GB2370503A

GB2370503A - Compositions comprising vitamin K

Info

Publication number: GB2370503A
Application number: GB0031693A
Authority: GB
Inventors: Diane Clayton; Cees Vermeer
Original assignee: Novartis Nutrition AG
Current assignee: Novartis AG
Priority date: 2000-12-27
Filing date: 2000-12-27
Publication date: 2002-07-03
Also published as: GB0031693D0

Abstract

Use of vitamin K in the preparation of a composition for counteracting a vitamin K-deficiency in bone of a healthy child and/or adolescent, for increasing peak bone mass or decreasing bone fracture risk. Vitamin K serves as a cofactor for posttranslational carboxylation of glutamate residues into q -carboxyglutamate (Gla), in proteins such as osteocalcin, produced in bone tissue. Despite having serum vitamin K level in the same range as healthy adults, non-supplemented healthy children and adolescents show substantially undercarboxylated osteocalcin, i.e. unexpected vitamin K-deficiency in bone. It has been shown that supplementation of vitamin K is effective to increase carboxylated osteocalcin. Vitamin K refers in particular to phylloquinones, dihydrophylloquinones, and menaquinones, and/or their pharmaceutically or nutritionally acceptable salts.

Description

Organic Compounds The present invention concerns the use of vitamin K to supplement vitamin K deficiency in bone of healthy children and adolescents.

The most immediate health issue relating to vitamin K concerns the risks and benefits of neonatal vitamin K prophylaxis. In the neonate, in whom bleeding due to spontaneous vitamin K deficiency is a well-known hazard, routine vitamin K prophylaxis was introduced in the 1950s. Another line of vitamin K research has been the discovery of a diverse group of proteins dependent on vitamin K, which are implicated in calcium homeostasis. Their discovery closely followed the elucidation of the role of vitamin K in promoting the conversion of protein-bound glutamate residues to y-carboxyglutamate (Gla). Vitamin K serves as a cofactor for y-glutamylcarboxylase, an endoplasmic enzyme involved in the posttranslational carboxylation of glutamate residues into y-carboxyglutamate. The resulting Gia residues are found in a limited number of proteins, and in these proteins only at certain well-defined positions. Gla-containing proteins are now known to occur in a wide variety of tissues such as bone, liver, kidney, placenta, pancreas, spleen and lungs. In most tissues these proteins have not yet been fully characterized. In bone three Gla proteins are presently known: osteocalcin, matrix Gla protein (MGP) and protein S. All three bone Gia proteins are synthesized by the osteoblasts (the bone forming cells), but only osteocalcin is a unique product of bone tissue. Protein S and MGP are also synthesized in a number of soft tissues including the vessel wall. Osteocalcin forms about 20% of the noncollagenous proteins in bone and is one of the most abundant proteins in humans. After its synthesis it is secreted by the osteoblasts and a major part is directly bound to the hydroxyapatite matrix of the bone. About 25% of the de novo synthesized osteocalcin, however, is set free in the circulation where it can be detected with commercially available test kits. So the serum concentration of osteocalcin forms a reflection of the osteoblast activity, which makes it an important and widely used marker for bone formation. During episodes of poor vitamin Kstatus, tissues produce undercarboxylated Gla-proteins. In this way it may be concluded that the occurrence of undercarboxylated prothrombin (PIVKA-II) is indicative for a poor vitamin K status of the liver, whereas undercarboxylated osteocalcin (ucOC) is a measure for the vitamin K status of bone.

From a number of studies it is known that among all Gla-proteins known, osteocalcin is the most sensitive for low vitamin K intake and poor vitamin K status. Consequently, a significant fraction (about 20%) of circulating osteocalcin occurs in the undercarboxylated state in healthy adults. Increased vitamin K intake results in a decrease of the fraction of ucOC. Gla residues form calcium-binding groups in proteins, so the main physicochemical difference between normal and undercarboxylated proteins is their large difference in both binding of calcium from solution and the adsorption of these proteins to insoluble calcium salts.

Osteocalcin may occur in a series of related forms: 3-Gla (= fully carboxylated), 2-Gla, 1-Gla, and 0-Gla. All forms with less than 3 Gla-residues are called undercarboxylated. The most sensitive test for bone vitamin K status is a recently developed set of kits, one exclusively

detecting O-Gla osteocalcin (0-Gla-OC), the other one 3-Gla osteocalcin (3-Gla-OC). The ratio between both values (0-Gla-OC/3-Gla-OC) is used as a measure to estimate the vitamin K status of bone. The major part of osteocalcin occurs in the 2-Gla-form, and this form is not detected by either assay. Therefore, also the total osteocalcin should be measured using an independent test kit.

Booth SL et al. (1999), J Am Diet Assoc; 99 (9): 1072-6 estimated the dietary intakes of vitamin K (phylloquinone) in a representative sample of the American population using 14

day food diaries and found that in men and women in the 18-to 44-year-old groups mean phylloquinone intakes were below the current Recommended Daily Allowance (current RDA = 1) J. g/kg body weight/day). The intake of phylloquinone was highest in children aged 2-to 5years, still above the current RDA for children aged 6-to 12-years and at the current RDA for children aged 13-to 17-years.

In postmenopausal women elevated ucOC levels and low circulating vitamin K levels (phylloquinone and menaquinone) were demonstrated to be associated with low bone mass and increased fracture risk. Children suffering from cystic fibrosis show a poor absorption of fat-soluble vitamins. This is also seen in Example 1 below, where children with cystic fibrosis show lower circulating vitamin K levels. At 20 years of age cystic fibrosis children are reported to be at high risk for bone fractures.

The present inventors have now found that non-supplemented healthy children and adolescents show an unexpected vitamin K-deficiency in bone, as evidenced by a higher 0 Gla-OC to 3-Gla-OC ratio than seen in healthy adults. In other words all healthy children and adolescents appear to have substantially undercarboxylated osteocalcin despite having serum vitamin K levels in the same range as healthy adults.

Therefore, the present invention provides the use of vitamin K in the preparation of a composition for counteracting a vitamin K-deficiency in bone of a healthy child and/or adolescent.

Also provided is the use of vitamin K to counteract vitamin-K deficiency in bone of a healthy child and/or adolescent.

Further provided is a method for counteracting vitamin K-deficiency in bone of a healthy child and/or adolescent, comprising the administration of an effective amount of vitamin K to counteract the vitamin K-deficiency in bone to a child and/or adolescent in need of such treatment.

By the term"child and/or adolescent", as defined herein, infants up to the age of 6 months are excluded. Preferably the term refers to children or adolescents aged 1 to 18, particularly preferred to children or adolescents aged 3 to 16.

The vitamin K-deficit in bone is counteracted when the ratio of O-Gla-OC/3-Gla-OC in serum is within the normal range seen in healthy adults. The normal range of this ratio in serum usually seen in healthy adults lies within 0.1-0. 8. Effective amounts to obtain this normalization of vitamin K status in the bone in healthy children and/or adolescents will vary depending on the degree of deficiency observed in a particular child or adolescent but will usually lie in the range of 0.5 to 50 jg/kg body weight/day, preferably 0.75 to 25 g/kg body weight/day, more preferred 1 to 15 jug/kg body weight/day.

Also provided according to the present invention is the use of vitamin K in the preparation of a composition for a child and/or adolescent for increasing the peak bone mass.

Without wanting to be bound by theory, it is believed that the mechanism by which vitamin K supplementation increases peak bone mass is the following: Two of the bone Gia proteins, osteocalcin and MGP, are not optimally functional in case of vitamin K deficiency. As they are involved in the deposition of calcium in the bone (noncarboxylated osteocalcin does not bind to bone hydroxyapatite), less bone will be accreted in a vitamin K deficient state. In a specific animal model for vitamin K deficiency (Pastoureau et al., J. Bone Miner Res 1993,8 (12) : 1417-26) the bone of growing lambs was examined and a decrease in bone formation in growing animals was found. Growing children normally have a high bone-turn-over with formation being higher than resorption, this results in a positive net accumulation of bone. Increasing peak bone mass is one of the ways to prevent the occurrence of osteoporosis in later life.

The invention provides furthermore the use of vitamin K in the preparation of a composition for a child and/or adolescent for decreasing bone fracture risk.

As set out above, a high serum ucOC concentration has been associated with skeletal turnover, low bone mineral densitiy, and an increased risk of osteoporotic fracture.

Additionally, clinical use of vitamin K antagonists as anticoagulants has been related to low bone mineral density and increased risk of fracture. There observations imply that vitamin K insufficiency contributes to increased fracture risk. By reverse analogy supplementation of a vitamin K deficiency in bone will decrease the fracture risk.

Also provided is the use of vitamin K for increasing the level of osteocalcin carboxylation in a healthy child and/or adolescent from a low level to a normal level.

Low level of osteocalcin carboxylation typically means a ratio of O-Gla-OC/3-Gla-OC of > 0.8, normal level means ratio of O-Gla-OC/3-Gla-OC of < 0.8.

Preferably carboxylation is increased such by vitamin K supplementation that a ratio of 0 Gla-OC/3-Gla-OC of 0. 4 is obtained. More preferred the ratio to be obtained is : 50. 3, particularly preferred it is : 50. 2.

Vitamin K as used herein refers to one or more compounds of Formula 1, and/or their pharmaceutically or nutritionally acceptable salts,

wherein R is hydrogen, a substituted or unsubstituted straight-or branched chain Ci to C30 alkyl or alkenyl group, an aliphatic or aromatic substituted or unsubstituted straight or branched Ci to Cio thio alkyl or alkenyl group, an aliphatic or aromatic substituted or unsubstituted straight or branched Ci to Cio mercapto alkyl or alkenyl group, dithiothreitol or dithioerythritol or a sulphate containing group. The alkyl or alkenyl groups may be substituted by one or more of the following groups: hydroxy, amino, carboxy and methyl groups.

Preferably vitamin K refers to one or more compounds of Formula 1', and/or their

pharmaceutically or nutritionally acceptable salts, Formula l'

in which n is an integer from 1 to 12 ; and in which the broken lines indicate the optional presence of a double bond.

Vitamin K as used herein refers in particular to phylloquinone (also known as vitamin Ksi), dihydrophylloquinone ; menaquinone-4 (MK-4) and the long chain menaquinones. It is generally accepted that the naphtoquinone is the functional group, so that the mechanism of action is similar for all K vitamins. Differences may be expected, however, with respect to intestinal absorption, transport, tissue distribution, and bioavailability. For use in the present invention, phylloquinone and MK-4 are preferred, phylloquinone is particularly preferred.

Sources of vitamin K which can be used according to the present invention include the following : phyolloquinone from natural sources such as vegetable extracts, fats and oils, synthetic phylloquinone, synthetic vitamin K3 (menadione), different forms of vitamin K2: synthetic MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11, MK-12 and MK-13, natto (food prepared from fermented soy-bean, rich in MK-7), and other fermented foods or dairy products.

Healthy children as referred to in the context of the present invention means children who are not suffering from malabsorption syndromes, such as cystic fibrosis, sprue, celiac disease, ulcerative cholitis, regional ileitis, ascaris infection, short bowel syndrome or cholestasis, or from metabolic diseases, such as phenylketonuria or galactosemia, or children suffering from liver disease, or children who are on antibiotics for prolonged periods of time.

Preferably,"healthy children", as used herein, furthermore refers to children who have normal serum vitamin K concentrations in the range of 150-1500 nanogram/L, preferably 400-1000 nanogram/L.

Achieving a higher peak bone mass is likely to reduce the risk of osteoporotic fracture in later life. Since peak bone mass is achieved by late adolescence or early adulthood, intervention in childhood and adolescence may be important. Several studies suggest that exercise in prepubertal children and dietary calcium supplementation has positive effects on bone density. However, to our knowledge vitamin K supplementation of healthy children has never been suggested in connection with increasing peak bone mass.

The compositions for administering the vitamin K according to the present invention preferably additionally contain one or more of the following active ingredients: vitamin D, calcium, magnesium, zinc, CPP and prebiotics. Typical daily dosages of these are 0-50 pg/day for vitamin D, 0-2.5 g/day for calcium, 0-350 mg/day for magnesium, 0-15 mg/day for zinc, 0-5 g/day for CPP and 0-15 g/day for prebiotics. CPP (casein phosphopeptide) is obtainable from milk.

Compositions suitable for incorporating the vitamin K to be administered according to the present invention include pharmaceutical compositions, dietary supplements, nutritional compositions such as medical nutrition, functional food or weaning food, which in addition to the active ingredient (s) contain at least one pharmaceutically or nutritionally acceptable carrier.

Preferred product formats include the following : beverage products such as orange juice, other fruit juices, milk or yogurt drinks, smoothies, sports drinks, mineral water, soy beverages, hot chocolate, malt drinks; food products such as (cereal or dairy) bar, breakfast cereals, snack-foods, milk snack, cookies, filled biscuits, crackers (e. g. with filling), chocolate candies, toffees, desserts and yogurts; and dietary supplements such as pills and capsules.

The invention is further illustrated with reference to the following examples.

Examples Example 1 Measurement of 0-Gla-OC to 3-Gla-OC ratio in children Methods: Subjects. The initial cohort consists of 19 apparently healthy children (boys and girls) between 3 and 16 years old (mean age: 8.9 years) who visit the Department of Pediatry for various reasons. The judgment"healthy"is made by an experienced pediatrician. A second cohort consists of 20 children suffering from cystic fibrosis with pancreatic insufficiency (= fat malabsoprtion). Informed consent is obtained from parents in all cases. Data from these children are compared with baseline data from a group of 12 healthy adults between 25 and 31 years old (mean age 28 years) who participate in a non-related other study. The study design is approved by the local Medical Ethics Committee.

Sample handling. Blood (2 ml, if possible) is drawn by venipuncture between 8 and 9 a. m.

Serum samples are stored at-80 C until serial testing. Variables measured: total osteocalcin (Biosource, Nivelles, Belgium), bone alkaline phosphatase (BAP: Metra

Biosystems, Mount View, CA), O-Gla-QC and 3-Gla-OC (Takara, Tokyo, Japan), PTH (Sangui Biotech, Santa Ana, CA), type I collagen N-terminal crosslinks (NTX : Ostex International, Seattle, WA), PIVKA-II and vitamin K.

Results : The high metabolic state of children (as compared to healthy adults) is given in Table 1 (upper part). A differentiation IS made between non-supplemented cystic fibrosis (low vitamin K-status), cystic fibrosis supplemented with vitamin K, non-supplemented children and adults. PIVKA-II is at or below the lower detection limit in all cases.

Table 1 : Metabolic state of bone in children and healthy adults

Variable CF low K CF high K Healthy child Healthy adult (n=16) (n=4) (n=19) (n=12) Age (years) 10. 6+3. 45. 8+4. 98. 96+3. 527. 8+1. 8 Total OC (g/L) 27. 1+13. 0 46. 7+8. 8 30. 4+16. 1 14. 9+1. 2 BAP (U/L) 108. 6+47. 5 120. 233. 0 121. 9+46. 0 24. 2+6. 3 NTX (nmol/L) 45. 1+13. 5 55. 9+6. 2 55. 0 9. 7 13. 5+4. 7 O-Gla-OCI 3-Gla-OC ratio 5. 5+2. 6 0. 3 0. 1 2. 3 1. 2 0. 4 0. 2 Vitamin K (g/L) 0. 38+0. 17 1. 85+1. 58 0. 56+0. 22 0. 55+0. 18

Abbreviations used : OC, osteocalcin ; BAP, bone alkaline phosphatase ; NTX, type 1 collagen N-terminal crosslinks ; O-Gla-OC and 3-Gla-OC, undercarboxylated and carboxylated osteocalcin.

Both markers for bone formation (OC and BAP) and those for bone resorption are high in all children as compared to adults. Difference between healthy children and CF children are not statistically significant. The vitamin K status of the subjects is given by the serum vitamin K concentrations, whereas the O-Gla-OC I 3-Gla-OC ratio is a measure for the vitamin K status of bone tissue. Circulating vitamin K levels of all children are within the normal range, although in cystic fibrosis the levels are lower than in healthy children. This is consistent with the known poor absorption in cystic fibrosis patients. The absence of PIVKA-II both in healthy and diseased subjects is consistent with the serum vitamin K profile. Obviously very high values of serum vitamin K are observed in supplemented children.

A most striking difference is observed between non-supplemented children and adults with respect to their O-Gla-OC I 3-Gla-OC ratio. All healthy children appear to have substantially undercarboxylated osteocalcin. The difference between cystic fibrosis children and healthy ones is statistically significant (p < 0.05, Mann-Whitney rank sum test), which is consistent with their poor absorption of fat-soluble vitamins. It is suggestive in this respect that at 20 years of age cystic fibrosis children are reported to be at high risk for bone fractures.

Although the number of vitamin-K supplemented children is low (n = 4), we conclude that supplementation brings the O-Gla-OC/3-Gla-OC ratio to the normal adult level, which demonstrates that vitamin K supplements are effective in this respect.

Claims

Claims 1. The use of vitamin K in the preparation of a composition for counteracting a vitamin Kdeficiency in bone of a healthy child and/or adolescent.
2. The use of vitamin K in the preparation of a composition for a child and/or adolescent for increasing the peak bone mass.
3. The use of vitamin K in the preparation of a composition for a child and/or adolescent for decreasing bone fracture risk.
4. The use of vitamin K for increasing the level of osteocalcin carboxylation in a healthy child and/or adolescent from a low level, i. e. a ratio of O-Gla-QC/3-Gla-OC of > 0.8, to a normal level, i. e. a ratio of 0-Gla-OC/3-Gla-OC of < 0.8.
5. The use according to any one of the preceding claims wherein the child and/or adolescent is aged 3 to 16.
6. The use according to any one of the preceding claims wherein the child and/or adolescent is not suffering from malabsorption syndromes, such as cystic fibrosis, sprue, celiac disease, ulcerative colitis, regional ileitis, ascaris infection, short bowel syndrome or cholestasis, or from metabolic diseases, such as phenylketonuria or galactosemia, or children suffering from liver disease, or children who are on antibiotics for prolonged periods of time.
7. The use according to any one of the preceding claims wherein vitamin K means one or more compounds of Formula 1, and/or their pharmaceutically or nutritionally acceptable salts,

wherein R is hydrogen; a substituted or unsubstituted straight-or branched chain Ci to C30 alkyl or alkenyl group; an aliphatic or aromatic substituted or unsubstituted straight or branched Ci to Cio thio alkyl or alkenyl group; an aliphatic or aromatic substituted or unsubstituted straight or branched Ci to Cio mercapto alkyl or alkenyl group; dithiothreitol or dithioerythritol ; or a sulphate containing group.
8. The use according to any one of the preceding claims wherein the composition contains one or more of the following active ingredients: vitamin D, calcium, magnesium, zinc, CPP and prebiotics together with a pharmaceutically or nutritionally acceptable carrier.
9. The use according to any one of the preceding claims wherein the child and/or adolescent to be treated has normal serum vitamin K concentrations in the range of 1501500 nanogram/L.