitrate, chlorhidrate, malate, or acetate salts.

(6) D-fagomine has been demonstrated to produce an immunostimulating response of the innate immune system at the level of mucosa which is the first barrier that the pathogens face in the infective process. D-fagomine activates the response of the innate immune system at the level of all types of mucosa, namely the oral mucosa, the esophageal mucosa, the gastric mucosa, enteric mucosa, the colonic mucosa, the nasal mucosa, the bronchial mucosa, the urethral mucosa or the uterine mucosa.

(7) In a particularly preferred embodiment the activating response of the innate immune system takes place in the nasal mucosa. The activation of innate immune response in the nasal mucosa is helpful as coadyuvant for improving nasal vaccine efficacy against bacterium as Streptococcus pneumonia or virus as influenza. In another particularly preferred embodiment, the activating response of the innate immune system takes place in enteric mucosa. The activation of innate immune response in the enteric mucosa is helpful as coadyuvant for improving oral vaccine efficacy against some microrganisms such as vibrio strains, tuberculosis or poliovirus.

(8) In the most preferred embodiment, the activating response of the innate immune system takes place at the level of the colon mucosa. The use of D-fagomine is especially interesting to trigger the innate immune response locally at the level of the colon for different reasons. A first one is that the colon is one of the regions with a more abundant concentration and presence of bacteria and also one of the regions with the presence of more opportunistic pathogens, including E coli that is ubiquitous in humans. In this sense, also the colon is one of the regions more sensible and with more likelihood of infection, hence it is important that the innate immune system is activated and under surveillance at this level of the gastrointestinal tract. The second reason is that while other kind of carbohydrates may have a similar kind of immunostimulating activity of the innate immune system, they normally do not reach the colon as they are consumed or degraded by the enteric bacteria during their transit through the gastrointestinal tract. On the contrary, D-fagomine is an iminosugar that cannot be metabolized by the enteric bacteria and as such it is capable of reaching the colon where it can exert its immunostimulating function at a local level, even at very low concentrations.

(9) In fact, although the presence of D-fagomine in concentrations of 5000 g/ml or less is sufficient to produce an immunostimulating effect of the innate immune system in the mucosa, it has been surprisingly observed that the lower is the concentration of D-fagomine the more potent is innate immune response. In fact, in a preferred embodiment D-fagomine is used in concentrations of 500 g/ml or less. In a still more preferred embodiment D-fagomine is used in concentrations of 50 g/ml or less. At concentrations of 50 g/ml or less the production of the cytokine TNF- is surprisingly increased. The production of TNF- is a direct indication of the activation of the innate immune system, that is of the immunostimulation thereof.

(10) As an agent capable of maintaining the innate immune system under alert, the regular consumption of D-fagomine contributes to the prophylaxis and prevention of inflammatory processes related with opportunistic pathogens. In particular, it can be very useful for preventing situations of chronic or subchronic inflammation caused by bacteria capable of escaping from the immune system by the formation of biofilms that can hide the colonies from the immune system. This kind of situation tends to facilitate infection and triggers immune responses not only at a mucosal level but also at a serosal level. The existence of this type of serosal responses is the source of the chronic inflammatory state.

(11) In a particular and preferred embodiment of the invention D-fagomine contributes to the prevention of inflammatory processes related with bacteria selected from gamma proteobacteria such as E. coli, Salmonella tiphymurium, Pseudomonas aeruginosa or Streptoccocci such as S. mutans, S. mitis, S. oralis, S. pneumonia, S. pyogenes, S. agalactiae or Enteroccoccus faecalis.

(12) In the preferred embodiment of the invention D-fagomine is useful for the prevention of inflammatory processes related to overactivation of the humoral response by certain pathogenic strains of Escherichia Coli which have very much tendency to escape to the immune system by forming biofilms or by other strategies and to produce chronic clinical and subclinical inflammation processes.

(13) Another aspect of the invention relates to a composition comprising D-fagomine or a pharmaceutical acceptable salt thereof, or a veterinary salt thereof or an edible salt thereof, and at least one additive for use as immunostimulating agent of innate immune system at a mucosal level and as an agent for the prevention of inflammatory processes associated to opportunistic pathogens.

(14) Non-limiting D-fagomine salts which may be used in the composition of the invention are tartrate, hemitartrate, citrate, hemicitrate, chlorhidrate, malate, or acetate salts.

(15) The composition of the invention may be in solid form or in the form of a liquid composition, preferably an aqueous composition.

(16) In the case of liquid or preferably aqueous composition D-fagomine is in a concentration of 5000 g/ml or less, preferably of 500 g/ml or less, and more preferably of 50 g/ml or less.

(17) D-fagomine in the composition of the invention can be synthetic or it can be of natural origin, in the form of enriched extract from a natural source. In the case of an enriched extract from natural sources D-fagomine is preferably extracted or food produced from the seeds of Fagopyrum esculentum or Fagopyrum tataricum, leaves of Morus bombycis or Morus alba, roots of Lycium chinense, roots and leaves of Xanthoceris zambesiaca, fruits of Angylocalyx pinaertii, leaves of Baphia nitida, seeds of Castanospermum austral or roots of Lonchocarpus latifolius.

(18) In a particularly preferred embodiment the composition of the invention comprises D-fagomine of an extract from seeds of Fagopyrum esculentum.

(19) The composition of the invention can be formulated either as a pharmaceutical, veterinary or food composition, the latter being the preferred type of composition.

(20) In a preferred embodiment the pharmaceutical composition is a mouthwash, a gel, a liquid dental cleaning lotion, a tooth paste, a chewing gum, a denture cleaner or a prothesis adherence cream. In a more preferred embodiment, the pharmaceutical composition is a chewing gum.

(21) Furthermore, it is possible to use D-fagomine as part of a nutritional composition food, pet food and feedstuff. The food composition of the invention can also be presented as a food or a beverage additive to produce a functional food or a functional beverage. In this form it can be added to liquid food products or concentrates or powders, such as milk and liquid milk like products, various beverage including juices, soft drinks, sport drinks, alcoholic beverages, and the like. It is especially useful to have a composition of D-fagomine according to the invention as part of a food for an infant, preferably as a part of a infant formula. Also, when the D-fagomine composition comes from a buckwheat (Fagopyrum esculentum) purified extract it can be part of a beer, non-alcohol beer, tea like drink, milk like drink, yogurt, pasta, biscuits, cookies, cereal bars, swollen grains, bread, crepes, cakes, creams, desserts, breakfast cereals and others. The food composition or food additive of the invention can also be produced in a natural way, e.g. by having a domestic animal such a cow or other animal that produce D-fagomine in its milk. This can be accomplished by feeding the animal with Fagopyrum esculentum, Fagopyrum tataricum, Morus alba or purified D-fagomine.

(22) In another embodiment, the composition of the invention also comprises rutin or other flavonol polyphenols in its formulation (i.e. hesperidin). Rutin, is the glycoside between the flavonol quercetin and the disaccharide rutinose (-L-rhamnopyranosyl-(1.fwdarw.6))--D-glucopyranose) and has the following formula.

(23) ##STR00002##

(24) When rutin is administered orally it tends to be stable in the upper intestine, but rapidly deglycosylated to liberate the quercetin at the level of the colon (Kim H. et al. Metabolic and pharmacological Properties of rutin, a dietary quercetin glycoside for treatment of inflammatory bowel disease Pharmaceutical research, 2005, Vol. 22 N 9). Quercetin has been taught to have anti-inflammatory effect, especially in the treatment of chronic inflammatory states such as inflammatory bowel disease and as such the use of rutine in the composition of the invention as a source of quercetin provides a synergistic effect in the treatment and prevention of inflammation especially at the level of the colon where on the one hand quercetin is liberated and on the other hand D-fagomine is able to arrive an exert its immunostimulating effect. The presence of rutin helps somehow to maintain under control the immune response triggered by D-fagomine.

(25) It is moreover noted that rutin is, as D-fagomine, present in buckwheat (Fagopyrum esculentum) and also Fagopyrum tataricum that usually presents higher amounts of rutin that the F. esculentum. In this sense, in the preferred embodiment of the invention, the composition of the invention is elaborated from an extract of Fagopyrum esculentum enriched in D-fagomine and rutin.

(26) Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

Example 1: Biological Activity of D-Fagomine in Human Colon Sample

(27) The aim of this investigation was to determine the effects ex vivo of D-fagomine on the adhesion of E. coli to human colon mucosal samples and their possible influence on the release of inflammatory mediators on a range of doses as an indication of the activation of the innate immune response.

(28) The approach, included the incubation of an E. coli strain with mucosal colonic samples obtained by surgery. The intention was to preserve the whole structure of mucosal samples including multiple cell populations such as epithelial cells and immunocompetent cells (macrophages, dendritic cells, plasma cells and lymphocytes), all of them keeping their original structure and disposition for cell to cell interaction. In other words, the approach was intended for mimicking the in vivo conditions of the colon mucosa.

(29) In the design of the study doses of 0, 50, 500 and 5000 g/ml of D-fagomine were tested.

(30) Pathogenic strains of E coli were used. The main differences of pathogenic strains of E coli vs. other non pathogenic are related to their capacity to avoid the immune system.

(31) Materials and Methods

(32) Organ Culture:

(33) Samples of macroscopically normal colonic mucosa were obtained at surgery from patients undergoing colectomy for colonic cancer. Areas free of disease were collected in ice-cold saline and transferred immediately to the Laboratory of the Digestive Research Unit of Hospital's Vail d'Hebron (Barcelona). After washing with saline, the mucosa was dissected from the muscularis layer and pieces between 25 and 30 mg were placed surface up in culture wells (15 mm diameter wells with 500 m bottom mesh, Netwell culture systems, Costar, Cambridge, Mass.), and orientated so that the epithelial surface was upper-most. Filters were suspended over wells containing 1.5 ml of RPMI edium (CanSera, Rexdale, Ontario, Canada) supplemented with 10% foetal calf serum (FCS; Gibco-BRL, Eggenstein, Germany), glutamine 2 mM and antibiotics: 100 U/ml penicillin, 100 microg/ml streptomycin and 50 microg/ml gentamycin, (all from Normon, Madrid, Spain). This volume is just for maintaining the humidity of the surface but not for sinking beneath the surface.

(34) Biopsies were incubated at 37 C. in a 95% O2+5% CO2 atmosphere and stimulated during 1 h with PMA (phorbol-12-myristate-13-acetate) and lonomycin (100/1000 ng/mL), both available from Sigma.

(35) Thereafter, medium was replaced by fresh RPMI 1640 medium (without glucose) supplemented with 10% FCS and sodium bicarbonate at 24 mmol/L.

(36) Bacteria strain E. coli (10-8 CFU/ml) and fagomine (50, 500 and 5000 g/mL) were added carefully to the explants. After 4 h of culture of tissues with E. coli, supernatants were collected for measurement of E. coli recovery, release of TNF-, LDH (viability) and pH. Moreover, the tissues were rinsed, homogenized and assayed for E. coli recovery and LDH (to study tissue viability).

(37) Bacteria Strains:

(38) The pathogenic E. coli reference strain (CECT 434, (Migula 1895) Castellani and Chalmers 1919) was provided by Dr. Maria Angeles Calvo (Microbiology Laboratory, Faculty of Veterinary, Autonomous University of Barcelona, Spain)

(39) Inoculum of E. coli:

(40) The day before of the experiment with tissue, the E. coli strain was inoculated in Luria Bertani broth (Pharmacy, Hospital Vail d'Hebron, Autonomous University of Barcelona, Spain) at 37 C. under aerobic conditions for 24 h. The day of the experiment bacteria were harvested in the stationary phase, cell counts in the bacterial suspension were estimated by optical density at 600 nm absorbance, and bacteria were added to the tissue-culture wells at the appropriate dilution.

(41) Rinse of Tissue:

(42) In order to quantify only the adherent E. coli bacteria, at the end of the culture, tissues were recovered and rinsed with 2 ml of saline in an orbital mixer at low speed during 2 minutes. After rinsing, each tissue was homogenized in 1 ml of saline and dilutions were prepared to quantify the recovery of E. coli in solid agar selective media.

(43) Recovery of E. coli at the End of the Experiment:

(44) Supernatant and tissue recovery of E. coli was quantified immediately after the experiment by culturing in selective solid media for Enterobacteriaceae. At least 3 different dilutions were carried out for each sample. The number of colonies was manually counted 24 hours later. The results have been normalized to a standard size of 30 mg.

(45) TNF- Quantification:

(46) Concentration of TNF- in the supernatants was measured using a commercially available ELISA assay system for human citoquine TNF- (Ready to Set, e-Bioscience). Cytokine concentration is expressed as pg (TNF-) per 30 mg of tissue.

(47) Colon Viability:

(48) To estimate tissue viability, we calculated LDH (lactate-dehydrogenase) activity release in the supernatant. This method was validated by Finnie (Gut 1995). The ratio of LDH activity in the supernatant over total LDH activity in tissue homogenates was calculated and used to estimate the percentage of viable tissue. Tissue samples were homogenized in Tris/HCl (100 mmol/L, pH 7.4) and supernatants were analysed for LDH using the spectrophotometric method. Tissue viability was assessed as the release of LDH per mg of tissue.

(49) Statistical Analysis:

(50) Results are expressed by the mean and sem, or by individual data in plots.

(51) Results

(52) Recovery of E. coli in Tissue at the End of the Experiment:

(53) The result in the recovery of E. coli in tissue samples are provided in table 1 and FIG. 1:

(54) TABLE-US-00001 TABLE 1 Control Ec Fag 50 Fag 500 Fag 5000 E coli 10 6 1 8.2 4 2.4 1.6 Recovery on 2 4.4 0.8 1.6 tissue E coli 10 6 1 7.45 4.12 2.21 1.4 Standarized 30 mg 2 4.31 0.9 1.55 Average 7.45 4.22 1.56 1.47 SEM 0.09 0.65 0.07 Average % over control 100.0% 56.6% 21.0% 19.8% SEM % over 1.3% 8.7% 1.0% control

(55) As can be clearly observed the higher presence of E. coli in colon tissue is shown in the control sample and the presence of bacteria adhered to the tissue decreases as the concentration of D-fagomine increases. In other words it can be inferred that D-fagomine has an antiadherent effect against E. coli on the colon epithelium. The antiadherent effect is more potent while the concentration of D-fagomine increases.

(56) Cytokine Release:

(57) The results of cytokine release are shown in table 2 and FIG. 2:

(58) TABLE-US-00002 TABLE 2 Control B Control Ec Fag 50 Fag 500 Fag 5000 TNF-alfa/ml 1 12.9 0.7 106.2 20.7 13.8 2 25.2 0.4 98.8 18.6 5.7 mg (tissue 1 31.2 33.0 29.1 32.6 34.3 weight) 2 26.5 33.6 30.6 26.2 31.0 TNF-alfa 1 18.6 0.9 164.2 28.6 18.1 (1.5 ml standarized 2 42.8 0.5 145.4 31.9 8.2 30 mg Average 30.7 0.7 154.8 30.3 13.2 SEM 8.5 0.1 6.7 1.2 3.5

(59) The results clearly demonstrate that the presence of D-fagomine has the effect of stimulating the release of TNF-alfa either with respect to the control with E. coli or with respect to the control without E. coli. In addition, it is noticeable that the release of TNF-alfa is dependent on D-fagomine concentration. It is interesting to see that the lowest is the concentration of D-fagomine the more potent is the cytokine release. TNF-alfa release at the dosage of 50 g/mL is surprisingly potent as it induces a TNF-alfa release which is 5 times higher to that of the dose of 500 g/mL and nearly 8 times higher to that of the dose of 5000 g/mL.

(60) In brief this assay demonstrates that D-fagomine induces the activation of the innate immune response in a dose dependent form and that said induction is more potent at lower concentrations of 50 g/mL or less.

(61) Control on Viability:

(62) The results of c viability of colon tissue are represented in table 3:

(63) TABLE-US-00003 TABLE 3 Viability LDH pH Blank 93.25% 7.71 Ec 94.16% 7.62 Ec Fago 50 88.20% 7.65 Ec Fago 500 91.47% 7.59 Ec Fago 5000 83.88% 7.65

(64) The results indicate that the coculture with E. coli or fagomine does not affect the viability of the tissue.

Example 2: Compositions Comparing D-Fagomine

(65) The following compositions with D-fagomine were prepared: Drink beverage supplemented with D-fagomine (50 mg) and rutin (500 mg). Tablet supplemented with D-fagomine (50 mg) and rutin (500 mg). Biscuits made of Fagopyrum esculentum flour which naturally comprises D-fagomine, rutin and a preparation of prebiotics containing plant fibers, FOS (Fructooligosacharides) and XOS (Xylooligoscharides) Alcohol free beer made from Fagopyrum esculentum with a content of 8 ppm of D-fagomine and 50 ppm rutin. Infant formula, based on milk from cows feeded with buckwheat showing a content of D-fagomine 10 ppm. Sugar free chewing gum with D-fagomine (50 mg) for stimulating the innate immune response in the oral mucosa and preventing caries and periodontal diseases. Candy with D-fagomine (20 mg) as coajyuvant in processes of bacterial infection in the throat or in guts. Pellet, micro-encapsulation preparation with D-fagomine (50 mg) and rutin (500 mg) as additive for the preparation of functional foods or pharmaceutical formulations, to improve bioavailability in the colon. Nasal spray with D-fagomine (10 mg) as coadjuvant for nasal vaccines in processes of immunization against Streptococcus pneumoniae, influenza virus etc. Oral powder formulation of 50-100 mg D-fagomine as coadjuvant in processes of enteric infections, together with probiotics (lactobacillus, bifidobacteria).