ESPECIALLY TO BEIGE ADIPOSE TISSUE

This application relates to vitamin K2 or prodrugs thereof, in particular MK- 7 to enhance metabolism of fat, in particular in the transformation of white adipose tissue to brown or more preferably beige adipose tissue. It is perceived that this transfer will enhance the metabolism of fat and hence reduce the detrimental effects of enhanced fat deposits. The compounds of the invention may also reduce the circulation of unhealthy lipoproteins which are a result of the fat. The compounds of the invention may therefore lead to improvements in the health status of individuals with enhanced visceral adiposity, metabolic syndrome, high blood pressure, type 2 diabetes, or the like.

Background 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-l,4-naphthoquinone derivatives.

Vitamin K is not a single compound, rather it is a series of related analogues. Vitamin Kl is called phylloquinone and has the systematic name all-E-2-methyl-3- (3,7,1 l,15-tetramethylhexadec-2-enyl)naphthalene-l,4-dione. Vitamin K2

(menaquinone) is normally produced by bacteria in the intestines, and dietary deficiency is extremely rare unless the intestines are heavily damaged or are unable to absorb the molecule.

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.

MK-7

All members of the vitamin K group of vitamins share a methylated naphthoquinone ring structure, and vary in the aliphatic side chain attached at the 3- position.

Vitamin K2 has been proposed for use in the treatment of various conditions. There are strong indications that vitamin K2 has a beneficial effect on bone diseases such as osteoporosis. Other authors have also suggested that vitamin K2 has beneficial effects on cardiovascular health (EP1153548). Vitamin K2 may help reduce calcification of blood vessels or assist with arterial elasticity. There is interest therefore in the investigation of various vitamin K2 type compounds for inhibitory effects in a variety of conditions such as osteoporosis, as potential anticancer drugs and for beneficial cardio -vascular activity.

The present invention relates to the use of compounds of the invention in the treatment of conditions related to the deposition of fat. The epidemic of obesity and type 2 diabetes presents a serious challenge to scientific and biomedical

communities worldwide. While this epidemic was first obvious in the United States and other highly developed countries of Europe, it has spread across Asia, Africa, and Oceania.

The most important single idea in the field of metabolic disease is the concept of energy balance. This means that, with the rare exception of

malabsorption of nutrients, an animal cannot gain or lose weight unless there is an imbalance between food intake and energy expenditure. When energy intake chronically exceeds energy expenditure, weight gain and obesity result. This excess weight is stored in adipose tissue, which consists of fat cells, or adipocytes, which have an incredible capacity for storing surplus energy in the form of lipids. This tissue is not just a passive storage depot, but also an endocrine organ, secreting molecules like leptin that can regulate appetite and whole-body metabolism. In addition to these well-described energy-storing fat cells, adipocytes also exist that are highly effective at transforming chemical energy into heat. Brown adipocytes, which get their name from their high number of iron-containing mitochondria, are specialized to dissipate energy in the form of heat,

a process called non-shivering thermogenesis. The thermogenic gene program of classical brown and beige fat cells (those brown cells that can emerge in white fat deposits under certain conditions) can increase whole-body energy expenditure and therefore can protect against obesity and diabetes. This role of brown (and now beige) adipose cells in increasing whole-body metabolic rates has driven much of the interest in these cell types.

The majority of adipose tissue in the body is white adipose tissue (WAT). WAT is mainly involved in energy storage and mobilization. Excess fat

accumulation in WAT, particularly in visceral WAT in the abdomen, is highly associated with metabolic diseases such as type 2 diabetes and cardiovascular complications. Coexisting with WAT, brown adipose tissue (BAT) is specialized in energy expenditure. Brown adipose tissue (BAT) was first described in small mammals and infants as an adaptation to defend against the cold. It was originally referred to as the hibernating organ due to its function in maintaining body temperature in hibernating animals.

Due to the thermogenic properties of BAT, its presence in certain anatomical locations in adult humans suggests a beneficial role in preventing the development of metabolic diseases. "Browning" of WAT has recently been reported to transform the energy storage capability of WAT to BAT-like energy expenditure

characteristics. Adipocytes with browning ability in WAT are so-called beige adipocytes, having characteristics of adipocytes in classic BAT. There are therefore two types of brown adipocytes: classical brown adipocytes and brown adipocyte-like cells, so-called beige/brite cells, which arise in white adipose tissue in response to cold and hormonal stimuli. These cells may derive from distinct origins, and while functionally similar, have different gene signatures.

Thus, one area of relative promise centres on targeting brown adipose tissue

(BAT) to increase energy expenditure. Differing from white adipose tissue (WAT), which stores excess energy from the diet as triglycerides, BAT is a thermogenic organ, metabolizing both fatty acids and glucose to produce heat to maintain homeothermy. The thermogenic activity of BAT is possible due to the presence of a BAT-specific mitochondrial proton transport protein, uncoupling protein 1 (UCP1), which uncouples the mitochondrial proton gradient from ATP synthesis and generates heat. While existence of BAT in infants as well as discrete populations of adult humans had been shown many years ago, recent findings using 18fluoro 2- deoxyglucose positron emission tomography (FDG-PET) verified the presence of metabolically active BAT in human adults. Thus, developing BAT may provide an attractive new target for prevention/ treatment of obesity.

Since the discovery of brown adipose tissue in adult humans, significant scientific efforts are being pursued to identify the molecular mechanisms to promote a phenotypic change of white adipocytes into brown-like cells, a process called "browning". It is well documented that white adipose tissue (WAT) mass and factors released from WAT influence the vascular function and positively correlate with cardiac arrest, stroke, and other cardiovascular complications.

The present inventors have now shown that vitamin K2 is involved in energy metabolism (e.g. carbohydrate and fat metabolism). The inventors also propose that vitamin K2 (e.g. MK-7) is able to influence (or change) the adipocyte phenotype characteristics from so-called white to beige, i.e. MK-7 enables the transition of white adipose cells into beige adipocytes, which are smaller, contain more mitochondria, thus metabolizing more lipids.

Thus, the consequence of Vitamin K2 (MK-7) ingestion on a regular basis may result in an improved day-to-day health status, either preventing detrimental adiposity occurring, or actually normalizing the fat turnover and/or blood lipoprotein profiles in need of corrections or normalization thereof.

The present invention therefore relates to the effect of vitamin K2 (MK-7) on energy metabolism. The benefits of enhanced energy consumption are wide spread. It will be clear to the skilled person that the conversion of white to beige adipose cells leads to health benefits such as reduction of obesity, treatment of diabetes type 2, reduction of high blood pressure, reduction of metabolic syndrome, improved cardiovascular outcomes and general improvement of any condition in which a reduction in fat or an increase in the burning of adipose tissue would provide a health benefit.

Our results demonstrate a physiologically significant role of BAT in whole- body energy expenditure, glucose homeostasis, and insulin sensitivity in humans and support the notion that BAT may function as an anti-diabetic tissue in humans.

There are also desirable health benefits associated with white to brown/beige fat conversion which are independent of health benefits traditionally associated with fat loss. For example, the improvement in glucose homeostasis and insulin sensitivity mentioned earlier suggests that anyone with impaired insulin function might benefit from BAT activation.

However there is a broader application given research showing even mildly elevated blood glucose in healthy non-diabetic humans is associated with damage over time of many organs such as eyes, brain, tendons, endothelial/cardio vascular system and brain, and results in higher levels of damaging advanced glycation end products.

Beige fat has also been associated with higher bone density, suggesting that brown fat interacts with bone growth (American Journal of Physical Anthropology.

156: 98-115, Osteoporosis International. 24 (4): 1513-1518. ). Moreover, BAT activation through cold exposure increases circulating irisin. As well as improving insulin sensitivity and being involved in weight reduction, vitmain K2 may be involved in increasing bone quality and quantity and is involved in the building of lean muscle mass.

It has been found that white fat cells secrete important hormone-like molecules such as leptin, adiponectin, and adipsin to influence processes such as food intake, insulin sensitivity, and insulin secretion. Brown fat, on the other hand, dissipates chemical energy in the form of heat, thereby defending against inter alia hypothermia.

Moreover, adipose tissue is a central metabolic organ in the regulation of whole-body energy homeostasis. The white adipose tissue functions as a key energy reservoir for other organs, whereas the brown adipose tissue accumulates lipids for cold-induced adaptive thermogenesis. Adipose tissues secrete various hormones, cytokines, and metabolites (termed as adipokines) that control systemic energy balance by regulating appetitive signals from the central nerve system as well as metabolic activity in peripheral tissues. The conversion of WAT to BAT cells may reduce appetite. The conversion of white to brown/beige fat is therefore associated with myriad health benefits, some of which are associated with fat loss, some of which are not. The reduction in body fat in a subject generally has beneficial impacts on certain conditions related to unhealthy levels of body fat (cardiovascular disease, diabetes etc), but the general health of a subject would be improved as a result of increased brown to white or beige to white fat cell ratios because of the numerous additional benefits described above. The core of the invention does not lie in the elucidation of the underpinning mechanism for fat loss, but in the surprising observation that vitamin K2 or its prodrugs are effective in the transformation of white adipose tissue to brown or more preferably beige adipose tissue. This has wide ranging health benefits.

The inventors are aware that vitamin K2 has been implicated in all manner of different conditions some of which would benefit from a reduction in fat. As previously noted, vitamin K2 has been suggested for improving cardiovascular health and it could be argued that cardiovascular health is generally improved by a reduction in fat in the body. Cardiovascular health is a totally generic concept however and disclosure of vitamin K2 in cardiovascular health is not in our view relevant to our invention.

Vitamin K2 has been suggested for helping in the treatment of obesity (see US20130267606). There is no discussion of white to beige transformation. Others have linked vitamin K2 and diabetes via increased energy levels (see

WO2010/103545). WO2010/103545 in fact makes a series of far-fetched claims regarding the numerous diseases which it proposes to treat. No one however, has proposed vitamin K2 for the enhancement of the metabolism of fat and shown the conversion of white to beige fat cells as we demonstrate in our examples. No one has suggested vitamin K2 in a process for enhancing energy expenditure, glucose homeostasis and insulin sensitivity. This new mode of action enables all sorts of therapeutic benefits to be realised. The results we present here open the door for a significant general improvement in human health. Summary of Invention

Thus, viewed from one aspect the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in enhancing the metabolism of fat, in particular by the transformation of white adipose tissue to brown adipose tissue, especially to beige adipose tissue.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in the transformation of white adipose tissue to brown adipose tissue, especially to beige adipose tissue.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in the transformation of white adipose tissue to brown adipose tissue, especially to beige adipose tissue so as to treat a condition in which a reduction of fat is beneficial.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in increasing thermogenic tissue in a person.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in the transformation of white adipose tissue to brown adipose tissue, especially to beige adipose tissue to increase thermogenic tissue in a person.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in enhancing energy usage in a person.

Viewed from another aspect, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in the transformation of white adipose tissue to brown adipose tissue, especially to beige adipose tissue to enhance energy usage in a person.

It will be appreciated that the method of the invention opens the door to the improvement of many medical conditions such as type-2 diabetes, overweight, obesity, metabolic syndrome and so on. Viewed from another aspect therefore, the invention provides a composition comprising vitamin K2 or a provitamin thereof for use in the treatment or prevention of overweight, obesity, diabetes mellitus type 2, metabolic syndrome, cardiovascular outcomes and any condition that will benefit from a reduction in white adipose tissue comprising administering to the patient an effective amount of said composition to transform white adipose tissue to brown adipose tissue, especially to beige adipose tissue.

The invention requires administration of an effective amount of the vitamin K2 or a provitamin thereof to a patient in need of the treatment in question, such as a human.

Viewed from another aspect the invention provides a method comprising administering to a human in need thereof, an effective amount of vitamin K2 or a provitamin thereof so as to transform white adipose tissue to brown adipose tissue, especially to beige adipose tissue.

Viewed from another aspect the invention provides a method comprising administering to a human in need thereof, an effective amount of vitamin K2 or a provitamin thereof so as to enhance thermogeneration.

Viewed from another aspect the invention provides use of vitamin K2 or a provitamin thereof in the manufacture of a medicament for transforming white adipose tissue to brown adipose tissue, especially to beige adipose tissue comprising administering to a mammal in need thereof, an effective mount of vitamin K2 or a prodrug thereof.

In a preferred embodiment, the vitamin K2 is administered as MK-4 or more referably MK-7:

Definitions By treating or treatment is meant inhibiting a disease i.e. arresting, reducing or delaying the development of the disease. Vitamin K2 (VK2) is ideally administered to slow down, stop or reverse the patient's condition. It may also be that VK2 can help prevent onset of the condition in question. Most preferably, the VK2 is administered to patients who require white to brown/beige cell transfer.

The term vitamin K2 is used herein to define the use either of naturally occurring vitamin K2 or a synthetic vitamin K2. It will be appreciated that vitamin K2 occurs naturally as a mixture of menaquinone compounds typically having 4 to 8 isoprenoid side chains attached to the napthoquinone ring. A synthetic vitamin K2 compound may be based on a mixture of menaquinones or a single menaquinone such as MK-4 or MK-7. The use of MK-7 is especially preferred as it is perceived to be the most active menaquinone within vitamin K2.

The term the "compound of the invention" is used herein to define vitamin K2 or provitamins thereof.

The invention preferably relates to the administration of natural or synthetic vitamin K2. More preferably, the vitamin K2 is synthetic and made via organic chemistry or made using microorganisms. The vitamin K2 is ideally administered as a single menaquinone or blend of menaquinones. The use of MK-n

menaquinones where n is 2 to 10 is preferred. The subscript n represents the number of repeating isoprenoid units in the isoprenoid side chain of vitamin K2. Preferred menaquinones are MK-4, MK-5, MK-6, MK-7, MK-8 and MK-9, especially MK-4 and most especially MK-7.

Whilst the invention is most conveniently carried out using vitamin K2 itself or a menquinone that forms part of natural vitamin K2, the invention may also be carried out using prodrugs/provitamins. The term prodrug/provitamin is used interchangeably herein to refer to a compound that breaks down to vitamin K2 in vivo. Prodrug compounds of the invention are preferably analogues of MK-6, MK-7 or MK-8. MK-9 is also an option. Most preferably, they are analogues of MK-7. It is thus preferred if the long chain isoprenoid at position 2 on the naphthoquinone ring is

The provitamin compounds can be mono or disubstituted analog

formula (I)

wherein each R is independently hydrogen, a -Ρ^) _γ group wherein y is 2 or 3, -SO ₂ R ⁴ , -COOH, -CO(CH ₂ ) _p Ar, -COOCi_ ₆ alkyl, -CON(R ² ) ₂ , COAr, -COCi_ ₆ alkyl group; -CO(CH ₂ ) _p COOR ³ , CO(CH ₂ ) _p CON(R ² ) ₂ or -CO(CHR ⁶ ) _p N(R ⁵ ) ₂ wherein at least one R group is not hydrogen;

each R ¹ is independently OH, halo, Ci_6-alkyl, OPh, Obenzyl, OCi_6-alkyl or oxo such that the valency of the P atom is 3 or 5;

each R ² group is independently hydrogen or Ci_6-alkyl;

R ³ is H, Ci_ ₆ -alkyl, Ar, or (CH ₂ ) _p Ar;

R ⁴ is OH, Ci_6 alkyl, Ph, CF ₃ , or tolyl;

each R ⁵ is H, an amino protecting group such as Boc, or CI -6 alkyl;

each R ⁶ is H or CI -6 alkyl;

any Ci_6-alkyl group is optionally substituted by one or more groups selected from -OR ² , N(R ² ) ₂ or COOR ² ;

each Ar is an optionally substituted phenyl or naphthyl group, said substituent being a CI -6 alkyl, CHalH ₂ , CHal ₂ H, CHal ₃ , (where Hal is halide), OH, OCi_ ₆ -alkyl, COOR ⁶ ;

each p is 1 to 4;

and n is 2 to 8, such as 3 to 8; or a salt or solvate thereof. Thus, both R groups cannot be hydrogen. Where the prodrug compounds of the invention are monosubstituted, the substituent can be present on either ketone position on the naphthoquinone ring (the 1 or 4 positions, where the 1 -position is adjacent the isoprenoid chain and 4-position adjacent the methyl group). It is preferred however, if the compounds of the invention are disubstituted. In one embodiment of the invention both R groups are acetate (thus forming -OCOCH ₃ at the 1 and 4 position). It is however, also within the scope of the invention for both R groups not to be acetate.

It is within the scope of the invention for the substituent groups R used in a compounds of formula (I) to be the same or different however, it is preferred if these are the same. Bis substituted compounds are generally more stable.

The invention further provides therefore a composition comprising a monosubstituted compound of formula (I) above (i.e. where one R group is H) and an MK-n compound, ideally the composition, e.g. a nutraceutical or pharmaceutical composition, comprises the MK-n compound corresponding to the monosubstituted compound of formula (I). In particular, a composition might comprise MK-7 and a monosubstituted compound of formula (I) where n is 5.

In one embodiment, at least one R is a phosphorus containing -P(R ¹ ) _y group, i.e. such that the O atom bonds to the phosphorus atom. The phosphorus atom can be in its 3 or 5 valency state, preferably the 5 valency state. Where the P is 5-valent, y is 3 and one R ¹ group represents oxo thus forming the P=0 group. A preferred P group is therefore P(0)(R ⁷ ) ₂ wherein each R ⁷ is Ci_6-alkyl, halo, OH, or OCi_ ₆ alkyl. Ideally, this group is PO(OH) ₂ . In another embodiment, it may be

alkyl) ₂ such as P=0(OEt ₂ ).

Where the P atom is in the 3 valent state, y is 2 and R ¹ should not be oxo. R ¹ is preferably OH, Ci_6-alkyl or OCi_ ₆ alkyl. Especially preferably, the 3-valent group is -P(OCi_ ₆ -alkyl) ₂ or P(OH) ₂ .

In an alternative preferred embodiment, the provitamin compounds of the invention are mono or diesters. Preferred ester groups are methyl esters, ethyl esters or phenyl esters. The combination of an ester and a phosphorus containing -P(R ¹ ) _y group is a further preferred option.

A further option is compounds in which R is -CO(CH ₂ ) _p Ar, such as benzyl. A further preferred option is the use of mono or dicarbonates or carbamates, i.e. where in the R group is -COOH or -COOCi_ ₆ alkyl or where in R group is CON(R ² ) ₂ .

A further preferred embodiment is the use of a sulphate or derivative thereof, i.e. where R is S0 ₂ R ⁴ . R ⁴ is preferably OH or represents methyl or tolyl (thus forming mesylate and tosylate).

In any embodiment of the invention the group R ² is preferably hydrogen. Any amino group is therefore preferably NH ₂ .

We have also found that the use of R groups of formula -CO(CHR ⁶ ) _p N(R ⁵ ) ₂ are preferred. R ⁶ is preferably H or a CI -6 alkyl such as CI -4 alkyl group. At least one R ⁵ is preferably H. The other R ⁵ is preferably a protecting group such as Boc (tButyloxycarbonyl). The subscript p is preferably 1 or 2. A preferred group is therefore -CO(CHR ⁶ )i/ ₂ NH(R ⁵ ) where R ⁵ is a protecting group for the amino, e.g. Boc and R ⁶ is H or a CI -6 alkyl group.

The use of -CO(CH ₂ ) _p COOR ³ is a further preferred option, especially where

R ³ is H. The subscript p may preferably be 1-3 in this embodiment.

Ar is preferably Ph or 4-CF ₃ -PI1-.

Where the provitamin compounds of the invention comprise an alkyl chain, e.g. as part of the ester or as part of an amino group, this alkyl chain may contain a substituent selected from -OR ² , N(R ² ) ₂ , or COOR ² . This substituent therefore provides polarity to the molecule and aids its dissolution in the body. If present, preferably one such group should be present. Preferably, that group should be OH. Preferably no such substituent is present.

In a further preferred embodiment however, the provitamin compounds of the invention comprise at least one ester OCO- at the OR position. Preferred compounds are of formula (la) wherein each R is independently hydrogen, a -P(R ) _y group wherein y is 2 or 3, COAr, -CO(CH ₂ ) _p Ar, -COCi_ ₆ alkyl group; -CO(CH ₂ ) _p COOH; or - CO(CHR ⁶ ) _p N(R ⁵ ) ₂ wherein at least one R group is not hydrogen and preferably R groups are the same;

each R ¹ is independently OH, halo, Ci_6-alkyl, OPh, Obenzyl, OCi_6-alkyl or oxo such that the valency of the P atom is 3 or 5;

each R ² group is independently hydrogen or Ci_ ₆ -alkyl;

each R ⁵ is H, an amino protecting group such as Boc, or CI -6 alkyl;

each R ⁶ is H or CI -6 alkyl;

any Ci_6-alkyl group is optionally substituted by one or more groups selected from -OR ² , N(R ² ) ₂ or COOR ² ;

each Ar is an optionally substituted phenyl or naphthyl group, said substituent being a Ci_ ₆ alkyl CHalH ₂ , CHal ₂ H, CHal ₃ , OH, OCi_ ₆ -alkyl, COOR ⁶ ; each p is 1 to 4;

and n is 4 to 7; or a salt or solvate thereof. Hal is halide, preferably CI or F. Preferred provitamin compounds of the invention are of formula (II) to (IV):

ĨIV) where n is 3 to 8, such as 4 to 7, preferably 4 to 6;

or the monosubstituted analogues of these compounds.

Further preferred provitamin compounds are those of formula (V)

wherein both R groups are COCH ₂ Ar, COAr or -COCi_ ₆ alkyl group;

each Ar is an optionally substituted phenyl or naphthyl group, said substitutent being a CI -6 alkyl, CHalH ₂ , CHal ₂ H, CHal ₃ , OH, OCl-6-alkyl, COOR ⁶ R ⁶ is H or CI -6 alkyl;

and n is 4 to 7; or a salt or solvate thereof.

The use of a diethyl ester where the R groups are COCH ₂ CH ₃ is highly preferred.

It has surprisingly been found that the provitamin compounds of the invention have a much longer shelf life than their corresponding diketone vitamin K2 analogues. Without wishing to be limited by theory, it is envisaged that the claimed compounds are less susceptible to oxidation.

It is important however, that the OR group is capable of hydrolysis and oxidation within the body to yield the native MK-n analogue and hence vitamin K2 type structure. The claimed structures are all based on readily hydrolysable ester type linkages.

The compounds (II) and (IV) above, and in particular the compound

(X) or

(XI)

where n is where n is 3 to 8, such as 4 to 7, preferably 4 to 6; especially 5 are interesting.

It is important to note that vitamin K2 and especially MK-7 passes the blood brain barrier making these products ideally suited for use in the treatment of the mental health conditions discussed herein.

Synthesis Vitamin K2 or menaquinones such as MK-7 can be purchased commercially from suppliers. MK-7 is available from Kappa Bioscience and a synthesis is described in WO2010/035000. The techniques therein can be adapted to synthesis a range of MK-n analogues.

The prodrug compounds of the invention can be synthesized from the corresponding menaquinone compound, e.g. MK-7. Naturally occurring vitamin K2 could also be used here. It will be appreciated therefore that the starting

menaquinone reactant might contain a mixture of different MK-n compounds (where n is the chain length). Naturally occurring vitamin K2 is formed from chains of differing lengths.

WO2013/128037 describes suitable synthetic protocols for the prodrugs of the invention.

The compounds of the invention may also be administered as salts. Salts of the compounds of the invention are those wherein the counterion is

pharmaceutically acceptable. However, salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are included within the ambit of the present invention.

The pharmaceutically acceptable salts are defined to comprise the therapeutically active non-toxic acid addition salt forms that the compounds according to formula (I) are able to form. Said salts can be obtained by treating the base form of the compounds according to formula (I) with appropriate acids, for example inorganic acids, for example hydrohalic acid, in particular hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid and phosphoric acid ; organic acids, for example acetic acid, hydroxyacetic acid, propanoic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid and pamoic acid. Conversely said acid salt forms can be converted into the free base form by treatment with an appropriate base.

The compounds according to the invention containing acidic protons may also be converted into their therapeutically active non-toxic base salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkaline and earth alkaline metal salts, in particular lithium, sodium, potassium, magnesium and calcium salts, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hybramine salts, and salts with amino acids, for example arginine and lysine.

Conversely, said base salt forms can be converted into the free acid forms by treatment with an appropriate acid.

It will of course be possible to use a mixture of compounds of the invention.

Compositions

The compositions of the invention may be pharmaceuticals or nutraceuticals. They will be safe for administration to a mammal such as a human. Compositions of the invention may comprise 0.0001 to 10 wt% of the compound of the invention, such as 0.001 to 1 wt% of the compound, or 0.01 to 1 wt%. Typically, there is 10 to 2000 microg, such as 10 to 500 microg of the compound of the invention, preferably 25 to 200 microg in the composition of the invention, especially 50 to 150 microg. However, much higher dosages of the compounds of the invention can also be used. For example, dosages of 1 mg to 500 mg, such as 5 to 200 mg are also possible.

It is also worth noting that the active ingredients proposed herein, in particular MK-7, are not toxic and can be taken safely in high dosages.

In particular, the compositions of the invention can be administered either solely or as a plurality of dosage forms so as to deliver a dosage of 10 to 2000 microg per day of the vitamin K2 or provitamin therefore, such as 50 to 1000 microg per day, especially 50 to 500 microg/day.

In some embodiment however larger doses might be desired to increase the clinical effect. The compositions of the invention can be administered either solely or as a plurality of dosage forms so as to deliver a dosage of 2 to 1000 mg per day of the vitamin K2 or provitamin therefore, such as 5 to 500 mg per day, especially 10 to 250 mg/day.

The compounds of the invention can be combined in any composition of the invention with other active ingredients. For example, the compositions may contain at least one Li, Na, Mg, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, or Se salt.

Preferably the salt is a calcium or magnesium salt. A mixture of salts may also be used.

The salt can preferably be any pharmaceutically acceptable salt such as a halide, nitrate, stearate, sulphate, carbonate, glycerophosphate, hydrogencarbonate, dihydro- or anhydro- phosphate.

In a most preferred embodiment, the salt is a calcium salt, ideally calcium carbonate or calcium phosphate. The use of a composition containing Ca ions and the vitamin K2 is preferred, especially one based on Ca ions, P ions and vitamin K2.

Other active ingredients of interest include other vitamins such as vitamin Kl and in particular vitamin D.

It will be appreciated that pharmaceutical compositions for use in accordance with the present invention may be in any suitable form but ideally, they are for oral administration, ideally in the form of tablets.

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

In one embodiment, the compound of the invention is freeze dried to form particles and then coated, e.g. spray coated, to form a composition of use in the invention. This improves the stability of the compound of the invention.

Examples of pharmaceutically 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, micro crystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminium silicates and crosslinked polyvinylpyrrolidone.

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

Examples of pharmaceutically acceptable fillers for oral compositions (other than the calcium salts required herein) include, but are not limited to, lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose).

Examples of pharmaceutically acceptable lubricants useful in the

compositions of the invention include, but are not limited to, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulfate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide.

Examples of suitable pharmaceutically 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 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 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 and dibasic sodium phosphate.

Suitable examples of pharmaceutically acceptable surfactants include, but are not limited to, sodium lauryl sulfate and polysorbates.

Suitable examples of pharmaceutically 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 stabilizers and

antioxidants include, but are not limited to, ethylenediaminetetriacetic acid (EDTA), thiourea, tocopherol and butyl hydroxyanisole.

The compounds of the invention may be taken/applied once a day, twice a day, more often or less often depending on the purpose of administration, preferably once a day. It is particularly preferred that MK-7 can be administered once a day whereas analogues of other menaquinones such as MK-4 cannot.

Conditions

The present inventors have appreciated that the compounds of the invention can be used to convert white adipose tissue into brown adipose tissue, especially beige adipose fat. As brown/beige adipose tissue is burnt more rapidly by the body than white adipose tissue, this conversion leads to an overall reduction in the fat deposits in an individual. Thus, the invention relates to the metabolism of fat in a person.

Specifically, the conversion WAT to BAT leads to an increase in beige fat. The invention enables a reduction in WAT levels in a patient relative to those that would have been observed without vitamin K2 supplementation.

In general therefore, the invention relates to an increase in the metabolism of fat (as WAT) or a reduction in the deposition of WAT and the improvements in health which result from that change. Health improvements owing to a reduction in body fat will be clear to the physician.

For example, conversion of white to brown/beige fat allows adaptive thermogenesis, inducible thermogenesis, reduction in fat cell side and lipid storage capacity, enhanced fatty acid turnover, diminuition of lipid droplet number and size, epigenetically inducible fat turnover.

Clinically therefore change leads to reduced lipid/fat storage capacity, enhanced/induced fat burning capacity, health promoting fat/lipid storage and turnover, reduction in adipose tissue size, enhanced lipid turnover and fatty acid metabolism.

We perceive that the administration of the compounds of the invention may help reduce blood pressure. The combination of high blood pressure, overweight and diabetes is called metabolic syndrome and hence the compounds of the invention may target metabolic syndrome as well.

We perceive that the compounds of the invention can reduce visceral adiposity in a person.

We suggest that the compounds of the invention may help with conditions such as insulin sensitivity, glucose homeostatic and diabetes.

We suggest that the compounds of the invention may help with increasing bone density.

We believe that the newly formed cell phenotype displays altered

metabolism via enhanced fatty acid turnover. Without wishing to be limited by theory, the inventors postulate the following mechanism of action. The liver provides lipid-derived energy sources to the body. Hepatic lipid metabolism is under the tight control of balance between insulin, glucagon and nutritional conditions. The inventors previously found that treatment with PCN decreased the mRNA levels of carnitine palmitoyltransferase 1 a (Cptla) and 3-hydroxy-3-methylglutarate-CoA synthase 2 (Hmgcs2) in livers of starved wild-type mice, but not in those of PXR- KO mice (Nakamura et al., 2007). CPT la plays an essential role in the overall mitochondrial β-oxidation by mediating the transport of long-chain fatty acids to mitochondria (Louet et al., 2001). The mitochondrial enzyme HMGCS2 catalyzes the first reaction of ketogenesis (Hegardt, 1999). Moreover, the activation of PXR by PCN is found to increase the mRNA level of stearoyl-CoA desaturase 1 (Scdl) only in livers of starved wild-type mice. SCD1, a key enzyme in hepatic lipogenesis, catalyzes the rate-limiting step in the synthesis of unsaturated fatty acids (Flowers & Ntambi, 2008). The blood levels of 3-hydroxybutylate were decreased whereas the hepatic level of triglyceride (TG) was increased by PCN treatment in wild-type mice under the assay conditions. Interestingly, neither TG nor cholesterol levels in blood changed in those mice in spite of a significantly elevated hepatic TG accumulation. Thus, in survival during fasting, PXR is thought to regulate hepatic lipid metabolism through repressing β-oxidation and ketogenesis and inducing lipogenesis at transcription levels in a similar manner as insulin dose . In short, as hepatic lipid homeostasis is tightly maintained by the balance between uptake, lipogenesis, β- oxidation and secretion, PXR seems to disrupt it and cause hepatic steatosis characterized by a marked accumulation of TG in mouse liver.

The Akt-regulated forkhead transcription factor FoxA2 (forkhead box A2), a well-documented mediator of insulin-dependent regulation of β-oxidation and ketogenesis, activates the expression of both CPTla and HMGCS2 gene (Wolfrum et al, 2003, 2004). Insulin activates the PI3K/Akt signaling pathway to

phosphorylate FoxA2. Phosphorylated FoxA2 is translocated from the nucleus to cytosol and is deactivated, thereby resulting in down-regulation of both genes. Our group has described the direct interaction between PXR and FoxA2 as an underlying mechanism by which PXR represses the transcription of Cptla and Hmgcs2 genes in mouse liver (Nakamura et al., 2007). Mechanistically, the PXR LBD and the DNA- binding domain of FoxA2 seem to determine the interaction, and ligand-activated PXR interferes with the FoxA2 recruitment into the promoter regions of the Cptla and Hmgcs2 genes in the liver of starved mice . However, the molecular mechanism of the PXR-mediated activation of Scdl gene remains to be determined. Consistent with these findings, Zhou et al. (2006b) have also reported a significant increase in hepatic TG level as well as up-regulation of lipogenic genes including Scdl, cluster of differentiation 36 (Cd36) and long-chain free fatty acid elongase (Fae) in transgenic mice expressing VP-hPXR. The hepatic TG levels in these transgenic mice were nearly 20 times higher than those in the wild-type mice were. CD36 is a key protein involved in the uptake of long-chain fatty acids across the plasma membrane (Ibrahimi & Abumrad, 2002). FAE is associated with insulin resistance in fatty livers, catalyzing the elongation of long-chain saturated and unsaturated fatty acids (Matsuzaka et al, 2007)."

Various transcription factors and co-regulators have been identified as regulators in hepatic lipid metabolism, including peroxisome proliferator-activated receptors (PPARs), liver X receptor a (LXRa) and sterol regulatory element-binding proteins (SREBPs) (Weickert & Pfeiffer, 2006). In particular, SREBPlc, a dominant regulator of hepatic lipogenesis, is expressed under the control of LXRa and mediates the insulin- and fatty acids-dependent responses of lipogenic genes such as fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), SCD1 and FAE. PXR is suggested to up-regulate hepatic lipogenesis independent of SREBPlc action and is not associated with the expression of both Fas and Accl genes. Among those lipogenic genes, Cd36 is characterized as a direct target of PXR in mouse liver . Upon ligand stimulation, the receptor is recruited to a DR3-type PXR response element in its promoter region in mouse liver (Zhou et al, 2006b). Moreover, PXR is also suggested to be associated with up-regulation of Pparg gene, a potent regulator of lipogenic enzymes (Zhou et al, 2006b). Such a cross-talk among nuclear receptors could have a significant impact on lipid homeostasis. Roth et al. (2008), in an independent study, have described an opposite phenomenon in which hepatic TG levels decreased as an acute response to PXR activation by ligand in mice. They found that PXR activation leads to a decrease in active SREBPlc protein levels by inducing INSIG1, which blocks proteolytic activation of SREBPlc by signals such as sterol depletion and insulin. Therefore, the effect of PXR on lipid metabolism seems complicated and strongly dependent on the study conditions applied. Taken together, current findings have demonstrated the complex roles of PXR in energy homeostasis. Again, we have revealed that PXR suppresses gluconeogenesis, glyconeolysis, fatty acid β-oxidation and ketogenesis, and promotes lipogenesis through direct gene regulation or through cross-talk with insulin/glucagon-responsive transcription regulators. Patient Group

The patient or person on which the invention is carried out may be one with a body mass index of at least 25, such as at least 30.

The invention may be carried out on male patients with a WAT level of 20% or more or females with a WAT level of 25% or more.

In view of the general health benefits above, it is envisaged that

supplementation has value in patients with a normal weight, such as a BMI of 20 to 25. The invention relates therefore to the administration of the claimed composition to non overweight patients. The use of the supplement may be enhanced by the patient exercising to encourage brown cell metabolism.

The invention will now be described with reference to the following non limiting example and figures. Brief Description of the Figures

Figure 1 shows the cells tested in Example 1. Figure 2 depicts a putative working model showing how vitamin K2 may affect the hormonal signalling systems and transcription factors responsible for the transition of "white" adipose tissue adipocytes to "beige" adipocytes, thus blocking fat deposition and enhancing the production of heat from fatty acids. Example 1

Human adipose stem cells (hASCs) and [R179E]Gi2a-3T3-Ll mouse preadipocytes were differentiated in a standard liquid medium containing insulin and IBMX for 14 days. Thereafter, the monolayer cultures were exposed to 10 μΜ MK-7 for another 10 days. At the end of the second incubation period, they were colored with Oil-red- O using standard methods. This experiment indicates that both cell lines were differentiated into fat-depositing adipocytes, and that the addition of MK-7 into the growth medium reduced the ability of both cell lines to produce and store triglycerides. Results are presented in figure 1. The reduction in the ability of the cells to store triglycerides implies that MK-7 can be used to reduce fat deposition.

Example 2- Q-PCR of bio markers distinguishing between white and beige adipocytes. The data is presented in the table below as percentage change compared to control levels (100%) and shows the following: the first seven parameters ^-adrenoceptors, transcription factor FoxC2, PGCl = peroxisome proliferator-activator receptor, PPARa and γ (transcription factors), Dio2 = deiodinase, UCP1 = uncoupling protein 1), are upregulated by MK-7, while the last two parameters (Adipoq & Leptin = hormones secreted from adipocytes) were reduced in the presence of MK-7. This experiment indicates that MK-7 upregulates the expression of genes normally associated with brown adipose tissue, while downregulating expression of genes associated with white adipose tissue, i.e. the emerging adipose cell phenotype may be defined as "beige" adipocytes with an enhanced ability to metabolize fatty acids, instead of storing them as triglycerides in the cytosol. Table 1 contains a complete breakdown of the results.

Q-PCR of biomarkers differentiating between white end

brown/beige types of fat cells/tissues

Hyman adipose stem cells or 3T3-L1 cells with activating mutations in the 6-pretein &k, mere differentiated towards adipocytes in the absence or presence of vitamin K2.

Isolated mRNA levels were analysed by Q-PCR, and presented as percent ratio between cells grown in control medium or medium supplemented with MK-7,

Table 1 Example 3

Table 3 shows dose-response values of the concentration yielding half-maximal concentration (ED50) of the effect of MK-7 on selected parameters characterizing brown and white adipocytes (UCP1 and Leptin, respectively). The ED50-value is estimated to be approximately 0.8 (0.4 - 1.0) μΜ for both parameters. It is inferred that this is also the case for the other parameters characterizing the white vs the brown adipocyte phenotype. Results are presented in table 2 below. Dose-response curves for MK-7 ^* s impact on U P1 and Leptin in Human adipose stew cells (hAS s) end gene manipulated 3T3-L1 preadipocytes

Human ASCs or 3T3-L1 cells with activating mutations in the 6-protein Si _a were di ferentiated towards adipocytes in the absence or presence of varying doses of vitamin K2. Isolated mRNA levels were analysed b Q-PCR, and presented as percent ratio between cells grown in control medium or medium supplemented with MK-7.

Table 2

Example 4. Analyses of bio markers related to enzyme activities responsible for the steady state level of triglycerides in human adipose stem cells and the gene- manipulated 3T3-L1 cells as a function of exposure to MK-7. This experiment indicates that the enzymes responsible for modulating total triglyceride contents are affected in such a direction by the presence of MK-7 that the TG contents is driven to a minimum. Table 3 contains the results.

Analyses of biomarkers related to the steady state contents of triglycerides in brown/beige types of fat cells/tissues

Human adipose stem cells or 3T3-L1 cells with en activating mutation in the S-protein &i _m were differentiated towards adipocytes in the absence or presence of vitamin K2, Isolated mRNA levels of PalCoA and HSL were analysed by Q-PCR, while TRC were measured as nmof/pg DNA Figures represent percent ratios between cells grown in control medium or medium supplemented with MK-7.

Table 3

Example 5 Analyses of microR A species miR-133 and miR-155) deemed to be targeting two important markers (PRDM15 and C/ΕΒΡβ) for brown adipose tissue cells. The cell culture set up was performed as previously described, and mRNAs for both the miRNA species and their most important targets within the adipocytes were analyzed using Q-PCR. The present experiment shows that the two important microRNA species regulating the adipocyte markers in question were down- regulated to allow an up-regulation of the two brown adipocyte markers. This experiment shows that MK-7 is able to interfere with the epigenetic machinery, with its strong regulatory function on adipocyte differentiation and adipocyte

"phenotypic" characters in such a way that white adipocytes are "converted" beige adipocytes. Table 4 shows the results. Analyses of iRNAs and targets related to the steady state contents of triglycerides in brown/beige types of fat cells/tissues

Human adipose stem ceils or 3T3-L1 cells with en activating mutation in the S-pretein Si» were differentiated towards adipocytes in the absence or presence of vitamin K2. isolated mftNA levels of miR-133, and miR-155, as well as their targets PRDM16 and C/EBPfc, were analysed by Q-PCR. Figures represent percent ratios between cells grown in

control medium or medium supplemented with MK-7.

Table 4 Example 6

A putative model for the interference of vitamin K2 (MK-7) with the adaptation of white adipocytes to beige adipocytes (burning more and storing less fat). Vitamin K2 (MK4/7), binding to the transcription factor PXR (also known as SXR) to affect bioactive molecules leading to the production of heat from fatty acids,

concomitantly with a reduction in the storage of energy as triacylglycerol = fat. The influence of vitamin K2 on is ultimately involved in the activation of the adrenergic system, transcription factors and enzymes converging towards the upregulation of UCPl (uncoupling protein 1), which favors heat production from fatty acids instead of storing as triacylglycerols. Finally, it should be mentioned that bioinformatics emulation of the interaction between microRNAs as epigenetic stabilizers, using the Mir@nt@n computer program, indicates that there is a reciprocal regulatory loop involving hsa-Mir-155 and the transcription factor C/ΕΒΡβ. The existence of this loop indicates that, once it is in operation, it will stabilize a certain cell phenotype. The model is shown in figure 2. It seems therefore that the exposure of stem cells or pre-adipocytes to vitamin K2 (MK-7), after having been differentiated into white adipocytes, will alter their adipocyte phenotype to a more brown-like ("beige") adipocyte, which clearly favors fatty acid metabolism over triglyceride production and/or storage.