WO2015132304A1

WO2015132304A1 - Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders

Info

Publication number: WO2015132304A1
Application number: PCT/EP2015/054523
Authority: WO
Inventors: Werner Ernst Ludwig Georg MÜLLER; Heinrich-Christoph Wilhelm Friedrich SCHRÖDER; Xiaohong Wang
Original assignee: Müller Werner Ernst Ludwig Georg; Schröder Heinrich-Christoph Wilhelm Friedrich; Xiaohong Wang
Priority date: 2014-03-05
Filing date: 2015-03-04
Publication date: 2015-09-11
Also published as: GB201403899D0

Abstract

The invention concerns a composition containing quercetin and polyphosphate that acts synergistically on bone formation compared with the bone mineralization stimulating effects of the two components alone, and the application of this composition for treatment of bone disorders like osteoporosis. The effects of the composition containing quercetin and polyphosphate can further be increased by addition of monomeric or polymeric silicate or bicarbonate/calcium carbonate.

Description

SYNERGISTIC COMPOSITION COMPRISING QUERCETIN AND

POLYPHOSPHATE FOR TREATMENT OF BONE DISORDERS

The present invention relates to a composition comprising quercetin and polyphosphate that acts synergistically on bone formation compared with the bone mineralization stimulating effects of the two components alone, and the application of this composition for treatment of bone disorders like osteoporosis. The effects of the composition containing quercetin and polyphosphate can further be increased by addition of monomeric or polymeric silicate or bicarbonate/calcium carbonate.

Background of the Invention

Osteoporosis has become a major public health problem worldwide. This progressive skeletal disorder is characterized by a decrease in bone mass and a compromised bone strength predisposing to an increased risk of fracture. Osteoporosis is most common in women after menopause, but it occurs after the age of 75, in both females and males in an almost equal ratio. On cellular level, the disease is caused by an imbalance of bone resorbing (osteoclasts) and bone forming cells (osteoblasts). It is assumed that lifestyle changes, including diet, e.g. the intake of calcium or vitamin D, and exercise can decelerate the progression of the disease.

Quercetin - flavonoids

Flavonoids are a group of naturally occurring compounds. They are ubiquitous in plants, and are found, in particular, in medicinal plants. Flavonoids show a various pharmacological activities, including anti-inflammatory, antiallergic, antihepatotoxic, antitumor, antidiabetic and antiosteoporotic activities (Di Carlo G, Mascolo N, Izzo AA, Capasso F (1999) Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Sci 65:337-353). They can be subdivided into different groups and subgroups: Flavones (2-phenylchromen-4- ones), e.g. apigenin, flavonols (3 -hydroxy flavones or 3-hydroxy-2-phenylchromen-4-ones), e.g. quercetin, kaempferol and myricetin, and flavanones (2,3-dihydro-2-phenylchromen-4- ones), e.g. naringenin, flavanonols (3 -hydroxy flavanones or 2,3-dihydroflavonols or 3- hydroxy-2,3-dihydro-2-phenylchromen-4-ones), like taxifolin, and isoflavonoids, like genistein. Chrysin is a 5,7-dihydroxyflavone. Besides aglycons, glycosides with one or more sugar residues are found, e.g. isoquercitrin (quercetin 3-P-D-glucoside).

Some flavonols like quercetin have been proposed to prevent bone loss, and might be taken for prophylactic use in postmenopausal women (Branca F (2003) Dietary phyto-oestrogens and bone health. Proc Nutr Soc 62:877-887). Quercetin has been described to be a potent inhibitor, at a concentration of ~ 1-10 μΜ, of osteoclasts in vitro (Wattel A, Kamel S, Mentaverri R, Lorget F, Prouillet C, Petit JP, Fardelonne P, Brazier M (2003) Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochem Pharmacol 65:35-42), as well as of osteoblasts, likewise in vitro at ~ 10 μΜ (Notoya M, Tsukamoto Y, Nishimura H, Woo JT, Nagai K, Lee IS, Hagiwara H (2004) Quercetin, a flavonoid, inhibits the proliferation, differentiation, and mineralization of osteoblasts in vitro. Eur J Pharmacol 485:89-96). The latter effect is controversial, since it has been reported that quercetin, at 50 μΜ, is a strong activator of osteoblasts in vitro resulting in an increased mineralization (Prouillet C, Maziere JC, Maziere C, Wattel A, Brazier M, Kamel S (2004) Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochem Pharmacol 67: 1307-1313). This effect has been attributed to the interaction of quercetin with the estrogen receptor [ER], which causes stimulation both of the MAPK/ERK pathway and the alkaline phosphatase [ALP].

Quercetin causes an anabolic effect on human osteoblasts within the range of 1 and 5 μΜ (Prouillet C, Maziere JC, Maziere C, Wattel A, Brazier M, Kamel S (2004) Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochem Pharmacol 67: 1307-1313). The quercetin content of edible plants ranges from 10 to 100 mg per 100 g of edible portion (Bhagwat S, Haytowitz DB and Holden JM (2011) USD A Database for the Flavonoid Content of Selected Foods; Release 3. U.S. Department of Agriculture; Beltsville, Maryland 20705), while the concentration of isoquercitrin is about 30-fold higher (Kalinova J, Vrchotova N (2009) Level of catechin, myricetin, quercetin and isoquercitrin in buckwheat (Fagopyrum esculentum Moench), changes of their levels during vegetation and their effect on the growth of selected weeds. J Agric Food Chem 57:2719-2725). Administration of quercetin at doses of 40-1,900 mg/kg/day to rats revealed treatment-related effects on survival and no treatment-related clinical signs of toxicity, due to its poor absorption (Dunnick JK, Hailey JR (1992) Toxicity and carcinogenicity studies of quercetin, a natural component of foods. Fundam Appl Toxicol 19:423-431). Likewise of low toxicity is isoquercitrin with an acute toxicity (LD₅₀ [lethal dose]; intraperitoneal in mice) with > 5.000 mg/kg (PubChem Substance 2014).

Polyphosphate

Polyphosphate [polyP] is a natural inorganic polymer that consists of up to 1000 phosphate units linked by high-energy phosphodiester bonds. This polymer has been shown to induce bone formation (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). PolyP induces the expression of the genes encoding for the osteoclastogenesis inhibitory factor osteoprotegerin [OPG], bone morphogenetic protein 2 [BMP2], and alkaline phosphatase [ALP] (reviewed in: Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

PolyP, after hydrolysis to monomeric phosphate [Pi] by phosphatases like ALP (Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Muller WEG, Schroder HC (1998) Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 13:803-812; Lorenz B, Munkner J, Oliveira MP, Kuusksalu A, Leitao JM, Muller WEG, Schroder HC (1997) Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis. Biochim Biophys Acta 1335:51-60), has been proposed to be involved in regulation of mineralization processes occurring during vertebrate skeleton formation (Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD (2009) Control of vertebrate skeletal mineralization by polyphosphates. PLoS One 4:e5634). PolyP is a calcium chelator and might act as a source of Ca²⁺ ions and monomeric Pi during hydroxyapatite deposition after enzymatic degradation of the polyphosphate calcium complex (Omelon SJ, Grynpas MD (2008) Relationships between polyphosphate chemistry, biochemistry and apatite biomineralization. Chem Rev 108:4694- 4715). The ALP also hydrolyzes pyrophosphate [PPi] produced by the ectonucleotide pyrophosphatase/ phosphodiesterase- 1 [ENPP1], an ecto-enzyme that regulates the extracellular levels of PPi by cleveage of ATP. PPi is a well-known inhibitor of mineralization, while Pi is inhibiting the carbonic anhydrase which is likely involved in bio- seed formation during mineralization of bone cells (Miiller WEG, Schroder HC, Schlossmacher U, Grebenjuk VA, Ushijima H and Wang XH (2013) Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation. Biomaterials 34:8671-8680).

The extracellular level of phosphate can be modulated physiologically, by the action of the hydro lytically-acting enzymes, as mentioned above, or pharmacologically e.g. by quercetin which acts as an inducer of ALP (Prouillet C, Maziere JC, Maziere C, Wattel A, Brazier M, Kamel S (2004) Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ER and estrogen receptor pathway. Biochem Pharmacol 67: 1307-1313). In addition, the expression level of ALP can be modulated by polyP (Miiller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). PolyP is not toxic on cells in vitro up to concentrations of 100 μΜ. PolyP administered in a stoichiometric ratio of 2 M polyP : 1 M CaCl₂ [polyP (Ca²⁺ complex), also termed polyP (Ca²⁺ salt)], causes in osteoblast-like SaOS-2 cells on the level of gene transcription an increased expression of BMP 2 and in osteoclast-like RAW 264.7 cells via a solute mediator, an inhibition of proliferation through down-regulation of tartrate-resistant acid phosphatase [TRAP] and inhibition of phosphorylation of ΙκΒα by the respective kinase involved in the NF-KB signal transduction cascade (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, Schlossmacher U, Wiens M and Miiller WEG (2013) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Engin Regen Med 7:767-776).

The dietary phytoestrogen quercetin is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders.

The state of the art of the biological effects of polyP (effects on bone formation) has been described in: Wang XH, Schroder HC, Wiens M, Ushijima H, Miiller WEG (2012) Biosilica and bio-polyphosphate: applications in biomedicine (bone formation). Curr Opin Biotechnol 23:570-578.

In view of the above, it is an object of the present invention, to provide improved compositions for the prophylaxis and treatment of osteoporotic disorders.

This invention in a first aspect thereof relates to a composition comprising quercetin or isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyP, a naturally occurring inorganic polymer inducing bone formation, on mineralization of osteoblasts. Both compounds, quercetin and polyP, or isoquercitrin and polyP, administered as a calcium complex [polyP (Ca²⁺ complex)], if given alone at non-toxic concentrations induce the mineralization process of bone-forming cells. Unexpectedly, the composition according to this invention containing quercetin or isoquercitrin and polyP was found to act synergistically on bone formation compared with the effects of the two components alone. Co-incubation experiments revealed that quercetin or isoquercitrin, if given simultaneously with polyP (Ca²⁺ salt), amplifies the mineralization-enhancing effect of the inorganic polymer in a synergistic manner. This potentiating effect of quercetin or isoquercitrin on polyP (Ca²⁺ salt) was even found if this compound is given at a concentration which had no significant effect on mineralization. For example, at a concentration of 0.1 μΜ isoquercitrin, an about 1.5-fold increase in mineralization of bone-forming SaOS-2 cells caused by 3 μΜ polyP (Ca²⁺ salt) was detected. The potentiating effect of isoquercitrin on mineralization further increased by 2.4-fold if this flavonoid was added at higher concentrations (0.3 μΜ).

The inventive composition turned out to be biocompatible. The biomineralization process induced by the two components, quercetin and polyP (Ca²⁺ salt), or isoquercitrin and polyP (Ca²⁺ salt), is based on two different modes of action (Figure 1). Both compounds, quercetin and polyP (Ca²⁺ salt), or isoquercitrin and polyP (Ca²⁺ salt), cause a significant upregulation of the expression of the human Runt-related transcription factor 2 [RUNX2]. The expression of the two co-activators of RUNX2, activating transcription factor 6 [ATF6] and Ets oncogene homo log 1 [Etsl] becomes strongly increased in cells after exposure to quercetin or isoquercitrin. The assumption that the activating effect of quercetin or isoquercitrin occurs via a signal transduction pathway involving ATF6 that is independent from that induced by polyP (Ca²⁺ salt) is supported by the finding that quercetin or isoquercitrin but not polyP (Ca²⁺ salt) upregulates the expression of the gene encoding osteocalcin [OCAL]. The polyP (Ca²⁺ complex), on the other hand, strongly increases the expression of gene encoding for Etsl, as well as the expression of alkaline phosphatase, an enzyme that has been proposed to be involved in hydrolysis of polyP (Ca²⁺ salt).

Based on its unexpected properties, the composition according to this invention consisting of the two natural inorganic/organic compounds, polyP and quercetin or isoquercitrin, is superior to the two components, polyP and quercetin or isoquercitrin, alone. The inventors demonstrated that the combination of polyP and quercetin or isoquercitrin has a synergistic effect on bone mineral formation. The inventive composition is of therapeutic value for prevention/treatment of osteoporosis.

The effects of the composition containing quercetin and polyphosphate can further be increased by addition of monomeric or polymeric silicate or bicarbonate/calcium carbonate.

The inventors evaluated the effect of combinations of polyP and quercetin or isoquercitrin on bone mineral formation. Having identified that the mode of action of the two compounds is different, they could demonstrate that polyP and quercetin or isoquercitrin act synergistically on the mineralization process in SaOS-2 (sarcoma osteogenic) cells in vitro. SaOS-2 is a non- transformed cell line that is derived from primary osteosarcoma cells and provided with a (limited) differentiation capacity (Hausser and Brenner 2005).

The inventors determined the pharmacological effect of quercetin, acting as a stimulator for the mineralization process in vitro, and of polyP (Ca²⁺ salt), functioning as a supplier for Pi. In view of the findings that quercetin-3-O-glucoside [isoquercitrin], the 3-O-glucoside of quercetin, provides a better bioavailability than non-glucosylated quercetin (Paulke A, Eckert GP, Schubert-Zsilavecz M, Wurglics M (2012) Isoquercitrin provides better bioavailability than quercetin: comparison of quercetin metabolites in body tissue and brain sections after six days administration of isoquercitrin and quercetin. Pharmazie 67:991-996) isoquercitrin has been used in the described studies, but quercetin and other quercetin derivatives can also be used. Isoquercitrin [quercetin 3-P-D-glucoside] is a common secondary metabolite in plants (e.g. Selenge E, Murata T, Kobayashi K, Batkhuu J, Yoshizaki F (2013) Flavone tetraglycosides and benzyl alcohol glycosides from the Mongolian medicinal plant Dracocephalum ruyschiana. J Nat Prod 76: 186-193).

This invention concerns the unexpected property of combinations of the two natural products, polyP (Ca²⁺ salt) and quercetin or isoquercitrin, to act synergistically on the biomineralization of SaOS-2 cells. The two compounds, polyP (Ca²⁺ salt) and quercetin, or polyP (Ca²⁺ salt) and isoquercitrin, show negligible, or low, toxicity. At non-toxic concentrations the two natural compounds significantly upregulate the mineralization onto SaOS-2. Importantly, if given simultaneously, quercetin or isoquercitrin amplifies the anabolic effect of polyP (Ca²⁺ salt) in a synergistic manner, even if this phenolic compound is given at concentration at which it causes no significant effect on biomineralization.

An example for the surprising effect of the combination can be seen in Figure 5 of the present application, where at a concentration of 3 μΜ of polyphosphate the effect of the addition of 0.1 μΜ of isoquercetrin (itself having no effect on the mineralization without polyphosphate) is significant, and more than doubled upon the addition of 0.3 μΜ.

Since both of the compounds, polyP (Ca²⁺ salt) and quercetin, or polyP (Ca²⁺ salt) and isoquercitrin, are genuine components in the animal and plant food they are qualified to represent valuable nutritional components and useful therapeutic agents suitable for prevention/treatment of osteoporosis.

The chain lengths of the polyP molecules can be in the range 2 to 1000 phosphate units, preferentially in the range 4 to 100 phosphate units. Optimal results were achieved with polyP molecules with chain lengths of approximately 40 phosphate units.

Based on its properties, the composition can be used as a drug or injectable material for treatment of osteoporosis and other bone disorders.

Compositions containing quercetin instead of isoquercitrin are also effective. Compositions containing isoquercitrin are preferentially used because of the good bioavailablility.

Besides quercetin and isoquercitrin, similar synergistic effects of polyP (Ca²⁺ complex) are found for other flavonoids, including both flavonols and flavones, like chrysin, kaempferol, myricetin and apigenin, as well as flavanones and flavanonols, like naringenin and taxifolin, the glycosides of these compounds, and mixtures of various flavonoids present in vitamin P preparations/supplements. The concentration range of the compounds, other than the quercetin / isoquercitrin secondary metabolites is also in the range between 1 to 100 μg/ml (preferably as single dose).

In addition, compositions containing polyP instead of polyP (Ca²⁺ complex) are effective. Compositions containing polyP (Ca²⁺ complex) are preferentially used to avoid a depletion of calcium ions by complex formation with polyP, which may interfere with the results of in vitro experiments (cell culture) but is expected not or less to interfere with the results in vivo.

Another aspect of the invention then relates to a method of producing the composition according to the present invention, comprising either suitably combining of commercially available synthetic or purified components of said composition or of components that are isolated from natural sources (see examples). The producing also includes the formulation into pharmaceutically acceptable compositions, e.g. by adding the suitable excipients, such as buffers or carriers commonly used in pharmaceutical technology.

The preparation of the polyP (Ca²⁺ complex) is state of the art and has previously been described by the inventors (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671, 2011; Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Muller WEG (2012) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Engin Regen Med 7:767-776; as well as patent application EP 11152208.2 Food supplement and injectable material for prophylaxis and therapy of osteoporosis and other bone disease. Inventors: Muller WEG, Wang X, Schroder HC).

A further aspect of this invention concerns the combined application of one of the compositions described above with monomeric or polymeric silicic acid or one or more of the components (enzymes, proteins, and substrates) involved in their formation.

The silica precursor can consist of water glass, orthosilicic acid, orthosilicates, monoalkoxysilanetriols, dialkoxysilanediols, trialkoxysilanols, tetraalkoxysilanes, alkyl- silanetriols, alkyl-silanediols, alkyl-monoalkoxysilanediols, alkyl-monoalkoxysilanols, alkyl- dialkoxysilanols, or alkyl-trialkoxysilanes.

The compositions described above, either alone or combined with monomeric or polymeric silicic acid or one or more of the components (enzymes, proteins, and substrates) involved in their formation or the compositions described above, combined with calcium carbonate or an enzyme forming calcium carbonate in the presence of calcium ions, such as carbonic anhydrase, can be encapsulated in an organic polymer such as shellac, alginate, or poly(lactic acid), or poly(D,L-lactide)/polyvinyl pyrrolidone-based microspheres, following state-of-the art procedures, as previously described by the inventors; e.g. EP 11152208.2 (Food supplement and injectable material for prophylaxis and therapy of osteoporosis and other bone disease. Inventors: Muller WEG, Wang X, Schroder HC).

For example, the synergistic effect of the composition according to the invention on mineralization of bone- forming cells can further be increased after addition of 10 μΜ silicate (monomeric silicate; orthosilicate) or if that cells are grown on biosilica-coated culture plates, as described in European patent application EP10167744.1. EP 2409710 Al (Injectable material and material to be used as drug or food supplement for prophylaxis or treatment of osteoporosis; inventors: Muller WEG, Wang X, Wiens M).

The synergistic effect of the composition according to the invention on mineralization of bone-forming cells can also be increased if that composition is applied in the presence of calcium carbonate. For example, this can be demonstrated when SaOS-2 cells cultured in McCoy's medium, lacking Na-bicarbonate but containing 2 mM L-glutamine and 1 mM CaCl₂, in the absence or presence of the composition containing isoquercitrin and polyP (Ca²⁺ complex) according to the invention, are compared with SaOS-2 cells incubated under the same conditions but in the presence of 20 mM Na-bicarbonate (NaHC0₃). In this experiment, the medium/serum is buffered with 25 mM HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) to a pH of 7.4. Incubation of the cells in the presence of mineralization activation cocktail (MAC; containing β-glycerophosphate, ascorbic acid and dexamethasone) is performed as described in Patent application Great Britain-Europe No. 1319416.2 (Modulator of bone mineralization based on a combination of polyphosphate/carbonate and carbonic anhydrase activators; inventors: Miiller WEG, Schroder HC, Wang XH), but in the absence or presence of the composition containing isoquercitrin and polyP (Ca²⁺ complex) according to the invention. The results show that in the presence of 20 mM Na-bicarbonate, a 10-30% increase in the synergistic effect on mineralization of the composition containing isoquercitrin and polyP (Ca²⁺ complex) can be observed in the concentration range of 0.1 μΜ to 0.3 μΜ isoquercitrin and 3 μΜ to 10 μΜ polyP (Ca²⁺ complex).

A further aspect of the invention concerns the application of the composition described above or one of the combinations of the composition described above for the formulation of an orally or parenterally-administrable drug for therapy or prophylaxis of osteoporosis or other bone disorders.

The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures,

Figure 1 shows the different modes of action of polyP (Ca²⁺ salt) and isoquercitrin on the synthesis of master effector proteins, involved in biomineralization onto SaOS-2 cells. The gene encoding for one key effector protein, the enzyme alkaline phosphatase [ALP] is induced by polyP (Ca²⁺ salt). The most likely signal transduction pathway involves both BMP2 that undergoes an increased expression, as well as an upregulation of Etsl and RUNX2, two transcription factors that cause an increased expression of ALP. ALP in turn hydrolyses polyP (Ca²⁺ salt) under release of Ca²⁺ and phosphate that constitute the building blocks for the Ca-phosphate mineral deposits. The second effector protein, identified to be stimulated by isoquercitrin is osteocalcin [OCAL]. Isoquercitrin induces the expression of the protein encoding the transcription factor RUNX2 which in turn causes an increased expression of ATF6. The expressed protein, the ATF6 precursor protein ATF6p undergoes proteolytic cleavage at the ER after BMP2-caused stress. The binding of the mature factor ATF6 to the ATFRE the ATF6 response element, results in an increased expression of the gene OCAL. The protein OCAL synthesized, the pre-pro-OCAL, undergoes intracellular maturation via a signal peptidase and the subsequent C0₂-dependent carboxylase, and is finally transported to the extracellular space after propeptide cleavage. Further details are given in the text.

Figure 2 shows the effect of increasing concentrations of isoquercitrin and polyP (Ca²⁺ salt), as measured by the colorimetric cell proliferation (XTT) assay. SaOS-2 cells remained either untreated or were exposed to 0.3 to 10 μΜ isoquercitrin (open bars) or to 3 to 100 μΜ polyP (Ca²⁺ salt) (closed bars) for 72 h in McCoy's medium/serum. Subsequently the cultures were treated with XTT and the orange formazan solution formed was quantified spectrophotometrically. The increased number of living cells results in an increased overall activity of reference mitochondrial dehydrogenases. The staining intensity monitored by the absorbance values is given. Data represent the means ± SD of ten independent experiments (* < 0.01).

Figure 3 shows the mineralization by SaOS-2 cells after exposure to different concentrations of isoquercitrin [IQC], as measured on the basis of Alizarin Red S (AR) staining. The cells were grown in medium/serum for 7 days either in the absence (bar in white/black flow) or the presence of the activation cocktail (dexamethasone [DEX], ascorbic acid [AA] and β- glycerophosphate [β-GP]; open bars). Then the cells were collected and extracts were prepared, which were stained with Alizarin Red S. The extent of biomineralization is correlated with the DNA content of the cells in the assays. Values represent the means ± SD from 10 separate experiments each (* P <0.01).

Figure 4 shows the enhanced biomineralization of SaOS-2 cells in the presence of polyP (Ca²⁺ salt). The cells were incubated in the absence (bar in white/black flow) or presence of MAC (filled bars). Other details are as in legend to Figure 3.

Figure 5 shows the enhancement of the polyP (Ca²⁺ salt)-caused biomineralization process by isoquercitrin. The cells remained during the incubation period of 7 days without MAC (open bar). In the main set of experiments MAC was added to the cultures (indicated by the horizontal line and mentioning the three components of the MAC which are DEX, AA and B- GP). After addition of isoquercitrin, added alone to the MAC-exposed cells at concentrations of 0.1 or 0.3 μΜ no significant change of the mineralization is seen. Finally, SaOS-2 cells were incubated in medium/serum, supplemented with MAC, in the co-incubation period with 3 μΜ to 100 μΜ polyP (Ca²⁺ salt) either alone (filled bars) or together with 0.1 μΜ (crosshatched bars) or 0.3 μΜ isoquercitrin (horizontally hatched bars).

Figure 6 shows the effect of isoquercitrin on the polyP (Ca²⁺ salt)-caused increase in biomineralization onto SaOS-2 cells. The cultures were incubated in 24-well plates for 5 days in McCoy's medium/FCS. Then the cultures were supplemented with MAC and incubation was continued for additional 7 days. Some well remained free of any other component, or were incubated with polyP (Ca²⁺ salt) (polyP/Ca) or isoquercitrin (IQC) as marked at the respective well. The concentrations of the components are given. Where indicated the two components [polyP (Ca²⁺ salt) and isoquercitrin] were added together. Then, after removal of the incubation medium/serum the cells within the wells were stained with Alizarin Red S. The red staining reflects the extent of biomineralization.

Figure 7 shows the effect of 1 μΜ isoquercitrin [IQC] and 50 μΜ polyP (Ca²⁺ salt) on the mineralization in SaOS-2 cells. The technique of qRT-PCR was applied to determine the steady- state expression levels of RUNX2 and GAPDH (housekeeping gene, used as reference for normalization). The cells were kept first for 5 days in medium/serum and then transferred to the MAC and continued to be incubated for 5 days. Then RNA was extracted and qRT- PCR was performed. Open bars represent the effect of isoquercitrin on gene expression, while the closed bars show the influence of polyP (Ca²⁺ salt) on RUNX2 transcription level. The means ± are shown (5 experiments/time point); * P <0.01.

Figure 8 shows the differential effect of 1 μΜ isoquercitrin and 50 μΜ polyP (Ca²⁺ salt) on the expression level of ATF6. After transfer of the cells into medium/serum, supplemented with MAC, the cells were incubated for up to 5 days. The significant increase in ATF6 expression is seen in the assays exposed to isoquercitrin. Further details are given in legend to Figure 7.

Figure 9 shows the superior expression of OCAL in assays stimulated with 1 μΜ isoquercitrin, compared to those with 50 μΜ polyP (Ca²⁺ salt). For additional explanations, see Figure 8.

Figure 10 shows the strong and significant expression of the gene, encoding the transcription factor Etsl, after incubation with 50 μΜ polyP (Ca²⁺ salt). In contrast, isoquercitrin caused no significant change of the steady-state transcript level. Figure 11 shows the induction of the ALP gene expression in assays supplemented with either 1 μΜ isoquercitrin or 50 μΜ polyP (Ca²⁺ salt).

Examples

In the following examples, except for the first example, only the effects determined for isoquercitrin (quercetin 3-P-D-glucoside) and it combinations with polyP (Ca²⁺ salt) are shown. This quercetin derivative has been used because of its good bioavailability. Similar effects are found with quercetin.

Production of a composition comprising quercetin and polyP

The composition consisting of quercetin and polyP can either be prepared from commercially available, synthetic or purified components or isolated from natural sources, in particular fruits, vegetables, leaves or grains, some of them contain considerable amounts of quercetin (for example, raw capers: 233.84 mg/100 g; juniper berries, ripe: 69.05 mg/100 g; coriander leaves: 52.90 mg/100 g; fennel leaves: 48.80 mg/100 g; lovage leaves: 170.00 mg/100 g; Onions: 21.40 mg/100 g; Hot peppers, yellow: 50.63 mg/100 g; and spinach: 3.97 mg/100 g; Bhagwat S, Haytowitz DB and Holden JM (2011) USDA Database for the Flavonoid Content of Selected Foods; Release 3. U.S. Department of Agriculture; Beltsville, Maryland 20705). The polyP content in spinach leaves amounts to 2-3 mg/100 g (Miyachi S. (1961) J Biochem, Tokyo 50:367-371), similar to the quercetin content (see above).

In the following example, the extraction of the composition consisting of quercetin and polyP from leaves of spinach (Spinacea oleracea L.) is described. The composition can be extracted from other plant sources as well. One part of spinach leaf-blades is ground in a Waring blender for 2 min with one part of 0.01 M Tris-HCl pH 8. The homogenate is filtered through two layers of gauze and then centrifuged at 25,000g for 30 min. The composition is isolated by successive extraction of the pellet as follows. In order to prevent oxidation of quercetin, for example, ascorbic acid (e.g., 1 mM ascorbic acid) can be added to the solutions. In the first step (step 1), the pellet is extracted with ice-cold TCA (2% w/v). The suspension is then centrifuged (5 min, 4°C, 15000g; Eppendorf centrifuge) and the supernatant, containing short- chain polyPs (chain length <20 phosphate units) is discarded. In the next step (step 2), the pellet is extracted by suspension in TCA/acetone (0.5%/70% w/v) and centrifuged again (as above). In step 3, the newly obtained pellet is extracted with ice-cold acetone (70%) and centrifuged again (as above). The supernatants from steps 2 and 3 are combined (= Extract A). In step 4, the pellet from step 3 is suspended in 2 mM EDTA and titrated at room temperature with 0.2 M lithium hydroxide to a pH of about 7.5, as checked by using pH-paper. Then the suspension is centrifuged (as above). The resulting supernatant (= Extract B) is then combined with Extract A at a ratio of at least 2: 1 and a solution containing 1 mM Ca²⁺ is added until the precipitate consisting of the composition has been formed. If required samples can be concentrated by lyophilisation or binding to metal oxides (Lorenz B, Marme S, Miiller WEG, Unger K, Schroder HC (1994) Preparation and use of polyphosphate-modified zirconia for purification of nucleic acids and proteins. Anal Biochem 216: 118-126; DE4309248; Modifikation von Metalloxiden mit Polyphosphaten oder ahnlichen Verbindungen und ihre Anwendung; inventors: Lorenz B, Marme S, Unger K, Schroder HC, Miiller WEG). The polyP content of the extracts and composition can be determined by measuring the change in the absorption spectrum of toluidine blue at 530 nm and 630 nm due to the metachromatic effect produced by polyP (Lorenz B, Miiller WEG, Kulaev IS, Schroder HC (1994) Purification and characterization of an exopolyphosphatase from Saccharomyces cerevisiae. J Biol Chem 269:22198-22204). A calibration curve is used, which can be obtained by determination of the ratio of the absorbance values at 530 nm to the absorbance values at 630 nm for increasing amounts of a polyP standard (for example, Sigma polyP type 35 with average chain length of 35 phosphate residues). Quercetin can be detected by High- Performance Liquid Chromatography (HPLC). For example, reversed-phase HPLC using a monolith column CI 8 can be applied (Castellari M, Sartini E, Fabiani A, Arfelli G, Amati AJ (2002) Chromatogr A 973:221), whereby quercetin can be detected at 360-370 nm using photometric UV detectors.

The composition isolated from spinach leaves was biologically active if tested in the biomineralization assay described in Figure 5 (Alizarin Red S assay). Incubation of the composition isolated from spinach at a final polyP concentration of 10 μΜ (based on phosphate units) with SaOS-2 cells under the conditions described in Figure 5 in the presence of MAC and 5 μΜ CaCl₂ and an incubation period of 7 days resulted in a significant increase in mineralization (by about 17%) if compared to assays containing the same concentration of polyP (Ca²⁺ salt) (10 μΜ), obtained from commercial polyP.

Effect of isoquercitrin and polyP (Ca²⁺ salt) on growth of SaOS-2 cells

Applying the XTT cell proliferation assay the growth of SaOS-2 cells was determined in medium/serum at a concentration range between 0.3 to 10 μΜ isoquercitrin and 3 to 100 μΜ polyP (Ca²⁺ salt) (average chain length of approximately 40 phosphate units), respectively (Figure 2). While isoquercitrin was found to be significantly toxic only at the high concentration of 10 μΜ, polyP (Ca²⁺ salt) did not impair cell growth within the concentration range used.

Enhancement of biomineralization of SaOS-2 cells by isoquercitrin and polyP (Ca²⁺ salt) The cells were incubated with isoquercitrin for 7 days. In the absence of MAC (mineralization activation cocktail; see under Methods) the extent of mineralization is low with 0.27±0.05 nmoles^g cell DNA (Figure 3). After addition of the activation cocktail MAC the level of mineralization (Alizarin Red S staining) increases to 0.41±0.06 nmoles g. After exposure to low concentrations of isoquercitrin (0.1 or 0.3 μΜ) the mineralization does not change significantly; only at concentrations > 1 μΜ the degree increases significantly to 0.68±0.11 nmoles^g (1 μΜ) or 0.83±0.13 nmoles^g (3 μΜ), respectively.

The polymer polyP (Ca²⁺ salt) causes, in the presence of MAC, a significant enhancement of the level of biomineralization already at a concentration above 3 μΜ (Figure 4). The level of mineralization increases from 0.32±0.05 nmoles^g [absence of polyP (Ca²⁺ salt)] to 0.55±0.10 nmoles^g (presence of 3 μΜ of the polymer). At the higher level of 100 μΜ to extent reaches a value of 1.44=1=0.19 nmoles^g.

Potentiating effect of isoquercitrin on the mineralization-enhancing effect of polyP (Ca²⁺ salt) In order to determine a potential interaction of isoquercitrin with polyP (Ca²⁺ salt) during the mineralization process the cells were incubated first for a period of 5 days. Then the cultures were continued to be incubated for 7 days in the absence or the presence of MAC. Then, the extent of biomineralization, measured on the basis of Alizarin Red S staining, was determined.

In the absence of MAC the extent of mineralization remains low; a concentration of 0.11=1=0.02 nmoles^g DNA was measured (Figure 5). If the cultures were exposed during the 7 days to MAC a level of 0.36=1=0.05 nmoles^g DNA is measured. As expected, this amount remains unchanged after addition of 0.1 or 0.3 μΜ isoquercitrin. However, if polyP (Ca²⁺ salt) was added and co-incubated together with isoquercitrin for 7 days a significant amplification of mineralization, as reflected by an increased Alizarin Red S straining, was measured. If the non-stimulating dose of isoquercitrin (0.1 μΜ) is added to 3 μΜ polyP (Ca²⁺ salt) a significant increase of mineralization from 0.59±0.09 nmoles^g [polyP (Ca²⁺ salt) alone] to 0.87±0.15 nmoles/μg [polyP (Ca²⁺ salt) and isoquercitrin] is measured. Such a ~ 50% amplification of mineralization, caused by isoquercitrin, is also seen if 0.1 μΜ isoquercitrin is co-incubated with higher concentrations of polyP (Ca²⁺ salt).

Even more pronounced is the potentiating effect of isoquercitrin if this flavonoid is added at higher concentrations, at 0.3 μΜ (again a dose that does not act stimulating on mineralization), to the polyP (Ca²⁺ salt) polymer; Figure 5. In the presence of 0.3 μΜ isoquercitrin the degree of mineralization during co-incubation with 3 μΜ polyP (Ca²⁺ salt) increases from 0.59±0.09 nmoles^g [polyP (Ca²⁺ salt) alone] to 1.44±0.18 nmoles^g [isoquercitrin together with polyP (Ca²⁺ salt)], by 140%. This potentiating effect is slightly lower, but still significant, at the higher polyP (Ca²⁺ salt) concentrations of 10 μΜ and 30 μΜ with 70%) and 50%>, respectively. Only at 100 μΜ polyP (Ca²⁺ salt) the isoquercitrin-caused potentiating effect is not significant.

The amplifying effect of isoquercitrin on the polyP (Ca²⁺ salt)-caused mineralization of SaOS-2 cells can also be documented by direct cell culture staining with Alizarin Red S. The cells, after an initial incubation period of 5 days, were transferred to a medium/serum, supplemented with MAC, for additional 7 days. Subsequently, the cover slips, onto which the cultures were grown, were stained with Alizarin Red S for bio mineralization (Figure 6). Eye inspection of the cover slips revealed that the cultures exposed to 3 μΜ or 10 μΜ polyP (Ca²⁺ salt) showed a much stronger red color intensity. Addition isoquercitrin at the concentrations that do not change the extent of mineralization, 0.1 μΜ and 0.3 μΜ, strongly enhanced the intensities after Alizarin Red S straining.

From these data we conclude that isoquercitrin and polyP (Ca²⁺ salt) act synergistically on the biomineralization of SaOS-2 cells. Furthermore this finding suggests that isoquercitrin and polyP (Ca²⁺ salt) cause the mineralization-enhancing potency via two different modes of action.

Additional increase of the potentiating effect of isoquercitrin on the mineralization-enhancing effect of polyP (Ca²⁺ salt) in the presence of silicate

The synergistic effect of isoquercitrin and polyP (Ca²⁺ salt) on mineralization of SaOS-2 cells can further be increased by addition of silicate. As shown in Figure 5, if the non-stimulating dose of isoquercitrin (0.1 μΜ) is added to 3 μΜ polyP (Ca²⁺ salt) an increase of mineralization to 0.87±0.15 nmoles^g is measured after an incubation period of 7 days (Alizarin Red S straining). If the same experiments, incubation of the cells with 0.1 μΜ isoquercitrin and 3 μΜ polyP (Ca²⁺ salt), is performed in the presence of 10 μΜ silicate (monomeric silicate; orthosilicate), a further increase of mineralization to 1.21=1=0.23 nmoles^g DNA is determined. The increase in mineralization with silicate alone (same concentration) in the absence of isoquercitrin and polyP (Ca²⁺ salt) amounts to 0.41 ±0.14 nmoles^g DNA (incubation period, 7 days).

Additional increase of the potentiating effect of isoquercitrin on the mineralization-enhancing effect of polyP (Ca²⁺ salt) in the presence of bicarbonate / calcium carbonate

The synergistic effect of isoquercitrin and polyP (Ca²⁺ salt) on mineralization of SaOS-2 cells can also be increased if the composition according to the invention is applied together with calcium carbonate. The formation of calcium carbonate by the cells, maintained in McCoy's medium lacking Na-bicarbonate but containing 2 mM L-glutamine and 1 mM CaCl₂, is induced by addition of 20 mM Na-bicarbonate (NaHCOs). The medium/serum is buffered with 25 mM HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) to a pH of 7.4. Incubation of the cells in the presence of MAC results in a 10-30% increase in the effect on mineralization caused by the composition according to this invention containing isoquercitrin and polyP (Ca²⁺ complex) in the concentration range of 0.1 μΜ to 0.3 μΜ and 3 μΜ to 10 μΜ, respectively.

RUNX2 expression is under the control of isoquercitrin and polyP (Ca²⁺ salt)

RUNX2 [human Runt-related transcription factor 2] is an essential transcription factor controlling differentiation of osteoblasts from multipotent stromal cells. We have used for the mineralization stimulation experiments isoquercitrin and polyP (Ca²⁺ salt) at concentrations that cause a significant increase in inorganic mineral deposition onto the SaOS-2 cells; meaning 1 μΜ isoquercitrin (Figure 3) and 50 μΜ polyP (Ca²⁺ salt) (Figure 4). At those concentrations a significant increase in the extent mineralization is seen after 3 days of incubation, with a 4.2-fold increase for isoquercitrin and a 2.5-fold enhanced mineralization for polyP (Ca²⁺ salt) (Figure 7). At an extended incubation period for 5 days the increased level can still be measured.

Expression of the genes ATF6 and OCAL is strongly upregulated by isoquercitrin

The expressions of two genes become strongly upregulated in SaOS-2 cells after exposure to isoquercitrin; ATF6 [activating transcription factor 6] and OCAL [osteocalcin]. The induction effects after incubation to polyP (Ca²⁺ salt) were lower. For the analysis of the differential expression the following concentrations were used: 1 μΜ isoquercitrin and 50 μΜ polyP (Ca²⁺ salt). At these concentrations the compounds cause a significant increase in the mineralization of SaOS-2 cells (Figs. 3 and 4). After an initial incubation for 5 days the cells were transferred to MAC; after an additional incubation for 7 days the expression levels were determined. The technique of qRT-PCR was applied to quantify the transcript level of the respective genes. The steady-state expression levels were correlated with that of the housekeeping gene GAPDH [glyceraldehyde 3 -phosphate dehydrogenase].

The steady-state level of the gene encoding the transcription factor ATF6 which is known to cause a strong upregulation of OCAL expression via RUNX2 activation is significantly upregulated already after a one-day incubation period with 1 μΜ isoquercitrin (Figure 8) by about 3-fold, a value that increases to 5.3-fold after the total incubation period of 5 days. No or only a small effect was found for 50 μΜ polyP (Ca²⁺ salt).

A likewise strong inducing activity of 1 μΜ isoquercitrin is seen for the gene encoding OCAL [osteocalcin], compared to 50 μΜ polyP (Ca²⁺ salt). Both compounds display after a one day incubation period a significant ~ 50-80% increase in the expression of OCAL. However, after 3 days of incubation the inducing activity of isoquercitrin, by 310%), is much stronger, while the expression level of polyP (Ca²⁺ salt) is not significantly changed. A likewise significant upregulation for isoquercitrin is measured after 5 days (Figure 9).

Preferential expression of Etsl and ALP by polyP (Ca²⁺ salt)

The gene for the transcription factor Etsl is the primary target for the polyP (Ca²⁺ salt) polymer. While the expression is significantly upregulated by polyP (Ca²⁺ salt) between the 1 to 5 days' incubation period, no significant increase is seen in the cultures with isoquercitrin as a test component (Figure 10). The steady-state level of the Estl transcripts is, at day 3, 1.6- fold higher compared to the beginning of the incubation period.

A similar differential expression pattern is seen if the cells were studied by qRT-PCR with primer pairs directed against the ALP [human alkaline phosphatase] gene. While the inducing activity caused by isoquercitrin becomes significant after 3 days, by an increase of 2.2-fold, the induction in response to exposure to polyP (Ca²⁺ salt) measures an increase by 4.2-fold at this time point (Figure 11).

Methods

Cultivation of SaOS-2 cells

SaOS-2 cells (human osteogenic sarcoma cells) can be cultured in McCoy's medium containing 2 mM L-glutamine and 1 mM CaCl₂. The medium is supplemented with 10% heat- inactivated fetal calf serum (FCS) and 100 units/mL penicillin/ 100 μg/mL streptomycin. Routinely, the cells are incubated in 25 cm² flasks or in six-well plates (surface area 9.46 cm²) in a humidified incubator at 37°C; 3 · 10⁵ cells/well are added (total volume, 3 mL). To induce biomineralization the cultures are supplemented with the mineralization activation cocktail (MAC) which is composed, for example, of 5 mM β-glycerophosphate, 50 mM ascorbic acid and 10 nM dexamethasone. The mineralization activation cocktail is usually added 3 days after starting the experiments. Medium is changed every 3 days and new MAC is added; likewise the test assays are added with the test compounds after each medium change.

The test compounds [isoquercitrin and polyP (Ca²⁺ salt)] are usually added after a cultivation period of the cells for 5 days. At this time point also MAC is added to the cultures. The incubation is terminated after a total period of 12 days. PolyP is administered as a Ca²⁺ salt; for this, 2 M polyP (referred to the monomer unit in the polymer) is mixed together with 1 M CaCl₂ in a stoichiometric ratio 2: 1 , to compensate for the chelating function of polyP for Ca²⁺. Isoquercitrin is dissolved in a stock solution 300 μΜ in dimethyl sulfoxide [DMSO].

The cell density can be determined by the colorimetric method based on the tetrazolium salt XTT (Cell Proliferation Kit II; Roche). The absorbance of the samples is measured against a background control at a wavelength of 450 nm; the non-specific readings, measured at a wavelength of 690 nm, are subtracted.

Mineralization by SaOS-2 cells in vitro

The extent of mineralization by SaOS-2 cells can be quantitatively determined applying, for example, the Alizarin Red S spectrophotometric. The amount of bound Alizarin Red S shown in the Figures is given in nmoles. Values can be normalized to total DNA in the samples.

The cultures, growing into culture wells can also be stained directly on the coverslips with 10% Alizarin Red S, after fixation with ethanol.

Quantitative real-time RT-PCR (qRT-PCR) analysis of the transcripts

The technique of quantitative real-time RT [reverse transcription] -PCR (qRT-PCR) using suitable primer pairs can be applied to determine the levels of transcription of the following genes in SaOS-2 cells: ALP [human alkaline phosphatase; accession number NM 000478.4]; RUNX2 [human Runt-related transcription factor 2; variant-3; NM 004348.3]; Etsl [human v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1); NM 001143820]; ATF6 [human activating transcription factor 6 (ATF6); NM 007348]; and OCAL [human osteocalcin; NM 199173.4]. As the reference gene GAPDH [glyceraldehyde 3-phosphate dehydrogenase; NM 002046.3] can be used. Initially the cells are incubated in medium/serum for 5 days and then transferred to medium/serum, supplement with MAC. Parallel assays are additionally supplemented with either 1 μΜ isoquercitrin, or 50 μΜ polyP (Ca²⁺ salt). RNA is extracted, cDNA is prepared and the PCR reactions are performed using a thermocycler (e.g., iCycler; Bio-Rad). After determination of the Ct values the expression of the respective transcripts is calculated.

Additional methods

The results are statistically evaluated. The DNA content can be determined, for example, by application of the PicoGreen method (Schroder HC, Borejko A, Krasko A, Reiber A, Schwertner H, Miiller WEG (2005) Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces. J Biomed Mat Res Part B - Appl Biomater 75B:387-392) using calf thymus DNA as a standard.

Claims

1. Pharmaceutical composition, comprising quercetin, a quercetin derivative or another flavonoid, and inorganic polyP or a complex of inorganic polyP and divalent metal ions.

2. The composition according to claim 1, wherein said quercetin derivative is isoquercitrin.

3. A composition according to claim 1 or 2, wherein said flavonoid is a flavonol or flavone, such as, for example, selected from chrysin, kaempferol, myricetin and apigenin, or a flavanone or flavanonol, such as, for example, selected from naringenin and taxifolin, or a glycoside of these compounds, or a mixture of flavonoids, like vitamin P preparations/supplements.

4. The composition according to any of claims 1 to 3, wherein said divalent metal ions are calcium ions [polyP (Ca²⁺ complex)].

5. The composition according to any of claims 1 and 4, wherein the chain lengths of the inorganic polyP molecules are in the range of about 2 to about 1000 phosphate units, preferably in the range of about 4 to about 100 phosphate units, and most preferred about 40 phosphate units.

6. The composition according to any of claims 1 to 5, further containing monomeric silicic acid (orthosilicic acid) and/or polymeric silicic acid (silica).

7. The composition according to claim 6, wherein said polymeric silicic acid has been formed by an enzyme or protein involved in biosilica (amorphous, hydrated silicon oxide) metabolism, such as silicatein or a silicatein fusion protein or combinations thereof.

8. The composition according to any of claims 1 to 7, comprising silicatein or a silicatein fusion protein or combinations thereof, together with a suitable substrate.

9. The composition according to claim 7 or 8, wherein said silicatein polypeptide or silicatein fusion protein has been produced using a prokaryotic or eukaryotic expression system, or has been produced synthetically.

10. The composition according to any of claims 1 to 9, further comprising calcium carbonate, wherein said calcium carbonate preferably has been formed enzymatically using carbonic anhydrase in the presence of calcium ions.

11. The composition according to any of claims 1 to 10, wherein said composition is encapsulated in an organic polymer.

12. The composition according to claim 11, wherein said organic polymer consists of shellac, alginate, or poly(lactic acid), or poly(D,L-lactide)/polyvinyl pyrrolidone-based microspheres.

13. The composition according to any of claims 1 to 12 in the form of an injectable material, as an oral dosage form or as a parenterally-administrable dosage form.

14. The composition according to any of claims 1 to 13 for use in medicine.

15. The composition according to any of claims 1 to 13 for use in the prophylaxis and/or treatment of osteoporosis and bone disorders.