Field of the invention

The present invention relates to compositions which are beneficial for immune health. In particular, the compositions of the invention comprise vitamin K2 or a prodrug thereof and vitamin D, which act synergistically together to improve immune health. 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 immune conditions.

Background of the invention

Boosting immune health is desirable because of both the emergence of new diseases and the increasing immuno-resistance of known diseases. Immune health products are relevant in the nutraceutical as well as pharmaceutical fields.

There are a number of nutraceutical/pharmaceutical products on the market. Improving the efficacy of these compositions is clearly desirable. The present inventors aim to provide compositions which have a clear beneficial effect on immune health. TNF-alpha is a pleiotropic cytokine and mediates many activities in the body such as modulation of cell recruitment, cell proliferation, cell death and immune regulation. At sites of inflammation, the level of TNF-alpha is elevated, and methods for down regulation of TNF-alpha activity is shown to be beneficial for several conditions like arthritis, Chron’s disease, psoriasis, ulcerative colitis (for example, see Tracey, D., L. Klareskog, E. H. Sasso, J. G. Salfeld and P. P. Tak (2008). "Tumor necrosis factor antagonist mechanisms of action: a comprehensive review." Pharmacol Ther 117(21: 244-279).. The present inventors have surprisingly found that vitamin K2 and vitamin D can act synergistically to inhibit TNF-a. The compositions and products of the present invention therefore have highly desirable potential applications in immune health maintenance/improvement.

Summary of the invention

Viewed from a first aspect, the invention provides a composition comprising (I) vitamin K2 or prodrug thereof;

(II) vitamin D wherein the weight ratio of vitamin K2 or prodrug thereof to vitamin D is at least 44:1.

Viewed from another aspect, the invention provides a composition comprising

(I) MK-7 or prodrug thereof;

(II) vitamin D wherein the weight ratio of MK-7 or prodrug thereof to vitamin D is at least

37:1.

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 in the treatment or prevention of a condition associated with vitamin K deficiencies, such as bone disease, an immune condition, cardiovascular disease, or eye disease, preferably an immune condition.

Viewed from another 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 an immune condition.

Viewed from another aspect, the invention provides a method of preventing or treating bone disease, an immune condition, cardiovascular disease, or eye disease, preferably immune condition, comprising administering to a subject in need thereof an effective amount of a composition as defined herein.

Viewed from another aspect, the invention provides a method of maintaining or improving general immune health, comprising administering to a subject 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 of bone disease, an immune condition, cardiovascular disease, or eye disease, preferably an immune condition

Viewed from another aspect, the invention provides the use of a composition as defined herein as a nutraceutical product, preferably a nutraceutical product to promote immune health. Viewed from another aspect, the invention provides the use of a composition as defined herein for maintaining and/or improving the general immune 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 maintaining and/or improving immune health in particular.

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 improving immune health, 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 (which is preferably MK-7) 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, -COC1-6 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.

As is seen in the data below, vitamin K2, in combination with vitamin D, inhibits TNF-a synergistically.

Since one of the key ingredients is vitamin K2, it is envisaged that the compositions are not only useful in improving/maintaining immune health, but also in preventing or treating conditions associated with vitamin K2 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 K2. For example, it is known that vitamin K2 is relevant for bone health or conditions of the cardiovascular system such as arteriosclerosis. The advantage of using vitamin K2 is therefore two-fold. Firstly, it acts synergistically with vitamin D to maintain and/or improve immune health, as can be seen by TNF-alpha inhibition data. Secondly, the administration of vitamin K has benefits in terms of acting on other organs/biostructures which are affected by or associated with vitamin K2.

The ranges, amounts etc. disclosed herein or in the claims can refer to vitamin K2 or MK-7 (or prodrugs thereof).

In a particular embodiment, the weight ratio of vitamin K2 or prodrug thereof (e.g. MK-7 or prodrug thereof) to vitamin D is at least 37:1 , preferably at least 38:1 , preferably at least 39:1 , preferably at least 40:1 , preferably at least 41 :1 , preferably at least 42:1 , preferably at least 43:1 , preferably at least 44:1 , preferably at least 45:1 , preferably at least 50:1 , preferably at least 51 :1 , preferably at least 70:1 , preferably at least 71 :1 , preferably at least 72:1 , preferably at least 73:1 , preferably at least 74:1 , preferably at least 75:1 , preferably at least 76:1 , 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 a particular embodiment, the weight ratio of vitamin K2 or prodrug thereof (e.g. MK-7 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 a particular embodiment, the weight ratio of vitamin K2 or prodrug thereof (e.g. MK-7 or prodrug thereof) to vitamin D is in the range 37:1 to 50,000:1 , preferably 38:1 to 50,000:1 , preferably 39:1 to 50,000:1 , preferably 40:1 to 50,000:1 , preferably 41 :1 to 50,000:1 , 42:1 to 50,000:1 , preferably 43:1 to 50,000:1 , preferably 44:1 to 50,000:1 , preferably 45:1 to 50,000:1 , preferably 50:1 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 Other suitable ranges for the weight ratio of vitamin K2 or prodrug thereof (e.g. MK-7 or prodrug thereof) to vitamin D include 37:1 to 50,000:1 , preferably 37:1 to 25,000:1 , preferably 37:1 to 10,000:1 , preferably 37:1 to 5,000:1 , preferably 37:1 to 3,500:1 , preferably 37:1 to 2,000:1 , preferably 37:1 to 1 ,000:1 , preferably 37:1 to 500:1 , preferably 37:1 to 250:1 , preferably 37:1 to 250:1. Other suitable ranges include 44:1 to 50,000:1 , preferably 44:1 to 25,000:1 , preferably 44:1 to 10,000:1 , preferably 44:1 to 5,000:1 , preferably 44:1 to 3,500:1 , preferably 44:1 to 2,000:1 , preferably 44:1 to 1 ,000:1 , preferably 44:1 to 500:1 , preferably 44:1 to 250:1 , preferably 44:1 to 100:1 .

Typically, the vitamin K2 or prodrug thereof (e.g. MK-7 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, preferably at least 90 pg. In another embodiment, the vitamin K2 or prodrug thereof is present in the composition of the invention in an amount of at most 50,000 pg, preferably at most 10,000 pg, preferably at most 2000 pg, preferably at most 1000 pg, preferably at most 900 pg, preferably at most 750 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, preferably 90-500 pg, more preferably 90 to 300 pg.

The amounts given herein for vitamin K2 can also be 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).

Vitamin D is known as an immune regulator. It is surprising, however, that the combination of vitamin D with vitamin K2 results in a synergistic inhibition of TNF-alpha.

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 2 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 1000 pg, preferably at most 500 pg, preferably at most 250 pg, preferably at most 100 pg, preferably at most 75 pg, preferably at most 50 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 2 to 250 pg, preferably 5 to 100 pg, preferably 5 to 75 pg, preferably 5 to 50 pg.

The amounts given herein for vitamin D can also be daily dosages.

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

Compositions and applications

The compositions of the invention are particularly suited for improving immune health. 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 an immune condition.

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 immune health. In a particular embodiment, the uses/methods described herein are non-therapeutic.

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.

The composition can also comprise nutrients 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 an immune condition. By immune condition is typically meant a condition selected from type 1 diabetes, rheumathoid arthritis (RA), psoriasis, psoriatic arthritis, multiple sclerosis (MS), Systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison’s disease, Grave’s disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, Myasthenia gravis, Autoimmune vasculitis, Pernicious anemia, Celiac disease.

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. In a particular embodiment, the compositions are not in the form of capsules, e.g. soft-gel capsules.

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. In one embodiment the composition of the invention is microencapsulated, e.g. using techniques described in WO2015/169816.

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 50,000 pg/day is suitable, preferably 1 to 10,000 pg/day, 5 to 5,000 pg/day, preferably 5 to 5,000 pg/day, preferably 10 to 2,000 pg/day, preferably 25 to 1 ,000 pg/day, preferably 50 to 1 ,000 pg/day, preferably 100 to 1 ,000 pg/day, preferably 200 to 1 ,000 pg/day, preferably 240 to 1 ,000 pg/day, based on the combined amount of vitamin K2 and vitamin D.

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)), and for comparative composition 2 (vit D3). 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)-(ll). 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).

Examples

Composition The following compositions were investigated for TNF-a inhibition.

Table 1 : Mixes used for TNF-a inhibition studies. In vitro testing on immune health - TNF- a inhibition studies

TNF-a was selected as a target because of its well-known role in immune health. The toxicity of the compositions were tested by metabolic activity. Immuno-modulating activity of vitamin K2 (MK-7) and the metabolic active version of vitamin D3 (1a,25-(OH)2D3) 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 MO 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 , 2 and Tables 2, 3. Figure 1 and Table 2 show the effects on TNF-a production by macrophages of inventive composition 1 (mix) and the comparative compositions 1-2 (individual components). Figure 1 and Table 2 show the results of the inhibition studies on LPS-induced TNF-a production by macrophages, and Figure 2 and Table 3 show 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 comparative compositions 1 -2 (‘SUM’).

It can be seen that there is a synergistic effect for the combination of vitamin K2 and vitamin D since the inhibitory effect by the combination of these components is greater than the sum of the inhibitory effects of each component individually. Having more vitamin K2 than vitamin D is particularly beneficial.

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 1-2 (‘SUM’)

Example 2

The following composition was prepared: - MK-7: 220-500 meg

- Vit D3: 5-10 meg