WO2023203501A1 - 2'-fucosyllactose and 6'-sialyllactose for modulating the immune system - Google Patents

2'-fucosyllactose and 6'-sialyllactose for modulating the immune system Download PDF

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WO2023203501A1
WO2023203501A1 PCT/IB2023/053995 IB2023053995W WO2023203501A1 WO 2023203501 A1 WO2023203501 A1 WO 2023203501A1 IB 2023053995 W IB2023053995 W IB 2023053995W WO 2023203501 A1 WO2023203501 A1 WO 2023203501A1
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composition
combination
sialyllactose
use according
fucosyllactose
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PCT/IB2023/053995
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French (fr)
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Jonathan Lane
Clodagh WALSH
Rita HICKEY
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Health And Happiness (H&H) Hong Kong Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a combination of 2'-fucosyl lactose and 6'-sialyllactose for modulating the immune system of an individual.
  • the present invention pertains to the use of these compounds for preventing and/or treating immunological disorders.
  • Organisms of higher complexity have sophisticated defence mechanisms to maintain their own integrity against foreign invaders. These defence mechanisms, generically termed the immune system, are made up of a multifaceted network of proteins, cells, tissues and organs working together to provide protection. In mammals the immune system is very complex and divided into an innate and an acquired immune system, both parts being interconnected via messenger molecules, such as chemokines or cytokines. These messengers do not only provide for a communication between the two parts of the immune system but also within the same.
  • messenger molecules such as chemokines or cytokines.
  • a proper operation of the immune system may be perturbed by exogenous factors, such as stress, lack of nutrients, exposure to pathogens or drugs and/or by endogenous factors, such as a genetic predisposition. All of these perturbations may result in a low activity or also an over-activity or the immune system, both conditions making the individual susceptible to develop various disorders and/or diseases.
  • a low or even abnormally low activity of the immune system increases the individual's risks that pathogens successfully invade the organism, the infections potentially becoming chronic and even live threatening.
  • Well known examples for such conditions are infections with the human immunodeficiency virus and endogenous factors, e.g. CVID, where the individual lacks IgA antibodies and other B- and T-cell functions.
  • an over-activity of the immune system may be detrimental to the organisms, when the immune system's excessive reactions damage own cells and tissues
  • overreactive activities are allergic reactions, auto-immune diseases and also to some extent inflammatory reactions.
  • Inflammation is in principle part of an organism's response to the presence of foreign material, e.g. of a pathogen.
  • Cells present at the particular site secrete chemokines or cytokines that attract immune effector cells, i.e. macrophages or T-cells, which perform their designated task at the site, for example extinguishing the pathogen.
  • chemokines or cytokines that attract immune effector cells, i.e. macrophages or T-cells, which perform their designated task at the site, for example extinguishing the pathogen.
  • the inflammatory reactions come to a stop.
  • these inflammatory responses continue to prevail being accompanied by an unbalanced, excessive level or particular cytokines, attracting and stimulating more and more effector cells, which eventually damage healthy tissues.
  • Such inflammatory reactions also occur and continue to prevail in certain tissues based on a genetic predisposition, such as during rheumatoid arthritis in joints.
  • Over-activity of the immune system is conventionally treated by administering agents devised to suppress the immune reaction.
  • Their effect basically results from an inhibition of cell proliferation of immune cells, a reduction of the count of T-lymphocytes or interference with cytokines.
  • glucocorticoids prevent formation of classical inflammatory messengers, such as IL-1, IL-6, TNFa or interferons.
  • Antibodies such as basiliximab or adalimumab, are used to bind and scavenge interleukins or TNFa such interfering with immune responses.
  • Other agents such as cyclosporin A or pimecrolimus inhibit activation of T-lymphocytes, while compounds such as methotrexate or azathioprine inhibit DNA biosynthesis during proliferation of T-lymphocytes.
  • the above problem has been solved by providing a combination of 2'-Fucosyllactose and 6'- sialyllactose for modulating the immune system, thus providing an improved basis for preventing and/or treating immune disorders in an individual.
  • 2'-Fucosyllactose and 6'-sialyllactose are both members of of a family of glycans, which are the third most abundant fraction in human breast milk.
  • the most prevalent fucosylated and sia lylated glycans in human breast milk are 2'-FL and 6'-SL, respectively.
  • Both, 2'-fucosyllactose and 6'-sialyllactose are known as prebiotics, promoting growth of beneficial bacteria in the gut thus preventing adherence of toxins or pathogens to the gut epithelium.
  • IL-8, IL-1R, 11-6, IFN-y and TNFa are known pro-inflammatory messengers and are essentially abundantly present in immune disorders, such as rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease etc..
  • IL-8 is known to attract and recruit leucocytes into tissues, where in addition with e.g. IFN-y activates macrophages and cytotoxic T-cells to effect their damaging activities there.
  • IL-10 and TGF-R have been found to be downregulated in these disorders.
  • These cytokines primarily serve for reducing formation of pro- inflammatory cytokines as such but they also provide a prolonged life of B-lymphocytes, allowing an increased antibody production thereof. An increased IL-10 level thus also supports the immune system in its defence against foreign invaders.
  • the claimed combination balances immunological reactions, i.e. down-regulating expression etc. of pro- inflammatory chemokines/cytokines while at the same time up-regulating expression etc. of antiinflammatory cytokines, which concurrently also promote the activity of the immune system.
  • the present invention further provides pharmaceutical and food compositions containing 2'- fucosyllactose and 6'-sialyllactose for modulating the immune system of a subject.
  • Figure 1 Intestinal release of pro-inflammatory cytokine IL-8 (assessed using IL-8 ELISA assay) in an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments.
  • 2'-FL 2'- Fucosyllactose
  • 6'-SL 6'-sialyllactose.
  • Inflammation induced through exposure of THP-1 cells to I FNy and lipopolysaccharide meanan ⁇ s.d, a p ⁇ 0.05 versus HMO control, b p ⁇ 0.05 versus glucose control, c p ⁇ 0.05 versus negative control
  • FIG. 1 Concentration (in pg/mL) of interleukin-10 (IL-10), which acts as an anti-inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments.
  • Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ⁇ s.d, a p ⁇ 0.05 versus HMO control, b p ⁇ 0.05 versus glucose control).
  • IFNy Interferon gamma
  • HMO human milk oligosaccharide
  • Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ⁇ s.d, a p ⁇ 0.05 versus HMO control, b p ⁇ 0.05 versus glucose control).
  • TNFa Tumour Necrosis Factor alpha
  • HMO human milk oligosaccharide
  • FIG. 1 Concentration (in pg/mL) of interleukin 1 beta (I L-ip), which acts as a pro-inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments.
  • IL-ip concentration quantified using MagPix multiplex ELISA assay.
  • 2'-FL 2'- Fucosyllactose (used at concentration of 1.2g/L or O.6g/L);
  • 6'-SL 6'-Siallylactose (used at concentration of 0.4g/L, O.3g/L, O.2g/Lor O.15g/L).
  • Ratio of 2'-FL to 6'-SL when applied in combination 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ⁇ s.d, a p
  • FIG. 6 Concentration (in pg/mL) of interleukin 6 (IL-6), which acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments.
  • IL-6 concentration quantified using MagPix multiplex ELISA assay.
  • IL-8 interleukin 8
  • HMO human milk oligosaccharide
  • Ratio of 2'-FL to 6'-SL when applied in combination 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ⁇ s.d, a p ⁇ 0.05 versus HMO control, b p ⁇ 0.05 versus glucose control).
  • the present invention provides a combination of 2'-fucosyllactose and 6'-sialyllactose for modulating the immune system in a subject.
  • the phrase modulating the immune system shall designate the ability of the combination to return immunological reactions to a normal, adequate level, i.e. reducing an over-reaction but maintaining and/or even promoting an adequate reaction against foreign invaders, such as e.g. pathogens. Yet, the said term shall also designate the ability of the combination to reduce the level of particular chemokines and/or cytokines involved in immune- reactions, in particular in inflammatory reactions, but at the same time to also increase the level of other cytokines, such as cytokines reducing the level of pro-inflammatory cytokines and/or also promoting the activity of the immune system.
  • cytokines are IL-8, IL-1R, IL-6, IFN-y, TNFa, and IL-10 or TGF-R, preferably IL-1R as a central messenger.
  • the combination as proposed herein is thus useful in the prevention and treatment of diseases or disorders associated with an abnormal cytokine secretion and may be used in methods for the treatment of these diseases and disorders.
  • 2'-fucosyllactose and 6'- sialyllactose may be obtained from natural sources, i.e. from mammals, in particular humans.
  • the compounds may also be synthesized chemically or obtained by recombinant methods involving expression thereof in bacteria, such as E.coli.
  • Today 2'-fucosyllactose is commercially available.
  • 6'-sialyllactose also a prominent human milk oligosaccharide, which may likewise be isolated from milk or synthesized via chemical or recombinant means.
  • 6'-sialyllactose is commercially available.
  • the subject to be treated may be any individual requiring improvement of its general immune condition, i.e. individuals exposed to environmental conditions weakening the immune system, such as stress, coldness, psychological influences etc., or individuals exposed to pathogens or drugs.
  • the subject may likewise be an individual suffering from uncontrolled immune reactions, such as during allergic reactions, autoimmune diseases and inflammatory reactions.
  • Non limiting examples for subjects envisaged by the present invention are mammals, in particular humans or animals, e.g. pets.
  • Conditions to be prevented and/or treated are in general immune deficient conditions, e.g. the individual being at a higher risk to acquire infections based on life circumstances, for example during winter time, or suffering from opportunistic infections. Also conditions, where the immune system is derailed and over-active are embraced, such as allergic reactions, inflammatory reactions and auto-immune disorders, e.g. psoriasis, rheumatoid arthritis etc.. A prevention and/or treatment of inflammatory reactions and disorders and diseases associated therewith are particularly contemplated.
  • Non limiting examples for subjects envisaged by the present invention are mammals, in particular humans or animals, e.g. pets.
  • pathogen comprises any foreign material against which the immune system provides protection, in particular prions, transposons, retroelements, viroids, viruses, bacteria, fungi, protists and parasites.
  • pathogen infection relates to the invasion by, emergence and replication of a pathogen as defined above.
  • the pathogen is selected from the group comprising bacteria, viruses, viroids, protists or parasites.
  • the compounds may be administered as such, i.e. in singular doses each or together in a composition.
  • the concomitant formulation of the compounds in a composition has the advantage that the amount and the ratio of the compounds may be controlled already at the stage of production, so that the consumer does not have to care about the amount to be ingested or the ratio of the compounds.
  • the compounds 2'-fucosyllactose and 6'-sialyllactose may be mixed and provided in a ratio of from 1 : 1 to 3 : 1 to 4 : 1 to 6 : 1.
  • the ratio of 3 : 1 is preferred resembling the natural abundance of the compounds in mother's milk.
  • the compounds and compositions to be used according to the present invention may be prepared for any uptake by the subject but are preferably prepared to be suitable for oral administration.
  • 2'-fucosyllactose may be administered at a minimum daily dose of 35 mg/kg body weight, preferred from about 100 - about 600 mg/kg body weight, more preferably from about 125 - about 275 mg/kg body weight and even more preferred from about 150- about 200 mg/kg body weight.
  • the term about shall designate a deviation of 10 % at maximum. 6'-sialyllactose is admixed or administered in amounts to fulfil the above ratios.
  • the compounds or compositions, respectively, may be provided as pharmaceutical compositions.
  • the pharmaceutical compositions may be formulated in a manner allowing administration thereof, release of the compounds at a desired target site and protection from conditions prevailing in the gastrointestinal tract.
  • the pharmaceutical composition may be provided as tablets, capsules, powders, granules, syrups, aqueous suspensions or solutions to which carriers and or excipients are added depending in the needs.
  • carriers and or excipients are filling agents, solvents, emulsifier, buffer, thickeners, coatings, disintegrants, lubricating agents, flowing agents, preservatives, resorption accelerating agents etc..
  • the compounds or compositions, respectively, may also be provided to the subject together with other agents supporting the desired effect, e.g. known treatment regimens for the respective disorder or disease to be treated, e.g. immune suppressants, synthesized or of biologic origin.
  • immune stimulants such as plant extracts from coneflower or mistletoe, lectins, bacterial extracts, or vaccines or antibodies are embraced herewith.
  • active agents directed to killing the pathogens may be provided with the present combination or compositions, such as antibiotics, anti-mycotika, antiviral agents, anti-parasitic agents or any combination thereof.
  • the present combination may also be included in a food composition, which provides the advantage that individuals have a steady support for their immune system simply based on food to be ingested, which is in particular beneficial for elderly or other people suffering from an impaired or debilitated immune system.
  • the nature of the compounds, being members of human milk oligosaccharides, makes them particularly suitable for being simply included and added in/to any milk based product, such as milk itself, ice-cream, yogurt, kefir, cheese, fermented milk, infant formula, powder milk etc..
  • the compounds or compositions may be provided in any other known food product with the only proviso that the nature and/or amount of the compounds does not negatively interfere with the organoleptic properties of the food product and the expectation of the consumer in this respect.
  • Non limiting examples for food products are fruit or vegetable juices, cereals, bakery products, cereal-based products, nutritional supplements, soft drinks and/or dietary supplements.
  • the compounds or compositions as illustrated above are provided as a food additive, supplement or functional food.
  • the above described food products, additives are particularly useful in the prevention and treatment of disorders affecting the gastrointestinal tract, as in gastroenteritis, enterocolitis, enteritis and colitis.
  • the food product is ingested and may exert its effect its effect on immune reactions in the gastrointestinal tract itself. Since the present combination influences immune- logical reactions both, inflammatory disorders based on infections or based on chronic conditions, such as in Colitis ulcerosa or Morbus Crohn, may be treated and/or ameliorated.
  • a daily intake of these food products etc. the emergence, severity and duration of any inflammatory disorders in the gastro-intestinal tract may be addressed without the need of taking pharmaceuticals.
  • Example 1 Effect of a combination of 2'-fucosyllactose and 6'-sialyllactose on the secretion of cytokines by Caco2 cells 2'-fucosyllactose and 6'-sialyllactose had been obtained by bacterial fermentation with E. coli and purified to a purity of >94%.
  • Caco-2 cells obtained from ATCC culture collection were seeded on transwell inserts (Corning, USA) and maintained in Dulbecco Modified Eagle's Medium (DMEM) (Sigma Aldrich) supplemented with FCS for 18-21 days in an incubator at 37°C under conditions of 90 % humidity and 5 % C02 up to formation of a confluent monolayer.
  • DMEM Dulbecco Modified Eagle's Medium
  • THP-1 cells were seeded on 12-well plates (Sigma Aldrich) and differentiated with PMA (100 nM) for 48 hours (37°C, 90 % humidity, 5 % CO2) to form adherent macrophages.
  • the differentiated THP-1 cells were then stimulated with LPS and IFN-y (10ng/ mL each) for 4 hours (culture conditions as above) to activate the differentiated THP-1 cells and simulate an inflammatory condition.
  • the Caco-2 cells contained in the different transwells were pre-exposed to the compounds (2'-fucosyllactose and/or 6'-sialyllactose or a control containing no 2'-fucosyllactose and/or 6-sialyllactose) a defined concentration (1.2g/L 2'-fucosyl lactose and/or 0.4g/L 6'- sialyllactose) and at a defined ratio (2'-fucosyllactose : 6'-sialyllactose 3:1) for 4 hours (cultivation conditions as above) before addition to the primed THP-1 cells/activated macrophages. After addition the mixture was incubated for 24 hours under the condition as above.
  • the Caco-2 transwells were first transferred into the THP-1 containing wells and both cells were co-exposed to the compounds or the control (no compounds) in the given amounts and under the same conditions for 24 hours. More specifically, the compounds were co-exposed at various concentrations (O.6-1.2g/L 2'-fucosyl lactose and/or 0.15-0.4g/L 6'- sialyllactose) and at various ratios (2'-fucosyllactose : 6'-sialyllactose 3:1, 4:1 and 6:1) under the same conditions for 24 hours (see Table 3) As positive control Human Milk Oligossaccharides were used.
  • HPAEC-PAD High pH anion exchange chromatography with pulsed amperometric detection
  • Dulbecco Modified Eagle's Medium supplemented with glucose at an equivalent concentration to HMO, i.e.1.6 Or 0.8g/L was used.
  • 2'-FL, 6'-SL, combinations of 2'-FL and 6'-SL and the HMO control demonstrated immunomodulatory activities. That is the capacity to modulate cytokine specific expression upon induction of a pro-inflammatory response.
  • a combination of 2'-FL and 6'-SL demonstrated concentration dependent synergistic immunomodulatory activities similar to that of total HMO isolation from human breast milk. Specifically, this combination modulated the expression of IL-8, IL-1R, IL-6, TN Fa, and IL-10, similar to the control.

Abstract

The present invention relates to a combination of 2'-fucosyllactose and 6'-sialyllactose for stimulating and/or modulating the immune system of an individual. In particular, the present invention pertains to the use of these compounds for preventing and/or treating disorders and/or diseases associated with inflammatory reactions. A pharmaceutical and food composition is provided containing these compounds.

Description

2'-FUC0SYLLACT0SE AND 6'-SIALYLLACT0SE FOR MODULATING THE IMMUNE SYSTEM
Technical Field
The present invention relates to a combination of 2'-fucosyl lactose and 6'-sialyllactose for modulating the immune system of an individual. In particular, the present invention pertains to the use of these compounds for preventing and/or treating immunological disorders.
Background
Organisms of higher complexity have sophisticated defence mechanisms to maintain their own integrity against foreign invaders. These defence mechanisms, generically termed the immune system, are made up of a multifaceted network of proteins, cells, tissues and organs working together to provide protection. In mammals the immune system is very complex and divided into an innate and an acquired immune system, both parts being interconnected via messenger molecules, such as chemokines or cytokines. These messengers do not only provide for a communication between the two parts of the immune system but also within the same.
In some instances, a proper operation of the immune system may be perturbed by exogenous factors, such as stress, lack of nutrients, exposure to pathogens or drugs and/or by endogenous factors, such as a genetic predisposition. All of these perturbations may result in a low activity or also an over-activity or the immune system, both conditions making the individual susceptible to develop various disorders and/or diseases.
A low or even abnormally low activity of the immune system increases the individual's risks that pathogens successfully invade the organism, the infections potentially becoming chronic and even live threatening. Well known examples for such conditions are infections with the human immunodeficiency virus and endogenous factors, e.g. CVID, where the individual lacks IgA antibodies and other B- and T-cell functions.
On the other hand, also an over-activity of the immune system may be detrimental to the organisms, when the immune system's excessive reactions damage own cells and tissues Examples for such overreactive activities are allergic reactions, auto-immune diseases and also to some extent inflammatory reactions.
Inflammation is in principle part of an organism's response to the presence of foreign material, e.g. of a pathogen. Cells present at the particular site secrete chemokines or cytokines that attract immune effector cells, i.e. macrophages or T-cells, which perform their designated task at the site, for example extinguishing the pathogen. Upon completion of the task the inflammatory reactions come to a stop. In case of over-activity these inflammatory responses continue to prevail being accompanied by an unbalanced, excessive level or particular cytokines, attracting and stimulating more and more effector cells, which eventually damage healthy tissues. Such inflammatory reactions also occur and continue to prevail in certain tissues based on a genetic predisposition, such as during rheumatoid arthritis in joints.
Over-activity of the immune system is conventionally treated by administering agents devised to suppress the immune reaction. Their effect basically results from an inhibition of cell proliferation of immune cells, a reduction of the count of T-lymphocytes or interference with cytokines. For example, glucocorticoids prevent formation of classical inflammatory messengers, such as IL-1, IL-6, TNFa or interferons. Antibodies, such as basiliximab or adalimumab, are used to bind and scavenge interleukins or TNFa such interfering with immune responses. Other agents, such as cyclosporin A or pimecrolimus inhibit activation of T-lymphocytes, while compounds such as methotrexate or azathioprine inhibit DNA biosynthesis during proliferation of T-lymphocytes.
A disadvantage of all of these regimes resides in the severe side effects going along, with a general reduction of the body's own defence mechanisms being common to all of them, making the individual susceptible to infections. Thus, there is still a need for means suitable to prevent emergence of immune disorders and to modulate the immune system to restore a customary functionality.
Summary of the invention
The above problem has been solved by providing a combination of 2'-Fucosyllactose and 6'- sialyllactose for modulating the immune system, thus providing an improved basis for preventing and/or treating immune disorders in an individual.
2'-Fucosyllactose and 6'-sialyllactose are both members of of a family of glycans, which are the third most abundant fraction in human breast milk. The most prevalent fucosylated and sia lylated glycans in human breast milk are 2'-FL and 6'-SL, respectively. Both, 2'-fucosyllactose and 6'-sialyllactose are known as prebiotics, promoting growth of beneficial bacteria in the gut thus preventing adherence of toxins or pathogens to the gut epithelium.
In the course of the studies leading to the invention the present inventors have found that a combination of 2'-fucosyllactose and 6'-sialyllactose on the one hand reduces production of pro- inflammatory chemokines/cytokines, while at the same time increase production of antiinflammatory cytokines that also promote activity of the immune system.
IL-8, IL-1R, 11-6, IFN-y and TNFa are known pro-inflammatory messengers and are essentially abundantly present in immune disorders, such as rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease etc.. IL-8 is known to attract and recruit leucocytes into tissues, where in addition with e.g. IFN-y activates macrophages and cytotoxic T-cells to effect their damaging activities there. On the other hand IL-10 and TGF-R have been found to be downregulated in these disorders. These cytokines primarily serve for reducing formation of pro- inflammatory cytokines as such but they also provide a prolonged life of B-lymphocytes, allowing an increased antibody production thereof. An increased IL-10 level thus also supports the immune system in its defence against foreign invaders.
Without wishing to be bound by any theory it is presently contemplated that the claimed combination balances immunological reactions, i.e. down-regulating expression etc. of pro- inflammatory chemokines/cytokines while at the same time up-regulating expression etc. of antiinflammatory cytokines, which concurrently also promote the activity of the immune system.
The present invention further provides pharmaceutical and food compositions containing 2'- fucosyllactose and 6'-sialyllactose for modulating the immune system of a subject.
Figures
Figure 1. Intestinal release of pro-inflammatory cytokine IL-8 (assessed using IL-8 ELISA assay) in an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. 2'-FL= 2'- Fucosyllactose; 6'-SL= 6'-sialyllactose. Inflammation induced through exposure of THP-1 cells to I FNy and lipopolysaccharide (mean ± s.d, a p < 0.05 versus HMO control, b p < 0.05 versus glucose control, c p < 0.05 versus negative control)
Figure 2. Concentration (in pg/mL) of interleukin-10 (IL-10), which acts as an anti-inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. IFNy concentration quantified using MagPix multiplex ELISA assay.2'-FL= 2'- Fucosyllactose (used at concentration of 1.2g/L); 6'-SL= 6'-Siallylactose (used at concentration of 0.4g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p < 0.05 versus HMO control, b p < 0.05 versus glucose control).
Figure 3. Concentration (in pg/mL) of Interferon gamma (IFNy), which acts as a pro-inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. IFNy concentration quantified using MagPix multiplex ELISA assay.2'-FL= 2'- Fucosyllactose (used at concentration of 1.2g/L); 6'-SL= 6'-Siallylactose (used at concentration of 0.4g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p < 0.05 versus HMO control, b p < 0.05 versus glucose control).
Figure 4. Concentration (in pg/mL) of Tumour Necrosis Factor alpha (TNFa), which acts as a pro- inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. TNFa concentration quantified using MagPix multiplex ELISA assay. 2'-FL= 2'- Fucosyllactose (used at concentration of 1.2g/L or O.6g/L); 6'-SL= 6'-Siallylactose (used at concentration of 0.4g/L, O.3g/L, O.2g/Lor O.15g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p
< 0.05 versus HMO control, b p < 0.05 versus glucose control).
Figure 5. Concentration (in pg/mL) of interleukin 1 beta (I L-ip), which acts as a pro-inflammatory cytokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. IL-ip concentration quantified using MagPix multiplex ELISA assay. 2'-FL= 2'- Fucosyllactose (used at concentration of 1.2g/L or O.6g/L); 6'-SL= 6'-Siallylactose (used at concentration of 0.4g/L, O.3g/L, O.2g/Lor O.15g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p
< 0.05 versus HMO control, b p < 0.05 versus glucose control).
Figure 6. Concentration (in pg/mL) of interleukin 6 (IL-6), which acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. IL-6 concentration quantified using MagPix multiplex ELISA assay. 2'-FL= 2'-Fucosyllactose (used at concentration of 1.2g/L or O.6g/L); 6'-SL= 6'- Siallylactose (used at concentration of 0.4g/L, O.3g/L, O.2g/Lor O.15g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p < 0.05 versus HMO control, b p < 0.05 versus glucose control). Figure 7. Concentration (in pg/mL) of interleukin 8 (IL-8), which acts as a pro-inflammatory chemokine, released from an inflamed co-culture model of Caco-2 epithelial cells and THP-1 macrophage cells after 24 h exposure to controls and human milk oligosaccharide (HMO) treatments. IL-8 concentration quantified using MagPix multiplex ELISA assay. 2'-FL= 2'- Fucosyllactose (used at concentration of 1.2g/L or O.6g/L); 6'-SL= 6'-Siallylactose (used at concentration of 0.4g/L, O.3g/L, O.2g/Lor O.15g/L). Ratio of 2'-FL to 6'-SL when applied in combination= 3:1, 4:1 or 6:1. Details of treatments as outlined in Table 3. Inflammation induced through exposure of THP-1 cells to lOng/mL IFN-gamma and lipopolysaccharide (mean ± s.d, a p < 0.05 versus HMO control, b p < 0.05 versus glucose control).
Table 1. ELISA quantification of IL-8 in Caco-2 supernatants
Table 2. MagPix quantification of I FNy & IL-10 in Caco-2 supernatants (as summarised in Fig. 2 & Fig- 3)
Table 3. MagPix quantification of TNFa, IL1-P, IL-6, and IL-8 in Caco-2 supernatants (as summarised in Fig. 4-7)
Detailed Description of the Invention
The present invention provides a combination of 2'-fucosyllactose and 6'-sialyllactose for modulating the immune system in a subject.
In the context of the present invention the phrase modulating the immune system shall designate the ability of the combination to return immunological reactions to a normal, adequate level, i.e. reducing an over-reaction but maintaining and/or even promoting an adequate reaction against foreign invaders, such as e.g. pathogens. Yet, the said term shall also designate the ability of the combination to reduce the level of particular chemokines and/or cytokines involved in immune- reactions, in particular in inflammatory reactions, but at the same time to also increase the level of other cytokines, such as cytokines reducing the level of pro-inflammatory cytokines and/or also promoting the activity of the immune system. Non-limiting examples for such cytokines are IL-8, IL-1R, IL-6, IFN-y, TNFa, and IL-10 or TGF-R, preferably IL-1R as a central messenger.
The combination as proposed herein is thus useful in the prevention and treatment of diseases or disorders associated with an abnormal cytokine secretion and may be used in methods for the treatment of these diseases and disorders.
The compounds proposed for the claimed use or method, respectively, 2'-fucosyllactose and 6'- sialyllactose, may be obtained from natural sources, i.e. from mammals, in particular humans. The compounds may also be synthesized chemically or obtained by recombinant methods involving expression thereof in bacteria, such as E.coli. Today 2'-fucosyllactose is commercially available. Essentially the same applies to 6'-sialyllactose, also a prominent human milk oligosaccharide, which may likewise be isolated from milk or synthesized via chemical or recombinant means. Also 6'-sialyllactose is commercially available.
In principle, the subject to be treated may be any individual requiring improvement of its general immune condition, i.e. individuals exposed to environmental conditions weakening the immune system, such as stress, coldness, psychological influences etc., or individuals exposed to pathogens or drugs. The subject may likewise be an individual suffering from uncontrolled immune reactions, such as during allergic reactions, autoimmune diseases and inflammatory reactions. Non limiting examples for subjects envisaged by the present invention are mammals, in particular humans or animals, e.g. pets.
Conditions to be prevented and/or treated are in general immune deficient conditions, e.g. the individual being at a higher risk to acquire infections based on life circumstances, for example during winter time, or suffering from opportunistic infections. Also conditions, where the immune system is derailed and over-active are embraced, such as allergic reactions, inflammatory reactions and auto-immune disorders, e.g. psoriasis, rheumatoid arthritis etc.. A prevention and/or treatment of inflammatory reactions and disorders and diseases associated therewith are particularly contemplated. Non limiting examples for subjects envisaged by the present invention are mammals, in particular humans or animals, e.g. pets.
The combination of compounds is deemed to act by reducing disproportionate, i.e. excessive immune reactions, in particular inflammatory reactions, and also by enhancing the endogenous immune system's capability to combat foreign invaders, i.e. pathogens. In the context of the present invention the term pathogen comprises any foreign material against which the immune system provides protection, in particular prions, transposons, retroelements, viroids, viruses, bacteria, fungi, protists and parasites. In the context of the present invention the phrase pathogen infection relates to the invasion by, emergence and replication of a pathogen as defined above. According to a preferred embodiment the pathogen is selected from the group comprising bacteria, viruses, viroids, protists or parasites.
For modulating and/or also improving the immune condition in a subject the compounds may be administered as such, i.e. in singular doses each or together in a composition. The concomitant formulation of the compounds in a composition has the advantage that the amount and the ratio of the compounds may be controlled already at the stage of production, so that the consumer does not have to care about the amount to be ingested or the ratio of the compounds.
The compounds 2'-fucosyllactose and 6'-sialyllactose may be mixed and provided in a ratio of from 1 : 1 to 3 : 1 to 4 : 1 to 6 : 1. The ratio of 3 : 1 is preferred resembling the natural abundance of the compounds in mother's milk. The compounds and compositions to be used according to the present invention may be prepared for any uptake by the subject but are preferably prepared to be suitable for oral administration. As for mammals, in particular humans, 2'-fucosyllactose may be administered at a minimum daily dose of 35 mg/kg body weight, preferred from about 100 - about 600 mg/kg body weight, more preferably from about 125 - about 275 mg/kg body weight and even more preferred from about 150- about 200 mg/kg body weight. In the context ofthe present invention and the indication of amounts the term about shall designate a deviation of 10 % at maximum. 6'-sialyllactose is admixed or administered in amounts to fulfil the above ratios.
The compounds or compositions, respectively, may be provided as pharmaceutical compositions. The pharmaceutical compositions may be formulated in a manner allowing administration thereof, release of the compounds at a desired target site and protection from conditions prevailing in the gastrointestinal tract. In principle, the pharmaceutical composition may be provided as tablets, capsules, powders, granules, syrups, aqueous suspensions or solutions to which carriers and or excipients are added depending in the needs. Non-limiting examples for carriers and or excipients are filling agents, solvents, emulsifier, buffer, thickeners, coatings, disintegrants, lubricating agents, flowing agents, preservatives, resorption accelerating agents etc..
The compounds or compositions, respectively, may also be provided to the subject together with other agents supporting the desired effect, e.g. known treatment regimens for the respective disorder or disease to be treated, e.g. immune suppressants, synthesized or of biologic origin. Also immune stimulants, such as plant extracts from coneflower or mistletoe, lectins, bacterial extracts, or vaccines or antibodies are embraced herewith. Further, active agents directed to killing the pathogens may be provided with the present combination or compositions, such as antibiotics, anti-mycotika, antiviral agents, anti-parasitic agents or any combination thereof.
The present combination may also be included in a food composition, which provides the advantage that individuals have a steady support for their immune system simply based on food to be ingested, which is in particular beneficial for elderly or other people suffering from an impaired or debilitated immune system. The nature of the compounds, being members of human milk oligosaccharides, makes them particularly suitable for being simply included and added in/to any milk based product, such as milk itself, ice-cream, yogurt, kefir, cheese, fermented milk, infant formula, powder milk etc..
Also, the compounds or compositions may be provided in any other known food product with the only proviso that the nature and/or amount of the compounds does not negatively interfere with the organoleptic properties of the food product and the expectation of the consumer in this respect. Non limiting examples for food products are fruit or vegetable juices, cereals, bakery products, cereal-based products, nutritional supplements, soft drinks and/or dietary supplements.
According to a preferred embodiment the compounds or compositions as illustrated above are provided as a food additive, supplement or functional food.
The above described food products, additives are particularly useful in the prevention and treatment of disorders affecting the gastrointestinal tract, as in gastroenteritis, enterocolitis, enteritis and colitis. The food product is ingested and may exert its effect its effect on immune reactions in the gastrointestinal tract itself. Since the present combination influences immune- logical reactions both, inflammatory disorders based on infections or based on chronic conditions, such as in Colitis ulcerosa or Morbus Crohn, may be treated and/or ameliorated. By a daily intake of these food products etc. the emergence, severity and duration of any inflammatory disorders in the gastro-intestinal tract may be addressed without the need of taking pharmaceuticals.
The invention will now be described by the following examples which are not intended to limit the scope thereof.
Examples
Example 1: Effect of a combination of 2'-fucosyllactose and 6'-sialyllactose on the secretion of cytokines by Caco2 cells 2'-fucosyllactose and 6'-sialyllactose had been obtained by bacterial fermentation with E. coli and purified to a purity of >94%.
Caco-2 cells (obtained from ATCC culture collection) were seeded on transwell inserts (Corning, USA) and maintained in Dulbecco Modified Eagle's Medium (DMEM) (Sigma Aldrich) supplemented with FCS for 18-21 days in an incubator at 37°C under conditions of 90 % humidity and 5 % C02 up to formation of a confluent monolayer.
THP-1 cells were seeded on 12-well plates (Sigma Aldrich) and differentiated with PMA (100 nM) for 48 hours (37°C, 90 % humidity, 5 % CO2) to form adherent macrophages. The differentiated THP-1 cells were then stimulated with LPS and IFN-y (10ng/ mL each) for 4 hours (culture conditions as above) to activate the differentiated THP-1 cells and simulate an inflammatory condition.
Following this stimulation of the differentiated THP-1 cells, Caco-2 transwells were transferred into THP-1 containing wells.
Two different approaches were followed.
In a first assay the Caco-2 cells contained in the different transwells were pre-exposed to the compounds (2'-fucosyllactose and/or 6'-sialyllactose or a control containing no 2'-fucosyllactose and/or 6-sialyllactose) a defined concentration (1.2g/L 2'-fucosyl lactose and/or 0.4g/L 6'- sialyllactose) and at a defined ratio (2'-fucosyllactose : 6'-sialyllactose 3:1) for 4 hours (cultivation conditions as above) before addition to the primed THP-1 cells/activated macrophages. After addition the mixture was incubated for 24 hours under the condition as above.
In an alternative assay the Caco-2 transwells were first transferred into the THP-1 containing wells and both cells were co-exposed to the compounds or the control (no compounds) in the given amounts and under the same conditions for 24 hours. More specifically, the compounds were co-exposed at various concentrations (O.6-1.2g/L 2'-fucosyl lactose and/or 0.15-0.4g/L 6'- sialyllactose) and at various ratios (2'-fucosyllactose : 6'-sialyllactose 3:1, 4:1 and 6:1) under the same conditions for 24 hours (see Table 3) As positive control Human Milk Oligossaccharides were used. Donated human milk samples were pooled and immediately frozen and stored at -80 °C on arrival. Lipids and proteins were removed before applying samples to a BioGel P2 size exclusion column (92 x 5 cm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) to separate lactose and HMO components. High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used, to quantify lactose in the eluted fractions while a Pierce colorimetric peptide assay (Thermo Fisher Scientific, Reinach, Switzerland) was used to measure levels of peptides. Peptide-free and low-trace lactose (<80 mg/L) fractions were pooled and freeze-dried and the resultant HMO-enriched fraction was stored at 4 °C prior to use in the experiments.
As a negative control Dulbecco Modified Eagle's Medium supplemented with glucose at an equivalent concentration to HMO, i.e.1.6 Or 0.8g/L was used.
Following induction of inflammatory responses as outlined above, supernatants from the apical compartment were extracted and cytokines quantified using ELISA (IL-8) / Magpix (I L-1R, IL-6, IL- 8, TNF-a, IFNy, and IL-10).
As illustrated in Figs. 1-7 2'-FL, 6'-SL, combinations of 2'-FL and 6'-SL and the HMO control demonstrated immunomodulatory activities. That is the capacity to modulate cytokine specific expression upon induction of a pro-inflammatory response. A combination of 2'-FL and 6'-SL demonstrated concentration dependent synergistic immunomodulatory activities similar to that of total HMO isolation from human breast milk. Specifically, this combination modulated the expression of IL-8, IL-1R, IL-6, TN Fa, and IL-10, similar to the control.

Claims

Claims
1. A combination of 2'-fucosyllactose and 6'-sialyllactose for use in modulating the immune system in a subject.
2. A combination of 2'-fucosyl lactose and 6'-sialyllactose for use in treating and/or preventing a disorder in a subject associated with inflammatory reactions.
3. A composition containing 2'-fucosyllactose and 6'-sialyllactose for use in modulating the immune system in a subject.
4. A composition containing 2'-fucosyllactose and 6'-sialyllactose for use in treating and preventing a disorder in a subject associated with inflammatory reactions.
5. The combination or composition for use according to any of claims 1 to 4, wherein in the subject is a mammal.
6. The combination or composition for use according to any of claims 2, 4 or 5, wherein the disorder is selected from the group comprising allergic reactions, inflammatory reactions, opportunistic infections and auto-immune diseases .
7. The combination or composition according to any of the preceding claims, wherein the ratio of 2'-fucosyllactose to 6'-sialyllactose is between 6:1 and 3:1.
8. The combination or composition for use according to any of the preceding claims, wherein the combination or composition is suitable for oral administration.
9. The composition for use according to any of the claims 1 to 8, which is a pharmaceutical composition.
10. The composition for use according to claim 9, further comprising at least one pharmaceutically acceptable carrier and/or excipient.
11. The composition for use according to claim 9 or 10 in the form of a tablet, a capsule, a powder, granules, a syrup, aqueous suspensions or solutions.
12. The composition for use according to any of the claims 9 to 11 comprising an additional agent selected from the group comprising an immune suppressant and/or immune stimulants, an antibiotic, an anti-mycotikum, an antiviral agent, an anti-parasitic agent or any combination thereof.
13. The composition for use according to any of claims 1 - 8, which is a food composition.
14. The composition for use according to claim 13, selected from the group comprising fruit or vegetable juices, ice-cream, infant formula, milk, yogurt, cheese, fermented milk, powder milk, cereals, bakery products, milk and/or cereal-based products, nutritional supplements, soft drinks and/or dietary supplements.
15. The composition for use according to claim 14, wherein the composition is in form of a food additive, supplement or functional food.
PCT/IB2023/053995 2022-04-22 2023-04-19 2'-fucosyllactose and 6'-sialyllactose for modulating the immune system WO2023203501A1 (en)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20120172330A1 (en) * 2010-12-31 2012-07-05 Abbott Laboratories Human milk oligosaccharides for modulating inflammation
WO2015077233A1 (en) * 2013-11-19 2015-05-28 Abbott Laboratories Methods for preventing or mitigating acute allergic responses using human milk oligosaccharides
WO2019229711A1 (en) * 2018-05-31 2019-12-05 Glycom A/S Mixture of hmos for treating autoimmune diseases
WO2020126726A1 (en) * 2018-12-21 2020-06-25 Societe Des Produits Nestle S.A. A nutritional composition comprising 2'-fucosyllactose (2' fl) to improve the gastrointestinal barrier
US20210353653A1 (en) * 2020-05-13 2021-11-18 Glycosyn LLC 2'-fucosyllactose for the prevention and treatment of coronavirus-induced inflammation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120172330A1 (en) * 2010-12-31 2012-07-05 Abbott Laboratories Human milk oligosaccharides for modulating inflammation
WO2015077233A1 (en) * 2013-11-19 2015-05-28 Abbott Laboratories Methods for preventing or mitigating acute allergic responses using human milk oligosaccharides
WO2019229711A1 (en) * 2018-05-31 2019-12-05 Glycom A/S Mixture of hmos for treating autoimmune diseases
WO2020126726A1 (en) * 2018-12-21 2020-06-25 Societe Des Produits Nestle S.A. A nutritional composition comprising 2'-fucosyllactose (2' fl) to improve the gastrointestinal barrier
US20210353653A1 (en) * 2020-05-13 2021-11-18 Glycosyn LLC 2'-fucosyllactose for the prevention and treatment of coronavirus-induced inflammation

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