Field of the invention

The present invention relates to compositions which modulate the inflammation of the gut lining. In particular, the compositions of the invention comprise vitamin K2 or prodrug thereof, vitamin D, a xanthophyll and EPA and/or DHA which act synergistically together to modulate inflammation of the gut lining.

The compositions of the invention can be used as nutraceuticals, and can be used in the fortification of foods or simply as dietary supplements. They may also have utility as pharmaceuticals for the treatment of a variety of conditions where inflammation of the gut lining plays a key role. They can also be used as a composition to improve the absorption of nutrients or active agents through the gut lining.

Background of the invention

The intestinal tract represents the largest interface between the external environment and the human body. Nutrient uptake mostly happens in the intestinal tract, where the epithelial surface is constantly exposed to dietary antigens. Since inflammatory response toward these antigens may be deleterious for the host, a plethora of protective mechanisms take place to avoid or attenuate local damage. For instance, the intestinal barrier is able to elicit a dynamic response that either promotes or impairs luminal antigens adhesion and crossing (De Santis et al. Frontiers in Immunology, 2015, vol 6, Article 612). Regulation of the intestinal barrier is crucial to control intestinal permeability.

The present inventors aim to provide compositions which modulate/reduce inflammation of the gut lining. This can allow improved absorption of key nutrients and active ingredients at the gut wall. The compositions of the invention operate by regulating and/or minimising the inflammation of the gut lining. It is important when ingesting nutraceutical/pharmaceutical compositions for there to be good absorption of the nutrients/active ingredients in the product at the gut lining. Inflammation of the gut lining can hinder this absorption.

The present inventors have found that compositions comprising vitamin K2 or prodrug thereof, a vitamin D, a xanthophyll, and eicosapentaenoic acid (ΈRA’) and/or docosahexaenoic acid (‘DHA’) (or salt(s) thereof) may have various advantages, such as:

- supporting a healthy gut environment for better nutritional absorption

- managing inflammatory pathways at the gut wall

- supporting microbiome health and balance

- promoting nutrient transport (especially calcium through passive paracellular channels)

- helping support overall bone health and development.

It is known that elevated levels of TNF-a (Tumor Necrosis Factor alpha) are associated with increased inflammation of the gut (for example, see Gareb et al. ‘Review: Local Tumor Necrosis Factor-a Inhibition in Inflammatory Bowel Disease’, Pharmaceutics 2020, 12, 539). Inhibiting TNF-a can play a key role in reducing intestinal inflammation, which can be beneficial for general gut health as well as for inflammatory bowel diseases.

The present inventors have shown that the specific combination of ingredients described herein are surprisingly effective in inhibiting TNF-a. A synergistic improvement in TNF-a inhibition has been shown.

Since one of the key ingredients is vitamin K2, it is envisaged that the compositions are not only useful in modulating gut inflammation, but also in preventing or treating conditions associated with vitamin K deficiencies (e.g. bone health). This benefit can also be seen in relation to other organs or biostructures which are affected by or associated with vitamin K. For example, it is known that vitamin K2 is relevant for bone health or conditions of the cardiovascular system such as arteriosclerosis. By formulating vitamin K2 with other nutrients relevant for cardiovascular health, for example, the cardiovascular health is not only affected by the active action of the vitamin K, but also by its action as part of the composition of the invention on the gut lining which results in improved absorption of other nutrients relevant for cardiovascular health.

The specific formulation of the vitamin K with vitamin D, a xanthophyll and EPA and/or DHA results in marked improvements in regulating gut inflammation. The vitamin K and vitamin D act to manage inflammatory pathways at the level of the gut. The xanthophyll functions cooperatively to manage inflammatory chemical mediators at the level of the gut. The EPA/DHA helps to promote a healthy inflammatory response. The various ingredients work cooperatively to yield a composition with beneficial gut regulating properties.

Several prior art documents disclose compositions which comprise one or more of the components of the present composition. A number of documents describe various combinations of ingredients selected from vitamin K, vitamin D, fatty acids, xanthophylls, e.g. CN108125246, US 2016/220511 , W02007/076416 or US6579544, but there is no appreciation of the role of the combinations on gut inflammation, no disclosure of vitamin K2 specifically, and/or no anticipation of cooperative effects in TNF- a inhibition.

Summary of the invention

Viewed from one aspect the invention provides a composition comprising

(I) vitamin K2 or prodrug thereof;

(II) vitamin D;

(III) a xanthophyll; and

(IV) eicosapentaenoic acid (ΈRA’) and/or docosahexaenoic acid (‘DHA’), or salt(s) thereof.

Viewed from another aspect the invention provides a nutraceutical or pharmaceutical formulation comprising the composition as defined herein and an excipient.

Viewed from another aspect the invention provides an oral administration dosage form comprising a composition as defined herein.

Viewed from another aspect, the invention provides a composition as defined herein for use as a medicament.

Viewed from another aspect, the invention provides a composition as defined herein for use in the prevention or treatment of gut inflammation.

Viewed from another aspect the invention provides a composition as hereinbefore defined for use in the treatment of bone disease, cardiovascular disease, or eye disease.

Viewed from another aspect, the invention provides a method of preventing or treating inflammation of the gut lining, comprising administering to a patient in need thereof an effective amount of a composition as defined herein. Viewed from another aspect, the invention provides the use of a composition as defined herein for the manufacture of a medicament for the treatment or prevention of inflammation of the gut lining.

Viewed from another aspect, the invention provides a method of enhancing the absorption of a nutrient in a subject, comprising administering to a subject a composition as defined herein and said nutrient, wherein said nutrient is selected from osteopontin or salts of magnesium, calcium, phosphorus, magnesium, zinc, iron, copper, manganese, calcium, or combinations thereof, preferably salts of magnesium and/or calcium.

Viewed from another aspect, the invention provides a composition as defined herein for use in a method of enhancing the absorption of a nutrient in a subject, said method comprising administering to a subject a composition as defined herein and said nutrient, optionally wherein said nutrient is selected from osteopontin or salts of magnesium, calcium, phosphorus, magnesium, zinc, iron, copper, manganese, calcium, or combinations thereof, preferably salts of magnesium and/or calcium.

Viewed from another aspect, the invention provides a method of maintaining and/or improving the general gut health of a subject, comprising administering to said subject a composition as defined herein.

Viewed from another aspect the invention provides a method for modulating inflammation in the gut lining comprising administering to a patient in need thereof an effective amount of a composition as hereinbefore defined.

Viewed from another aspect, the invention provides the use of a composition as defined herein for maintaining and/or improving the general gut health of a subject.

The features of the aspects and/or embodiments indicated herein are usable individually and in combination in all aspects and embodiments of the invention where technically viable, unless otherwise indicated.

Detailed description of the invention

This invention relates to vitamin K2-containing compositions and their use in modulating inflammation in the gut lining. The compositions also have utility in the prevention or treatment of diseases such as inflammatory bowel disease, but also bone disease, cardiovascular disease, or eye disease. Vitamin K2 or prodrug thereof

Vitamin K denotes a group of lipophilic and hydrophobic vitamins that are needed for the post-translational modification of certain proteins, mostly required for blood coagulation. Chemically they are 2-methyl-1 , 4-naphthoquinone derivatives.

Vitamin K is not a single compound, rather it is a series of related homologues. Vitamin K1 is called phylloquinone and has the systematic name all- E-2-methyl-3-(3,7,11 ,15-tetramethylhexadec-2-enyl)naphthalene-1 ,4-dione. Vitamin K2 is a mixture of different molecules based on a naphthoquinone structure and varying lengths of isoprenoid chains. The compound MK-7 (i.e. 7 isoprenyl groups) is depicted below but other components of the vitamin have different numbers of isoprenoid links. Menaquinones have side chains composed of all-E polyprenyl residues; generally they are designated as MK-n, where n specifies the number of isoprenoid repeating units. The minimum value of n is 2. Whilst vitamin K2 occurs naturally in low concentrations in various fermented food products such as cheese, vitamin K2 can be produced by fermentation of soy beans, or can be produced synthetically.

The compositions of the invention comprise vitamin K2. Vitamin K2 is particularly beneficial over vitamin K1 for gut inflammation modulation, as it is better absorbed , and has a longer half-life. As an example, the half-life of vitamin K1 is 1.5 hours whereas the half-life of vitamin K2 MK-7 is 68 hours. In addition, the long chain menaquinones associate with LDL particles, and this allows a broader, extrahepatic distribution in the body compared to vitamin K1. Vitamin K2 is a specific subset of vitamin K compounds. Vitamin K1 , for example, has very different physiological data compared to vitamin K2. In a particular embodiment, the vitamin K2 or prodrug thereof makes up at least 50 wt%, preferably at least 75 wt%, preferably at least 90 wt%, preferably at least 95 wt%, preferably at least 99 wt% of any vitamin K in the composition. In a particular embodiment, the composition does not comprise any other vitamin K other than vitamin K2.

Preferably, the vitamin K2 in the compositions of the invention is a menaquinone MK-n, where n specifies the number of isoprenoid repeating units, and n is between 2 and 10, preferably between 4 and 8. Preferably, the vitamin K2 is MK-4 or MK-7. More preferably, the vitamin K2 is MK-7. The composition of the invention may also comprise a plurality of menaquinone MK-n compounds.

The use of MK-7 in particular is beneficial because that is more efficiently absorbed compared to both shorter and longer MK-n (like MK-4 and MK-9).

The vitamin K2 compounds of the invention may be in cis- or trans-forms, i.e. the polyisoprene chain may have cis- as well as trans- double bonds.

Preferably, the polysioprene chain on the MK-7 has all-trans geometry, however.

The composition may comprise a prodrug of the vitamin K2. In particular, vitamin K2 can suffer from stability issues with regards to oxygen and light. Racemisation of the double bonds in the isoprenoid chain leads to an inactive vitamin K2 analogue and these double bonds are obviously susceptible to oxidation. Also, the naphthoquinone ring itself is susceptible to oxidation. The use of prodrugs may be beneficial, for example, in compositions which require longer shelf-life or are particularly exposed to oxygen and light.

Useful prodrugs of vitamin K2 can be prepared from diester derivatives, where the ketone functionalities of the naphthoquinone ring are protected. The disubstituted vitamin K2 analogues are capable of undergoing hydrolysis and oxidation in the body to release the equivalent menaquinone type structure. Moreover, disubstituted compounds are more stable than the vitamin itself in solution and therefore have a longer shelf life. It is even envisaged that these compounds might also improve the bioavailability of the active component.

An advantage of the prodrugs is that they are more polar than MK-7 itself. That makes the compounds more easy to formulate and may increase their bioavailability within the body.

Prodrugs of vitamin K2 are preferably diester analogues of vitamin K2 such as those of formula (I): wherein each R is COAr, -COCi- ₆ alkyl group; each Ar is an optionally substituted phenyl, said substituent being a Ci- ₆ alkyl; and n is 2 to 8 such as 3 to 8, e.g. n=5. R is preferably -COMe. Both R groups should be the same.

The present inventors have found that the vitamin K2-containing compositions of the invention act to manage inflammatory chemical pathways at the level of the gut. For example, vitamin K2 reduces the activation of NFKB and upregulates IAP to support gut wall function and homeostasis.

With regards to NFKB, vitamin K2 reduces the activation of nuclear factor KB (NFKB) and inhibits the phosphorylation of IKKa/b. Reduction of epithelial NF-kB activation in Inflammatory Bowel Disease may repair defects in epithelial barrier function, reduce diarrhoea, and limit protein (e.g., serum albumin) losses. Epithelial NFKB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid efflux into the intestinal lumen.

Wth regards to Intestinal Alkaline Phosphatase (IAP), vitamin K2 up regulates expression of human intestinal alkaline phosphatase. IAP is an endogenous protein expressed by the intestinal epithelium that is believed to play a vital role in maintaining gut homeostasis. Loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation and subsequently systemic inflammation.

As is seen in the data below, vitamin K2, in combination with the other compounds of the present compositions, inhibits TNF-a. The use of vitamin K2 is thus beneficial as it regulates gut inflammation and thus can improve nutrient absorption. Other organs or biostructures which benefit from vitamin K2 supplementation can also be targeted. For example, it is known that vitamin K2 is relevant for conditions of the cardiovascular system such as arteriosclerosis. By formulating vitamin K2 with nutrients relevant for cardiovascular health, the cardiovascular health is not only affected by the active action of the vitamin K, but also by its action on the gut lining which results in improved absorption of other nutrients relevant for cardiovascular health.

In a particular embodiment, the composition comprises vitamin K2 in the range 0.01-50 wt%, such as 0.01-40 wt%, such as 0.01-30 wt%, 0.1-10 wt% or 0.5- 5 wt%, based on the total amount of components (l)-(IV) in the composition. A highly preferred range is 1.0 to 2.5 wt% based on the total amount of components (l)-(IV) in the composition. In another embodiment, the composition comprises vitamin K2 in an amount of at least 0.01 wt%, such as at least 0.1 wt% or at least 0.5 wt%, based on the total amount of components (l)-(IV) in the composition. In another embodiment, the composition comprises vitamin K2 in an amount of at most at most 50 wt%, such as at most 40 wt%, such as at most 30 wt%, such as at most 20 wt%, at most 10 wt% or at most 5 wt%, based on the total amount of components (l)-(IV) in the composition.

Throughout this entire disclosure, ‘amounts’ typically means weights, and ‘ratios’ means ratios by weight, unless indicated otherwise.

In a particular embodiment, the composition comprises vitamin K2 in the range 0.01-50 wt%, such as 0.01-40 wt%, such as 0.01-30 wt%, 0.1-10 wt% or 0.5- 5 wt%, based on the total weight of the composition. In another embodiment, the composition comprises vitamin K2 in an amount of at least 0.01 wt%, such as at least 0.02 wt%, such as at least 0.1 wt% or at least 0.5 wt%, based on the total weight of the composition. In another embodiment, the composition comprises vitamin K2 in an amount of at most at most 50 wt%, such as at most 40 wt%, such as at most 30 wt%, such as at most 20 wt%, at most 10 wt% or at most 5 wt%, based on the total weight of the composition.

Other suitable ranges for vitamin K2 include 10-70 wt%, such as 20-60 wt%, or 30-50 wt%, based on the amount of components (l)-(IV) of the composition. Other suitable ranges for vitamin K2 include 10-70 wt%, such as 20-60 wt%, or 30- 50 wt%, based on the weight of the composition.

Typically, the vitamin K2 or prodrug thereof is present in the composition of the invention in an amount of at least 20 pg, preferably at least 50 pg, preferably at least 80 pg. In another embodiment, the vitamin K2 or prodrug thereof is present in the composition of the invention in an amount of at most 10,000 pg, preferably at most 2000 pg, preferably at most 1000 pg, preferably at most 500 pg, preferably at most 300 pg. Suitable ranges include amounts of vitamin K2 or prodrug thereof of 20 to 10,000 pg, preferably 20 to 2000 pg, preferably 20 to 1000 pg, preferably 50 to 500 pg, more preferably 80 to 300 pg.

The amounts given herein for vitamin K2 and for the other components (II)- (IV) also apply for daily dosages.

Vitamin D

The composition of the present invention also comprises vitamin D. Preferably the vitamin D is vitamin D2 or D3, more preferably vitamin D3 (also known as cholecalciferol).

As well as promoting TNF-alpha inhibition, the vitamin D can promote calcium absorption by supporting the health of the channels that deliver calcium from the gut into the blood stream (see Assa et al, The Journal of Infectious Diseases, Volume 210, Issue 8, 15 October 2014, Pages 1296-1305).

In a particular embodiment, the composition comprises vitamin D in the range 0.001-10 wt%, such as 0.01-5 wt% or O.1-2 wt%, based on the total amount of components (l)-(IV) in the composition. In another embodiment, the composition comprises vitamin D in an amount of at least 0.001 wt%, such as at least 0.01 wt% or at least 0.1 wt%, based on the total amount of components (l)-(IV) in the composition. In another embodiment, the composition comprises vitamin D in an amount of at most 10 wt%, such as at most 5 wt% or at most 2 wt%, based on the total amount of components (l)-(IV) in the composition.

In a particular embodiment, the composition comprises vitamin D in the range 0.001-10 wt%, such as 0.01-5 wt% or 0.1-2 wt%, based on the total weight of the composition. In another embodiment, the composition comprises vitamin D in an amount of at least 0.001 wt%, such as at least 0.01 wt% or at least 0.1 wt%, based on the total weight of the composition. In another embodiment, the composition comprises vitamin D in an amount of at most 10 wt%, such as at most 5 wt% or at most 2 wt%, based on the total weight of the composition.

Other suitable ranges for the amount of vitamin D include 0.001-5 wt%, 0.001-2 wt%, 0.001-1 wt%, 0.001-0.1 wt%, 0.001-0.05 wt% based on the total amount of components (l)-(IV) in the composition. Other suitable ranges for the amount of vitamin D include 0.001-5 wt%, 0.001-2 wt%, 0.001-1 wt%, 0.001-0.1 wt%, 0.001-0.05 wt% based on the total weight of the composition.

Typically, the vitamin D is present in the compositions of the invention in an amount of at least 0.1 pg, preferably at least 1 pg, preferably at least 5 pg, more preferably at least 10 pg. Typically, the vitamin D is present in the compositions of the invention in an amount of at most 500 pg, preferably at most 250 pg, more preferably at 100 pg. Typically, the vitamin D is present in the compositions of the invention in an amount of 0.1 to 500 pg, preferably 1 to 500 pg, preferably 5 to 250 pg, more preferably 10 to 100 pg.

Unexpectedly, the compositions of the present invention show beneficial results at low amounts of vitamin D.

Xanthophyll

The composition of the present invention also comprises a xanthophyll. The xanthophyll can be selected from Canthaxanthin, Cryptoxanthin, Zeaxanthin, Astaxanthin, Lutein, Rubixanthin. Typically, however, the xanthophyll is astaxanthin or more preferably lutein.

As well as promoting TNF-alpha inhibition, lutein functions cooperatively to manage inflammatory chemical mediators at the level of the gut. It has a direct downstream impact on reactive oxygen species in the support of healthy bone building activities. Lutein shows inhibitory effects on H202-induced increases in intracellular ROS levels, activation of NF-KB, and IL-8 expression in AGS cells. Furthermore, supplementation of lutein may be beneficial for the treatment of oxidative stress-mediated gastric inflammation.

Lutein also supports bone health.

Typically, the composition of the present invention comprises an amount of xanthophyll in the range 5-99.9 wt%, preferably 10-99.9 wt%, preferably 20-99.9 wt%, preferably 50-99 wt%, preferably 75-98 wt%, preferably 90-97 wt%, based on the total amount of components (l)-(IV) in the composition. Typically, the composition of the present invention comprises an amount of xanthophyll in at least 5 wt%, preferably at least 10 wt%, preferably at least 20 wt%, preferably at least 50 wt%, preferably at least 75 wt%, preferably at least 90 wt%, based on the total amount of components (l)-(IV) in the composition. Typically, the composition of the present invention comprises an amount of xanthophyll in at most 99.9 wt%, preferably at most 99 wt%, preferably at most 98 wt%, preferably at most 97 wt%, based on the total amount of components (l)-(IV) in the composition.

Typically, the composition of the present invention comprises an amount of xanthophyll in the range 5-99.9 wt%, preferably 10-99.9 wt%, preferably 20-99.9 wt%, preferably 50-99 wt%, preferably 75-98 wt%, preferably 90-97 wt%, based on the total weight of the composition. Typically, the composition of the present invention comprises an amount of xanthophyll in at least 5 wt%, preferably at least 10 wt%, preferably at least 20 wt%, preferably at least 50 wt%, preferably at least 75 wt%, preferably at least 90 wt%, based on the total weight of the composition. Typically, the composition of the present invention comprises an amount of xanthophyll in at most 99.9 wt%, preferably at most 99 wt%, preferably at most 98 wt%, preferably at most 97 wt%, based on the total weight of the composition.

Other suitable ranges for the amount of xanthophyll in the composition include 1-40 wt%, 5-30 wt%, 10-20 wt% based on the total amount of components (l)-(IV) in the composition. Other suitable ranges for the amount of xanthophyll in the composition include 1-40 wt%, 5-30 wt%, 10-20 wt% based on the total weight of the composition.

Typically, the xanthophyll is present in the composition in an amount of amount of at least 0.1 mg, preferably at least 0.5 mg, more preferably at least 1 mg,. Typically, the xanthophyll is present in the composition in an amount of at most 250 mg, preferably at most 100 mg, more preferably at most 50 mg, more preferably at most 25 mg. Typically, the xanthophyll is present in the composition in an amount of 0.1 to 250 mg, preferably 0.5 to 100 mg more preferably 1 to 50 mg, more preferably 1 to 25 mg.

Eicosapentaenoic acid (ΈRA’) and/or docosahexaenoic acid (‘DHA’), or salt(s) thereof

The composition of the present invention comprises EPA and/or DHA or a salt thereof. EPA/DHA supplementation helps to promote a healthy inflammatory response. EPA/DHA supplementation in patients with inflammatory bowel diseases results in EPA/DHA incorporation into gut mucosal tissue and modification of inflammatory mediator profiles. If both EPA and DHA are present, the amount of component (IV) refers to the combined weight of both EPA and DHA. Preferably, the composition comprises both EPA and DHA.

Typically, the composition of the present invention comprises an amount of EPA and/or DHA in the range 5-60 wt%, preferably 10-55 wt%, preferably 15-50 wt%, preferably 20-45 wt%, preferably 20-40 wt%, based on the total amount of components (l)-(IV) in the composition. Typically, the composition of the present invention comprises an amount of EPA and/or DHA in an amount of at least 5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt%, based on the total amount of components (l)-(IV) in the composition. Typically, the composition of the present invention comprises an amount of EPA and/or DHA in an amount of at most 60 wt%, preferably at most 55 wt%, preferably at most 50 wt%, preferably at most 45 wt%, preferably at most 40 wt% based on the total amount of components (l)-(IV) in the composition.

Typically, the composition of the present invention comprises an amount of EPA and/or DHA in the range 5-60 wt%, preferably 10-55 wt%, preferably 15-50 wt%, preferably 20-45 wt%, preferably 20-40 wt%, based on the total weight of the composition . Typically, the composition of the present invention comprises an amount of EPA and/or DHA in an amount of at least 5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt%, based on the total weight of the composition. Typically, the composition of the present invention comprises an amount of EPA and/or DHA in an amount of at most 60 wt%, preferably at most 55 wt%, preferably at most 50 wt%, preferably at most 45 wt%, preferably at most 40 wt% based on the total weight of the composition.

Other suitable ranges for EPA and/or DHA include 30-60 wt%, 35-55 wt%, or 40-50 wt%, based on the total amount of components (l)-(IV) in the composition. Alternatively, suitable ranges include 30-60 wt%, 35-55 wt%, or 40-50 wt%, based on the total weight of the composition.

Preferably, the compositions of the invention comprise 20 to 1000 pg EPA or salt thereof, preferably 50 to 500 pg, more preferably 100 to 300 pg EPA or salt thereof, if present.

Preferably, the compositions of the invention comprise 1 to 500 pg DHA or salt thereof, preferably 10 to 500 pg, more preferably 20 to 150 pg DHA or salt thereof, if present. The EPA and DHA are typically present in a EPA:DHA weight ratio of 0.01 : 1 to 50:1 , preferably 0.1 :1 to 20:1 , preferably 0.5:1 to 10:1 , especially 3:1 to 1 :3..

Composition, health impacts/diseases to be treated

Preferably, there is more vitamin K2 in the composition than vitamin D (by weight). Preferably, the weight ratio of vitamin K2 or prodrug thereof to vitamin D is at least 1 .1 :1.0, preferably at least 1.5:1 , more preferably at least 2:1 , more preferably at least 2.5:1 , more preferably at least 3:1 , more preferably at least 3.1 :1 or at least 3.5:1 .

In another embodiment of the invention, the weight ratio of vitamin K2 or prodrug thereof to vitamin D is at least 44:1 , preferably at least 50:1 , preferably at least 51 :1 , preferably at least 100:1 , preferably at least 200:1 , more preferably at least 250:1 , more preferably at least 270:1 , more preferably at least 300:1 , more preferably at least 400:1 , more preferably at least 500:1 , more preferably at least 750:1 , more preferably at least 1000:1 , more preferably at least 1500:1 , more preferably at least 2000:1 , more preferably at least 2500:1 , more preferably at least 3000:1.

In another embodiment of the invention, the weight ratio of vitamin K2 or prodrug thereof to vitamin D is at most 65,000:1 , preferably at most 50,000:1 , preferably at most 25,000:1 , more preferably at most 10,000:1 , more preferably at most 9,000: 1 , more preferably at most 8,000: 1 , more preferably at most 7,000: 1 , more preferably at most 6,000:1 , more preferably at most 5,000:1 , more preferably at most 4,500:1 , more preferably at most 4,000:1 , more preferably at most 3,750:1.

In another embodiment of the invention, the weight ratio of vitamin K2 or prodrug thereof to vitamin D is in the range 44:1 to 50,000:1 , preferably 75:1 to 50,000:1 , preferably 100:1 to 50,000:1 , more preferably 200:1 to 50,000:1 , more preferably 250:1 to 25,000:1 , more preferably 300:1 to 10,000:1 , more preferably 400:1 to 9,000:1 , more preferably 500:1 to 8,000:1 , more preferably 750:1 to 7,000:1 , more preferably 1 ,000:1 to 6,000:1 , more preferably 1 ,500:1 to 5,500:1 , more preferably 2,000:1 to 5,000:1 , more preferably 2,500:1 to 4,000:1 , more preferably 3,000:1 to 4,000:1 , more preferably 3,000:1 to 3,750:1.

Typically, there is more EPA and/or DHA than vitamin D (by weight) in the composition of the invention. Typically, therefore, the weight ratio of EPA and/or DHA to vitamin D is at least 1.1 :1.0, preferably at least 1 .5:1 , more preferably at least 2:1 , more preferably at least 2.5:1 , more preferably at least 3:1 , more preferably at least 4:1. In another embodiment, the weight ratio can be at least 100:1 , preferably at least 500: 1 , preferably at least 1000: 1 , preferably at least 2000:1 , preferably at least 3500:1 . Suitable upper limits are 5000:1 , 4000:1 , 1000:1 , 100:1 , for example.

Typically, there is more xanthophyll than vitamin D (by weight) in the composition of the invention. Typically, therefore, the weight ratio of xanthophyll to vitamin D is at least 1.1 : 1.0, preferably at least 1.5:1 , more preferably at least 2:1 , more preferably at least 2.5:1 , more preferably at least 3:1 , more preferably at least 10:1 , more preferably at least 100:1 , more preferably at least 150:1. In another embodiment, the weight ratio can be at least 500:1, or at least 1000:1 . Suitable upper limits are 2000: 1 or 1500: 1 , for example.

In a particular embodiment, the weight ratio of component (I) to (III) is in the range 0.01 :1 to 50:1 , preferably 0.1 :1 to 10:1 , more preferably 0.5:1 to 8:1 , more preferably 1 :1 to 5:1 , more preferably 1.5:1 to 4:1.

In a particular embodiment, the vitamin D of the invention is the component in the smallest amount by weight out of components (l)-(IV). In a particular embodiment, the DHA and/or EPA is/are present in a greater amount than vitamin K2 (by weight). In another embodiment, the weight ratio of component (I) to (IV) is 0.01 :1 to 50:1 , preferably 0.1 :1 to 10:1 , more preferably 0.1 :1 to 8:1 , more preferably 0.5:1 to 5:1 more preferably 0.5:1 to 2:1 .

The compositions of the invention are particularly suited for modulating and/or reducing the inflammation of the gut lining. This can be seen through the effect of the compositions on TNF-a inhibition. In a particular aspect, the invention provides a composition as defined herein for use in the treatment or prevention of a condition associated with TNFa dysregulation, such as gut inflammation.

In a particular embodiment, the composition of the invention does not comprise any prebiotics.

The compositions of the invention are (or can be part of) nutraceutical products or pharmaceutical products. Preferably, the compositions are for use as nutraceutical products for maintaining and/or improving gut health. In a particular embodiment, the uses/methods described herein are non-therapeutic.

The reduction of gut inflammation can have additional benefits, especially in terms of nutrient absorption. By reducing gut inflammation, nutrients can be better absorbed into the blood system, and thus the combination of the compositions of the invention with additional nutrients can lead to beneficial effects associated with the increased absorption of additional nutrients. These additional nutrients can have advantages in many areas of human or animal health. The additional nutrient(s) can be administered simultaneously, separately, or sequentially with the composition of the present disclosure. By better/increased absorption is herein meant better/increased absorption through the gut lining.

Vitamin K2 (in particular MK-7) has well documented therapeutic applications and the compositions of the present invention are suitable for all known therapeutic applications of vitamin K2. They can also be used as a food supplement or in any nutraceutical product, e.g. as a vitamin supplement.

Conditions in which vitamin K2 administration may assist treatment include osteoporosis and bone related disorders, cardiovascular health in general such as arteriosclerosis, myocardial infarction, calcification of blood vessels, diabetes, male infertility, conditions associated with inflammation and so on.

Other conditions of interest relate to those which are affected by additional nutrients whose absorption his improved by the compositions of the invention. In a particular embodiment, the composition further comprises a nutrient for bone health, cardiovascular health and/or eye health. Nutrient for bone health is preferred. By nutrient for bone health is meant a nutrient selected from osteopontin or salts of magnesium, calcium, phosphorus, magnesium, zinc, iron, copper, manganese, calcium, or combinations thereof, preferably salts of magnesium and/or calcium.

In a further particular embodiment, the composition of the invention is for use in the prevention or treatment of bone disease, cardiovascular disease, or eye disease. By bone disease is meant any disease relating to the growth, mineralization, microarchitecture, bone organic matrix constituents, density, elasticity and/or strength of the bone, such as osteoporosis. A particular cardiovascular disease of interest is arteriosclerosis. A particular eye disease of interest is macular degeneration.

In a particular embodiment, the composition of the invention is for use as a medicament, in particular for use in the prevention or treatment of gut lining inflammation. In a particular embodiment, the composition of the invention is for use in the prevention or treatment of an inflammatory bowel disease, such as ulcerative colitis or Crohn’s disease. ‘Bowel’ can be used herein instead of ‘gut’, i.e. the compositions of the invention have uses in bowel inflammation regulation/reduction.

Any of the uses or methods described or claimed herein for the compositions are equally applicable to any formulations, dosage forms, products etc. comprising the composition of the invention.

Where it is described herein that the composition are for administration to a ‘subject’, said subject is an animal or human subject, preferably human.

The compounds of the present invention may be utilized alone or in combination with one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of diseases or conditions for which the compositions of the present invention may have utility.

The compositions of the present invention may therefore be formulated as pharmaceutically acceptable compositions or nutraceutically acceptable compositions using known excipients. The compositions of the invention may also be used in combination therapy with other active agents.

While it is possible that the compositions of the invention may be administered as the bulk substance, it is preferable to present the composition in a pharmaceutical or nutraceutical formulation, for example, wherein the composition is in admixture with an acceptable carrier/excipient. In a particular embodiment, therefore, the invention provides a pharmaceutical or nutraceutical formulation comprising the composition of the present invention and at least one excipient.

The term "carrier" or "excipient" refers to a diluent, and/or vehicle with which the composition is administered. The compositions of the invention may contain one carrier or combinations of more than one carrier. Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition. The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, in addition to, the carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s). It will be appreciated that compositions for use in accordance with the present invention may be in the form of oral (or other mucosally administered) suspensions, powders, capsules or tablets, which may be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients. Oral and enteral administration methods are preferred, preferably oral administration, e.g. in the form of a capsule (preferably a soft gel capsule), tablet, powder, powder for use in a beverage or food, gel, syrup, juice, food, food supplement, beverage, nutraceutical product, pharmaceutical product, veterinary product, a feed or feed supplement, a personal care product, preferably a nutraceutical or pharmaceutical product, preferably a nutraceutical product for oral administration. Tablet form and capsule form are particularly preferred administration forms.

There may be different composition/formulation requirements depending on the different delivery systems. As the composition comprises more than one active component, then those components may be administered by the same or different routes, preferably the same.

The pharmaceutical or nutraceutical formulations of the present invention can be liquids that are suitable for oral or mucosal administration, for example, drops, syrups, solutions, that are ready for use or are prepared by the dilution of a freeze-dried product but are preferably solid or semisolid as tablets, capsules, granules, powders, pellets or solutions, suspensions, emulsions, or other forms suitable for oral administration.

The composition of the invention can be administered for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.

In one aspect, oral compositions are slow, delayed or positioned release (e.g., enteric especially colonic release) tablets or capsules.

As mentioned above, oral administration is preferred. Examples of pharmaceutically acceptable or nutraceutically acceptable disintegrants for oral compositions useful in the present invention include, but are not limited to, starch, pre-gelatinized starch, sodium starch glycolate, sodium carboxymethylcellulose, croscarmellose sodium, microcrystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminium silicates and crosslinked polyvinylpyrrolidone.

Examples of pharmaceutically acceptable or nutraceutically acceptable binders for oral compositions useful herein include, but are not limited to, acacia; cellulose derivatives, such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose, dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, starch, pre-gelatinized starch, tragacanth, xanthane resin, alginates, magnesium- aluminum silicate, polyethylene glycol or bentonite.

Examples of pharmaceutically acceptable or nutraceutically acceptable fillers for oral compositions include, but are not limited to, lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose), dihydro- or anhydro- calcium phosphate, calcium carbonate and calcium sulfate.

Examples of pharmaceutically acceptable or nutraceutically acceptable lubricants useful in the compositions of the invention include, but are not limited to, magnesium stearate, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulfate, magnesium lauryl sulfate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide.

Examples of suitable pharmaceutically acceptable or nutraceutically acceptable odorants for the oral compositions include, but are not limited to, synthetic aromas and natural aromatic oils such as extracts of oils, flowers, fruits (e.g., banana, apple, sour cherry, peach) and combinations thereof, and similar aromas. Their use depends on many factors, the most important being the organoleptic acceptability for the population that will be taking the pharmaceutical compositions.

Examples of suitable pharmaceutically acceptable or nutraceutically acceptable dyes for the oral compositions include, but are not limited to, synthetic and natural dyes such as titanium dioxide, beta-carotene and extracts of grapefruit peel.

Suitable examples of pharmaceutically acceptable or nutraceutically acceptable sweeteners for the oral compositions include, but are not limited to, aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol, mannitol, sorbitol, lactose and sucrose. Suitable examples of pharmaceutically acceptable buffers include, but are not limited to, citric acid, sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate and magnesium hydroxide. Suitable examples of pharmaceutically acceptable or nutraceutically acceptable surfactants include, but are not limited to, sodium lauryl sulfate and polysorbates.

Suitable examples of pharmaceutically acceptable or nutraceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents such as solvents, for example ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methyl paraben, ethyl paraben, propyl paraben, etc.).

Suitable examples of pharmaceutically acceptable or nutraceutically acceptable stabilizers and antioxidants include, but are not limited to, ethylenediaminetetriacetic acid (EDTA), thiourea, tocopherol and butyl hydroxyanisole.

The compositions of the invention may be taken once a day, twice a day, more often or less often depending on the purpose of administration, preferably once a day.

The dose and the administration frequency will depend on the use in question, e.g. whether for clinical use or via a supplement. A dosage of 1 to 1000 mg/day is suitable, preferably 1-500 mg/day, preferably 1-100 mg/day, preferably 1- 50 mg/day, based on the combined amount of components (l)-(IV).

Brief description of figures

Figure 1 shows the inhibition of LPS-induced TNF-a production by macrophages for the cells only, for inventive composition 1 (MIX), for comparative composition 1 (Vit K2 (MK-7)), for comparative composition 2 (vit D3), for comparative composition 3 (Lutein), and for comparative composition 4 (EPA/DHA).

Figure 2 shows the TNF-a inhibitory effects of inventive composition 1 (‘MIX’) in comparison to the sum of the TNF-a inhibitory effects of components (l)-(IV). MIX: The delta of the effect (effect size) of the mix was calculated (=cells only - mix). SUM: The sum of the effect of the single components in the mix was calculated (=cells only - comparative composition 1) + (cells only - comparative composition 2) + (cells only - comparative composition 3) + (cells only - comparative composition 4). Examples

Example 1 Composition

The following compositions were investigated for TNF-a inhibition.

Table 1 : Mixes used for TNF-a inhibition studies.

In vitro testing on gut inflammation - TNF- a inhibition studies

TNF-a was selected as a target because of its well-known role in gut inflammation. The toxicity of the compositions were tested by metabolic activity.

Immuno-modulating activity of vitamin K2 (MK-7), the metabolic active version of vitamin D3 (1a,25-(OH)2D3), lutein and EPA/DHA was studied by following the cytokine production of THP-1 , macrophages. One batch of the THP-1 cell line was used for all the experiments and cells were cultured in a 5% C02 humidified atmosphere at 37°C, in cell culture medium (RPMI, Gibco/Thermo Fisher), supplemented with 10% (v/v) fetal bovine serum, and 1% penicillin-streptomycin (all obtained from Invitrogen, Breda, The Netherlands).

The human THP-1 cell line was cultured 1x10 ⁵ cells/well in 96-well plates in the presence of 100nM phorbol 12-myristate 13-acetate (PMA, Sigma) and incubated for 48 hours to induce differentiation of the THP-1 monocytes into M0 macrophages. Cells were washed and incubated for another 72 hours in culture medium. After this the cells were incubated for one hour with the test components (diluted in DMSO), after which the cells were incubated for another 16 hours with LPS (100 ng/ml, Sigma) in the presence of the test components. Supernatants were collected for analysis of TNF-alpha by ELISA assay (Life Technologies).

Metabolic activity of the cells was tested after collection of the supernatant using a WST-1 assay. This colorimetric assay assesses the metabolic activity of the cells. This activity can be used as indication for cell proliferation, cell viability and cytotoxicity. Upon collection of the supernatants for the TNF-alpha detection, the WST-1 reagent was added to 50 mI of cell culture medium which was left behind on the cells. OD450 was measured after 4h of incubation of the WST-1 reagent.

Results

The results are shown in Figures 1 and 2. Figure 1 shows the effects on TNF-a production by macrophages of inventive composition 1 (mix) and the comparative compositions 1-4 (individual components). Figure 1 shows the results of the inhibition studies on LPS-induced TNF-a production by macrophages, and Figure 2 shows the difference in TNF-a production between the cells only and inventive composition 1 (‘MIX’), and between the cells only and the sum of inhibitory effects of compositions 1-4 (‘SUM’).

It can be seen that there is a synergistic effect for the combination of vitamin K2, vitamin D, a xanthophyll and EPA/DHA, since the inhibitory effect by the combination of these components is greater than the sum of the inhibitory effects of each component individually.

Example 2

The following composition was prepared:

MK-7: 180 meg Vit D3: 50 meg Lutein: 10 mg EPA: 200 meg DHA: 80 meg

Table 2: Effects on TNF-a production by macrophages Table 3: Difference in TNF-a production between the cells only and inventive composition 1 (MIX), and between the cells only and the sum of inhibitory effects of comparative compositions 1-4 (SUM).