WO2010028432A1

WO2010028432A1 - Protocols for treating bone disorders

Info

Publication number: WO2010028432A1
Application number: PCT/AU2009/001177
Authority: WO
Inventors: Antonio Rajic; Dominic J. Autelitano; Ana K. Vrkic; Christopher G. Hosking; Leodevico L. Ilag
Original assignee: Healthlinx Limited
Priority date: 2008-09-11
Filing date: 2009-09-08
Publication date: 2010-03-18

Abstract

The present invention relates generally to the field of bone disorders and in particular to the control of osteoporosis and related conditions including bone resorption and complications arising therefrom.

Description

PROTOCOLS FOR TREATING BONE DISORDERS

FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2008904746, filed on 11 September, 2008, the entire contents of which are incorporated herein by reference.

FIELD

[0002] The present invention relates generally to the field of bone disorders and in particular to the control of osteoporosis and related conditions including bone resorption and complications arising therefrom.

BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Bone formation and bone resorption are closely coupled processes involved in normal remodeling of bone. Remodeling involves the removal of bone by osteoclasts (bone resorbing cells) and the subsequent replacement of the defect by osteoblasts (bone forming cells) and therefore maintaining bone homeostasis. When there is an imbalance in bone homeostasis this leads to the condition of "osteoporosis" resulting in more bone resorption than formation and a net loss of bone. Osteoporosis is a debilitating disease in humans and a major cause of morbidity and health expenditure in ageing populations by increasing susceptibility to fracture. Many medications reduce the risk of bone fractures in people with osteoporosis but the most commonly used drugs have not been proven more effective than conventional methods using calcium and vitamin supplements. There is a need to identify more efficacious agents for the treatment of bone disorders. [0005] α-Casein [(si) 23 IcDa and (s2) 25 kDa], β-casein (24 kDa) and κ-casein (19 kDa) are the most abundant proteins found in milk accounting for 80% of total protein. Bioactive peptides have been reported from α-casein and β-casein peptide fragments which have antiproliferative, pro-apoptotic, angiotensin-1-converting enzyme (ACE)- inhibition, immunomodulatory, opioid antagonists and pro-proliferative properties, whereas κ-casein bioactive peptides have been shown to be anti-thrombotic and opioid antagonists (Meisel, Curr Med Chem. 12(16):19Q5-1919, 2005; Meisel and Bockelmann, Antonie van Leeuwenhoek 76(l-4):207-2\5, 1999). Caseins are a slow-digesting source of amino acids as opposed to the fast-digesting whey protein; they also provide an extremely high source of glutamine (post-workout muscle building supplements).

[0006] The other abundant bovine milk proteins, whey, account for nearly 20% of total protein in milk. Alpha-lactalbumin, β-lactoglobulin, lactoperoxidase and lactoferrin are the major proteins found in whey. Alpha-lactalbumin and β-lactoglobulin are 14 kDa and 18 kDa bovine milk proteins respectively, making up 70% of total whey protein; they are a source of essential and branched amino acids. Both intact and hydrolyzed α-lactalbumin and β-lactoglobulin have very broad biological activity that include angiotensin-1- converting enzyme (ACE) inhibition, opioid agonists, immunomodulatory and anti- carcinogenic activities (Chatterton et al, J, Diary Journal 16(17): 1229-1240, 2006; Miesel, Curr Med Chem 12(16):\9O5-1919, 2005). The broad bioactivity of α-lactalbumin, β- lactoglobulin and their hydrolysates illustrates the nutritional value of these proteins. However, there had been no association published on the inhibitory bioactivity of hydrolysates of α-lactalbumin and β-lactoglobulin on bone resorption.

[0007] Lactoferrin is an 80 kDa iron binding glycoprotein found in bovine milk, making up 1-2% of total whey protein and is one of the most studied multifunctional proteins in milk. Lactoferrin and hydrolysates of lactoferrin have broad biological activities that include anti-oxidant, anti-bacterial, anti-viral, anti-fungal, immunomodulatory, and promote growth of beneficial bacteria (Marshall, Altern. Med. Rev. 90:136-156, 2004; Miesel, Curr Med Chem 12(16)ύ905-\9l9, 2005). As well as the aforementioned biological activity, lactoferrin has been extensively studied and found to be an anabolic factor in skeletal tissue able to stimulate skeletal growth and inhibit bone resorption (International Patent Publication No. WO 2003/082921).

[0008] Lactoperoxidase is a 78 kDa enzyme, making up 0.5% of total whey protein and has broad anti-microbial bioactivity and commonly added to products as a preservative. In addition to these anti-microbial effects, bovine lactoperoxidase is known to catalyze the oxidation of a number of organic molecules such as thiols, phenols, catechol amines, steroid hormones, halides and nitrite (Marshall, Altern. Med. Rev. 9(2):136-156, 2004; Miesel, Curr Med Chem 12(16):\905-\919, 2005).

[0009] It is proposed herein to use milk products and whey in a nutraceutical and medicinal approach to inhibit or reduce osteoclast activity in the treatment or prophylaxis of bone disorders such as osteoporosis and other bone resorption disorders.

SUMMARY

[0010] The present invention is predicated in part on the identification of regulators of osteoclast activity including osteoclast resorption which in turn regulate bone resorption. The regulators are identified from milk proteins including whey. However, the present invention extends to mimetics and agonists of these proteins from non-dairy sources. The regulators are, in particular, inhibitors of osteoclasts and bone resorption. The identification of the regulators of osteoclast and bone resorption enables a nutraceutical and therapeutic approach to controlling bone disorders such as osteoporosis and related conditions and complications arising therefrom including brittle bones and bone fractures. The regulators identified from milk proteins comprise α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase or crypteins or muteins thereof. The generation of crypteins is disclosed in International Publication No. WO 2004/008148 and review by Autelitano et al, Drug Discovery Today 77:306-314, 2006, which is incorporated herein by reference. It is proposed to utilize the osteoclast inhibitors in nutraceutical and pharmaceutical formulations, as food additives and supplements and as targets for the generation of mimetics and agonists to treat or prevent bone disorders.

[0011] Accordingly, one aspect herein contemplates a method for inhibiting osteoclasts or a process associated with osteoclast resorption in a subject, the method comprising administering to the subject an effective amount of an osteoclast regulator, the regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0012] Another aspect of the present invention provides a method for treating or preventing a bone disorder such as osteoporosis or a related condition or a complication arising therefrom in a subject, the method comprising administering to the subject an effective amount of an osteoclast regulator, the regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0013] Still another aspect of the present invention is directed to the use of a regulator of osteoclast resorption selected from the list consisting of (i) α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii); in the manufacture of a nutraceutical, or food supplement to control osteoporosis or a related condition in a subject.

[0014] Even yet another aspect of the present invention provides for the use of a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii) in the manufacture of a medicament to control osteoporosis or a related condition in a subject.

[0015] Another aspect of the present invention contemplates a nutraceutical formulation comprising a regulator of osteoporosis resorption selected from the list consisting of (i) α- casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0016] A therapeutic composition is also provided when used in the treatment of osteoporosis or a related condition or a complication arising therefrom in a subject, the composition comprising a regulator of osteoclast resorption selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii), the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.

[0017] Even yet another aspect of the present invention is directed to a food additive or supplement which reduces osteoporosis or a related condition or risk of development of osteoporosis or a related condition in a subject, the additive or supplement comprising a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0018] Generally, the nutraceutical formulations herein inhibit osteoclast resorption without overly affecting osteoclast numbers. The methods and compositions herein may be used alone or in combination with other therapeutic protocols for treating or managing bone disorders.

BRIEF DESCRIPTION OF THE FIGURES

[0019] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0020] Figure 1 shows the monitoring used in the generation of milk cryptein library A4 from AO. a) RP-HPLC chromatogram of 50 μg of AO; b) RP-HPLC chromatogram of 50 μg of A4; c) mass spectra of AO; d) mass spectra of A4 and e) SDS-PAGE of AO and A4, Lane 1) MW markers; Lane 2) 20 μg AO and Lane 3) 20 μg A4. (Abbreviations: A; α- lactalbumin; 0, native protein; 4, 24 hr chymotryptic digest; RP-HPLC, reverse phase high performance liquid chromatography).

[0021] Figure 2 is a graphical representation showing osteoclast assay data of dairy libraries A4 to G4. a) Osteoclast numbers and b) Osteoclast resorption activity. All samples were assayed at a final concentration of 0.42 mg/ml. (Abbreviations: A, α- lactalbumin; B, β-lactoglobulin; C, lactoferrin; D, lactoperoxidase; E, α-casein; F, β- casein; G, κ-casein; 4, 24 hr chymotryptic digest; CON; control).

[0022] Figure 3 shows the dose response relationship for osteoclast resorption in response to dairy libraries A4 to G4. Dairy libraries A4 to G4 were assayed at a) 0.42 mg/ml; b) 0.21 mg/ml and c) 0.08 mg/ml. (Abbreviations: A, α-lactalbumin; B, β-lactoglobulin; C, lactoferrin; D, lactoperoxidase; E, α-casein; F, β-casein; G, κ-casein; 4, 24 hr chymotryptic digest; CON; control; A4/2 to G4/2, assayed at 0.21 mg/ml; A4/5 to G4/5, assayed at 0.08 mg/ml).

[0023] Figure 4 is a graphical and photographic representation showing osteoclast bioassay data of milk proteins and milk cryptein libraries. Resorption and photomicrographs of a) parent milk proteins AO to GO; and b) milk cryptein libraries A4 to G4. All samples were assayed at a final concentration of 0.42 mg/ml. (Abbreviations: A, α-lactalbumin; B, β-lactoglobulin; C, lactoferrin; D, lactoperoxidase; E, α-casein; F, β- casein; G, κ-casein; 0, native protein; 4, 24 hr chymotryptic digest; CON; control; GC, granulocyte colony stimulating factor)

DETAILED DESCRIPTION

[0024] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0025] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0026] Unless otherwise indicated, the subject invention is not limited to specific formulation components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0027] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a single protein, as well as two or more proteins; reference to "an osteoclast inhibitor" includes a single osteoclast inhibitor, as well as two or more osteoclast inhibitors; reference to "the invention" includes a single or multiple aspects of an invention, and so forth.

[0028] Embodiments disclosed and described herein relate to the identification of proteins, and crypteins and muteins thereof and fractions comprising one or more proteins from milk or milk product which inhibit osteoclasts in a subject. Alternative sources of the proteins are also contemplated herein (e.g. soy milk). Other embodiments relate to mimetics and agonists of these proteins, crypteins and muteins from dairy and non-dairy sources.

[0029] Reference to "inhibit" in relation to osteoclasts includes retarding or otherwise delaying or reducing osteoclast resorption as well as processes associated with osteoclast resorption. The term may also be used in relation to inhibiting osteoclast activity. Generally, this occurs without affecting osteoclast numbers. The inhibition may be transient or permanent and may be total inhibition or partial inhibition. One consequence of osteoclast resorption is osteoporosis. In this condition, osteoclasts outperform osteoblasts resulting in more bone being removed than is laid down. The result is bone resorption and a thinning of the bone with an accompanying loss in bone strength and a greater risk of fracture. A thinning bone further results in a lower bone density or bone mass. Hence, reference to inhibition of osteoclasts can be considered, in one embodiment, as the inhibition of osteoclast development or activity or resorption. In a particular embodiment, osteoporosis is inhibited or otherwise controlled and this leads to inhibiting bone resorption.

[0030] Accordingly, one aspect herein contemplates a method for inhibiting osteoclasts or a process associated with osteoclast resorption in a subject, the method comprising administering to the subject an effective amount of an osteoclast regulator, the regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0031] Milk or a milk by-product or alternative source is subjected to fractionation and/or separation means to generate groups of proteins or single proteins which have an inhibitory effect on osteoclasts in an in vitro screening assay (e.g. osteoclast resorption bioassay). The fractionation and separation processes includes, but no limited to, ion exchange chromatography, size-based chromatography, affinity-binding chromatography including lectin-binding and immunoaffinity chromatography. The generation of crypteins is disclosed in International Publication No. WO 2004/008148 and reviewed by Autelitano et al, 2006 supra, which are incorporated herein by reference. The proteins or peptides or fractions or crypteins obtained may also be subjected to random or site-directed mutagenesis to generate mutated molecules referred to herein as "muteins". A "protein" herein includes a "glycoprotein" and other post translational modifications. The protein may also be regarded as a peptide (or glycopeptide). [0032] Hence, in an embodiment, bioactive molecules are identified on the basis of an in vitro assay of osteoclast resorption assay and are referred to herein as inter alia "osteoclast regulators", "osteoclast modulators", "osteoclast inhibitors", "osteoclast medicaments" or other like terms. Notwithstanding the osteoclast regulators may also have other biological activities such as inhibiting a process associated with osteoclast resorption. Generally, the osteoclast regulators do not affect osteoclast numbers. The regulators, modulators, inhibitors and medicaments referred to herein may be of dairy origin or non-dairy origin.

[0033] A method is provided for identifying an osteoclast regulator from milk or milk product or other alternative source, the method comprising providing a library of proteins, protein-containing fractions, crypteins and/or muteins from the milk or milk product and screening the library for an ability to inhibit osteoclast resorption in an in vitro assay, optionally subjecting protein or cryptein or mutein fractions to separation means to identify particular molecules or groups of molecules or sub-fractions which inhibit osteoclast resorption and then identifying the osteoclast regulator as an isolated protein, sub-fraction, cryptein or mutein.

[0034] As indicated above, the library is generated by fractionation of milk or a milk product or other alternative source by any one of a number of chromatographic and molecule-separating methods. Particular methods include, but not limited to, ion exchange chromatography, affinity chromatography such as immunoaffmity chromatography as well as lectin-based affinity chromatography.

[0035] The assay to identify the osteoclast regulators, in a particular embodiment, comprises subjecting a protein source such as but not limited to milk or milk product to digestion, cleavage and/or reduction to generate a series of digested or reduced fractions, subjecting each fraction to an osteoporosis bioassay assay to identify fractions having osteoporosis resorption inhibiting activity, and then optionally subjecting the active fractions to separation or purification means to generate single proteins, crypteins, muteins or enriched sub-fractions having osteoporosis regulator activity. [0036] The term "protein" as used herein shall be taken to refer to any polymer consisting of amino acids linked by covalent bonds and this term includes within its scope parts or fragments of full length proteins, such as, for example, polypeptides, peptides and shorter peptide sequences consisting of at least two amino acids, more particularly at least about 5 amino acid residues. The term "protein" includes all moieties containing one or more amino acids linked by a peptide bond. In addition, this term includes within its ambit polymers of modified amino acids, including amino acids which have been post- translationally modified, for example by chemical modification including but not restricted to glycosylation, phosphorylation, acetylation and/or sulphation reactions that effectively alter the basic peptide backbone. Accordingly, a protein herein may be derived from a naturally- occurring protein, and in particular may be derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, or proteases such as trypsin or chymotrypsin, amongst others. Alternatively, such peptides may be derived by chemical synthesis using well known peptide synthetic methods. In another alternative, the proteins are isolated following, for example, ion exchange or affinity chromatography.

[0037] Also included within the scope of the definition of a "protein" are amino acid sequence variants (referred to herein as "muteins"). These may contain one or more amino acid substitutions, deletions, or insertions in a naturally-occurring amino acid sequence. Such muteins may be synthesized by chemical peptide synthesis. Amino acid substitution reactions are well-known in the ait. Rules for making such substitutions are well known. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in their side chains. Genetically-encoded amino acids are generally divided into four groups: (1) acidic aspartate, glutamate; (2) basic=lysine, arginine, and histidine; (3) non- polat-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tyrosine and tryptophan are also jointly classified as aromatic amino acids.

[0038] Included within the scope of the definition of a "protein" are muteins which have undergone mutation including unnatural modifications such as but not limited to protection, carboxylation, and derivatization by amide and non-amide bonds as well as covalent and non-covalent modification.

[0039] The term "protein" also includes recombinantly generated polypeptides, oligopeptides or shorter peptide sequences. Protein which is produced in vitro or in a host cell by the expression of a genetic sequence encoding the protein, which genetic sequence is under the control of a suitable promoter, wherein a genetic manipulation has been performed in order to achieve said expression. Accordingly, the term "recombinant protein" clearly encompasses proteins produced by the expression of genetic sequences contained in viral vectors, cosmids or plasmids that have been introduced into prokaryotic or eukaryotic cells, tissues or organs. Genetic manipulations which may be used in this context will be known to those skilled in the art and include, but are not limited to, nucleic acid isolation, restriction endonuclease digestion, exonuclease digestion, end-filling using the Klenow fragment of E. colt DNA polymerase I to T4 DNA polymerase enzymes, blunt-ending of DNA molecules using T4 DNA polymerase or ExoIII enzymes, site- directed mutagenesis, ligation, and amplification reaction.

[0040] In the method herein, the initial library of proteins comprises a heterogeneous and unfractionated mixture of proteins derived from a precursor protein source (or protein mixture or protein-containing biological extract) such as milk which provides a comprehensive range of potentially bioactive proteins or crypteins or muteins.

[0041] The library is conveniently subjected to initial analysis or characterization to provide information on the activity or size or other characteristics of the component proteins, for example by osteoclast resorption bioassay or matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS).

[0042] Initial screening of the library to confirm that it includes bioactive proteins may particularly be carried out using an in vitro osteoclast resorption assay. [0043] After the library has been confirmed as including bioactive proteins, it is fractionated by suitable means of fractionation including but not limited to chromatographic methods such as, but not limited to, ion exchange, size exclusion, hydrophobic interaction and/or reverse phase-high performance liquid chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis in order to provide fractions of the library for subsequent further screening. This further screening may be carried out by any suitable screening assay or assays as discussed above so as to identify an active fraction or active fractions which include bioactive proteins.

[0044] Since such active fractions may include more than one protein, each fraction may, if desired, be subjected to one or more further cycles of fractionation by suitable means of fractionation including but not limited to chromatography, field-flow fractionation (including but not limited to sedimentation, flow, thermal and steric), and electrophoresis to form sub-fractions, followed by screening of each sub-fraction as described above so as to identify an active sub-fraction or active sub-fractions which include bioactive proteins (i.e. sub-fractions or proteins which have osteoclast regulator activity).

[0045] Each fraction or sub-fraction which is produced may also be subjected to analysis or characterization as described above, for example by osteoclast resorption bioassay or MALDI-ToF MS, so as to provide information on the activity, size or other characteristics of the component proteins in the fraction or sub-fraction.

[0046] Using this approach, α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase as well as crypteins and muteins thereof and fractions comprising same are proposed to be osteoporosis regulators useful in the inhibition of bone resorption and osteoporosis and related conditions. Non-dairy mimetics or agonists of the proteins and crypteins and muteins are also contemplated herein.

[0047] The source of the milk is not critical to the practice of the embodiments herein and may be from any mammal including a human, cow, sheep, goat, horse, pig, camel, laboratory test animal (e.g. mouse, rat, rabbit, guinea pig), companion animal (e.g. dog, cat) or captive wild animal (e.g. elephant, zebra, kangaroo). Once the protein has been identified as an inhibitor of osteoclasts it may also be sourced elsewhere from milk. The term "milk" includes soy milk and other legume milks.

[0048] The identification of proteins which inhibit osteoclasts enables their use to treat or prevent bone disorders and complications arising therefrom such as osteoporosis, weak bones and even some forms of bone cancers.

[0049] Accordingly, another aspect of the present invention provides a method for treating or preventing a bone disorder such as osteoporosis or a related condition or a complication arising therefrom in a subject, the method comprising administering to the subject an effective amount of an osteoclast regulator, the regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0050] In a related embodiment, provides for the use of a regulator of osteoporosis selected from the list consisting of (i) α-casein, β-casein, K-casein, α-lactalbumin, β- lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii) in the manufacture of a nutraceutical or food supplement to control osteoporosis or a related condition in a subject.

[0051] Even yet another aspect of the present invention provides for the use of a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α- lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii) in the manufacture of a medicament to control osteoporosis or a related condition in a subject.

[0052] The "subject" as used herein refers to an animal, particularly a mammal and more particularly a primate including a lower primate and even more particularly a human who can benefit from the formulations and methods of the present invention. A subject regardless of whether a human or non-human animal may be referred to as a subject, an individual, patient, animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.

[0053] As indicated above, particular subjects are humans, non-human primates such as marmosets, baboons, orangutangs, lower primates such as tupia, livestock animals, laboratory test animals, companion animals or captive wild animals. A human is the most preferred target. However, non-human animal models may be used.

[0054] Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses and donkeys.

[0055] The osteoclast regulators herein may be formulated from any convenient manner such as in a nutraceutical formulation or a pharmaceutical formulation using standard formulation technology.

[0056] Another aspect of the present invention contemplates a nutraceutical formulation comprising a regulator of osteoporosis selected from the list consisting of (i) α-casein, β- casein, ic-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0057] A therapeutic composition is also provided when used in the treatment of osteoporosis or a related condition or a complication arising therefrom in a subject is provided herein, the composition comprising a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii), the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.

[0058] Even yet another aspect of the present invention is directed to a food additive or supplement which reduces osteoporosis or risk of developing same in a subject, the additive or supplement comprising a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

[0059] Kits are contemplated herein such as comprising compartments or containers each containing one or more of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (iϊ) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii). The kits may be packaged for sale as food additives or supplements or may be in the form of nutraceutical or pharmaceutical packs. For example, the proteins may be in a freeze dried form, the contents of which may be reconstituted prior to use in food or as a medicament.

[0060] The present invention extends to the use of the methods and regulators alone or in combination with bone disorder therapy or prophylaxis including physiotherapy.

[0061] The present invention is further described by the following non-limiting Examples. In the Examples, crypteins were generated from milk as follows:

Step 1 - Digestion of protein(s)

[0062] Milk or milk product or a fraction of milk protein is cleaved into peptide components using, but not limited to, purified proteases, protease activity-containing extracts, chemical cleavage or other mechanism of protein fragmentation. The overall approach is to generate a library of peptides that comprises a heterogeneous and unfractionated mixture of peptides from the intact milk protein(s). A source other than milk may also be employed. Such a library can comprise partial, intermediate and/or complete digestions of milk proteins thus providing as comprehensive a range of protein fragments as possible. A wide range of fragments can be achieved by varying a range of conditions including digestion times, pH, buffer and temperature conditions and substrate to enzyme ratios. Additionally, cleavage of the proteins may be performed by a range of cleavage agents (including but not limited to protease and chemical cleavage) each having a defined and different protein cleavage specificity. Cleavage may also be performed either in series or in parallel, such that different combinations of these digestion methodologies can be used to generate distinct peptide species. Accordingly, each variation on a digestion condition will generate a distinct library of peptides from a particular protein source.

[0063] By way of example only, an automated procedure using a MALDI-ToF MS instrument for measuring the mass of the resulting peptides and an X-Y pipeting workstation controlled by a PC for the delivery of reagents and the extraction of reactants enables the operator to determine the optimal digest parameters to thus obtain the optimal peptide profile for further downstream experiments.

[0064] The method consists of the following steps: 1.

1. The protein or protein fraction to be cleaved is placed in several wells of a thermostated standard microtiter plate (MTP) at preset buffer conditions such as concentration and pH or other parameters relevant to the experiment in question.

2. The cleaving agent (i.e. enzyme or chemical) is added to the wells at time zero.

3. Aliquots (typically a few μl) are removed and deposited to a second MTP preloaded with quenching agent, typically a solution of dilute acid. Several aliquots are taken at successive time points as programmed into the PC controlling the pipeting station software for the duration of the experiment. Exponentially increasing time points are set starting at 5 minutes (e.g. 5, 10, 20, 40, 80, 160, 320, 640 and 1280 minutes-etc), however each time point can be set as required by the experiment in question. 4. Following the extraction of digest mixture at each time point, the X-Y pipeting workstation creates a mirror image of the second microtiter plate on an Anchorchip (Registered Trademark) target plate. The peptides are mixed with matrix, dried, washed and the matrix/sample mixture on each spot is re-crystallized prior to bioactivity or MALDI-ToF MS analysis.

5. Mass spectra of all reaction mixtures are automatically recorded using the MALDI- ToF MS instrument.

6. The distribution of peptide masses measured will thus yield an indication of the degree of cleavage that has progressed. For example, the presence of intact protein or large peptides is indicative of incomplete cleavage.

7. In the case of any ambiguous results the second microtiter plate contains sufficient sample for confirmative analyzes.

[0065] Classical monitoring of protein cleavage is performed using reversed phase HPLC where reduction in the peak corresponding to the protein and incremental increase in emerging peptide peaks is taken as an expression of the progress of the cleavage. However, the position of new emerging peptide peaks is hard to predict and although eluting time and peptide time may be correlated, it is far from an ideal method. Also, partial cleavages are hard to predict using HPLC based methodology. Furthermore, although the eluted peptides may be collected and recycled, in practice, the HPLC based method requires nanomoles or a few micrograms of protein sample which is often unrecoverable.

[0066] The proposed MALDI-ToF MS method consumes low to sub picomole amount of sample or, for a medium size protein of 50 kDa, a few nanograms per analysis. A modern MALDI-ToF MS instrument is able to analyze at least a hundred samples in automatic mode as soon as the sample is spotted onto the sample or target plate and the plate is loaded into the instrument. Step 2 - Selection of digestion conditions

[0067] Unfractionated peptide libraries may be subjected to analysis, including but not limited to an osteoclast resorption assay or mass spectrometry, an example of the latter is MALDI-ToF MS; this analysis provides precise information on the size and possible identity of the component peptides. In the case of MALDI-ToF MS analysis, spectra for a number of libraries may be compared and optimal sets of digestion condition parameters determined where marked changes in peptide profile occur. These sets of digestion condition parameters can be used to generate predictable "hot-spots" where digestion should be focussed to limit the number of libraries for subsequent fractionation. In addition, these MALDI-ToF MS spectra provide an exact record of the profile of each library of peptides, providing both a level of quality control and allowing the generation of reproducible preparations for down stream analysis in cases where cellular activity assays identify "hits" for bioactive peptides in the library of peptides.

Step 3 - Identification of fractions containing bioactivity

[0068] To reduce the number of libraries to be fractionated and screened, whole unfractionated digestion mixtures are preferably subjected to initial osteoclast resorption bioassays. This step then precedes fractionation and bioactive peptide identification and allows identification of digestion mixtures for fractionation, thus reducing the number of fractionations required for the identification of each bioactive peptide.

Step 4 - Initial fractionation of digestion mixtures

[0069] Each library of peptides found to contain a biological activity in Step 3 is then fractionated by chromatographic methods including but not limited to, size exclusion, ion exchange, hydrophobic interaction and/or reverse phase-high performance liquid chromatography. To interface directly with the biological screening assays detailed in Step 3, fractions (for example 2 ml in the first instance) are preferably collected in a format that is compatible with direct robot driven transfer into the assays for biological activity outlined in step 3. Whilst not restricted to any particular format, use of 96 deep-well plates is preferred as the aim, wherever possible, is to use chromatographic solvents that will be either compatible with subsequent bioassays or suitable for freeze-drying. At this point, the collected fractions may be freeze-dried and stored. When required, the freeze-dried material can be resuspended in a cell-compatible isotonic and buffered solution.

Step 5 - Assay of collected fractions for both analysis and biological activity

[0070] Fractions are re-assayed using the osteoclast bioassay.

Step 6/7 - Subsequent re-fractionation and assay of activity-containing fractions [0071] Since active fractions identified in Step 5 may be subjected to one or more further rounds of chromatography (second and subsequent dimensions) to form sub-fractions, with each round involving monitoring of the composition of each sub-fraction by MALDI-ToF MS, and identification of active sub-fractions using activity assays as described in Step 3.

Step 8-The full identification and analysis of the bioactive peptide(s)

[0072] Any peptide moiety found to have osteoclast regulator activity performed in Step 5 is subjected to further analysis. Peptide sequence identification of a given putative bioactive peptide can be achieved through a combination of MALDI-ToF MS-post source decay-MS data and alignments to the genome databases (e.g. for cow milk, the Bovine Genome Database). Putative bioactive peptides can be validated by synthesizing analogs and substitution/sequence reversal variants and examining their ability to replicate the initial osteoclast inhibiting activities observed in the osteoclast bioassay performed in Step 5.

[0073] As an example, α-lactalbumin (AO) was subjected to hydrolysis as described in WO2004/008148 to generate cryptein library (A4). Figure 1 shows the differences observed between AO and A4 as depicted by RP-HPLC, mass spectrometry and SDS- PAGE.

[0074] All RP-HPLC runs were performed on an Agilent HPLC using a Phenomenex Jupiter Cl 8 column (300 , 2.0 x 150 mm, particle size 5μm). Elution carried out using a linear gradient of 0-70% Buffer B over 35 mins after an initial 10 min wash at a flow rate of 0.3 ml/min, Buffer A consisted of 0.1% v/v TFA pH 2 and Buffer B 0.08% v/v TFA in acetonitrile, pH 2 and separation monitored by absorption at 214 run. The AO peak at retention time 39 min (Fig Ia) disappears after being digested with chymotrypsin for 24 hr and the generation of smaller peptides at retention times 20 to 40 min appear in cryptein library A4 (Figure Ib). Mass spectra were generated on a Bruker autoflex II ToF/ToF operated in linear mode, acquiring a mass range from 2,000 to 20,000 Da (Figure Ic) and reflectron mode, acquiring a mass range from 800 to 5,000 Da (Figure Id). Samples were analyzed in HCCA (α-cyano-4-hydroxycinnamic acid) matrix with a total of 700 laser shots summed per sample. The major AO mass at approximately 14 kDa (Figure Ic) represents α-lactalbumin, the emergence of smaller peptides varying in the range of 800 to 5,000 Da of cryptein library A4 appear post 24 hr chymotryptic digestion (Figure Id). Samples were further analyzed by SDS-PAGE (Figure Ie). Both AO and A4 were resolved on 4-12% Bis-Tris gels with the NuPAGE MOPS SDS running buffer in a Novex Mini- Cell system at 200 volts and visualized by staining with SimplyBlue safe stain. The same pattern is observed by SDS-PAGE analysis with the disappearance of the major AO band at approximately 14 kDa and the emergence of lower molecular weight bands of cryptein library A4 appear post 24 hr chymotryptic digestion (Figure Ie).

[0075] The milk cryptein library A4 can be monitored by RP-HPLC, mass spectrometry and SDS-PAGE. The clear differences from parent protein, AO, to the generated cryptein library, A4, serve as a QC measure and show the diverse range of crypteins able to be generated from a single milk protein.

EXAMPLE 1

Screening of dairy libraries in osteoclast bioassays

[0076] Milk or milk protein was subjected to fractionation and separation by ion exchange chromatography or chymotryptic digestion and fractions assayed in an osteoporosis resorption bioassay.

[0077] The assay is a modification of the published method described in Hodge et al, J. Bone Miner Res 19(2): 190-199, 2004 and is described below. All fractions (referred to as libraries) are assayed at a final concentration of 0.42 mg/ml.

[0078] Figure 2 show the result of (a) osteoclast numbers and (b) osteoclast resorption activity of milk crypteins A4 to G4. Human umbilical cord mononuclear cells incubated for 10 days in methylcellulose containing granulocyte macrophage-colony stimulating factor (GM-CSF, 10 ng/ml), interleukin 3 (IL-3, 10 ng/ml), and stem cell factor (SCF, 50 ng/ml) to generate colony forming unit-granulocyte macrophage (CFU-GM). The CFU- GM derived cells (osteoclast precursor cells) are incubated for 14 days in a 96-well plates containing 4 x 4 x 0.1 mm slices of sperm whale dentine in medium containing soluble receptor activator of NF-κB ligand (RANKL, 125 ng/ml) and human macrophage-colony stimulating factor (M-CSF, 25 ng/ml) either alone as control or with 10 μl of test libraries. Medium and test libraries are changed bi-weekly, osteoclast cells were fixed with 1% formalin and reacted for tartrate-resistance acid phosphatase (TRAP). Osteoclast number and size quantitified by a semi-automated image analysis and dentine slice stripped of osteoclasts and treated with black ink to identify resorption pits. Resorbed area, number of resorption pits, pit size and resorption/osteoclast quantified by image analysis. Bar represents mean ± SEM (n=6) for control and test libraries. Groups with different superscripts are significantly different (p < 0.0001; one-way ANOVA; Fishers multiple comparison test).

[0079] The results in Figure 2 refer to 24 hr chymotryptic digests of α-lactalbumin (A4), β-lactoglobulin (B4), lactoferrin (C4), lactoperoxidase (D4), α-casein (E4), β-casein (F4) and K-casein (G4) that were generated as outlined in WO2004/008148. Libr aries α- lactalbumin (A4), β-lactoglobulin (B4), lactoperoxidase (D4), α-casein (E4), β-casein (F4), K-casein (G4) as inhibitors of osteoclast resorption. All libraries decreased osteoclast numbers. Libraries A4, B4, and G4 decreased osteoclast numbers by 50%, C4 by 80% and D4 to F4 by 25% (Figure 2a). As well as decreasing osteoclast numbers all libraries reduced osteoclast resorption. Libraries A4 to D4 reduced osteoclast resorption proportionally to their reduction in osteoclast number, while libraries E4 to G4 disproportionately decreased osteoclast resorption compared to their reduction in osteoclast number (i.e. comparing libraries D4 and E4; both reduced osteoclast numbers by approximately 25% but libraries D4 and E4 reduced resorption by approximately 50% and 80% respectively). The libraries A4, B4, E4, F4, G4 decreased osteoclast resorption by >80%, D4 by 50% (Figure 2b), and libraries E4 to G4 inhibit activity of osteoclasts more potently than osteoclast generation. Thus, libraries A4, B4, D4, E4, F4, and G4 have the potential to regulate of osteoporosis. Library C4 is a lactoferrin hydrolysate, similar to a preparation that has been described previously to inhibit bone resorption (International patent Publication No. WO 2003/082921).

EXAMPLE 2

Osteoclast bioassay of milk crypteins

[0080] In Example 3, milk libraries A4 to G4 were re-screened and a dose response curve established. Figures 3 (a) to (c) show a repeat of the osteoclast resorption bioassay data of milk libraries A4 to G4 at a final concentration of (a) 0.42 mg/ml; (b) 0.21 mg/ml; and (c) 0.08 mg/ml. Libraries A4 to G4 are 24 hour chymotryptic hydrolysates of proteins α- lactalbumin (A4), β-lactoglobulin (B4), lactoferrin (C4), lactoperoxidase (D4), α-casein (EA), β-casein (F4) and κ-casein (G4) generated as outlined in WO2004/008148. Libraries were assayed in osteoclast assays as outlined in Example 2 and bars represents mean ± SEM (n=6) for control and test libraries. Groups with different superscripts are significantly different (p < 0.0001; one-way ANOVA; Fishers multiple comparison test). At the highest concentration 0.42 mg/ml all libraries A4 to G4 variously decreased osteoclast resorption ranging from 34 % to 73 % (Figure 3a), libraries B4, C4, F4 inhibited resorption by 50% and libraries E4 and G4 inhibited resorption by 75% when assayed at 0.42 mg/ml. To further verify anti-resorptive activity all libraries were assayed at concentrations of 0.21 mg/ml (A4/2 to G4/2) and 0.08 mg/ml (A4/5 to G4/5) [Figure 3b, c]. All libraries A4 to G4 inhibit osteoclast resorption in a dose dependent fashion when assayed at three different concentrations with all osteoclast resorption activity lost at the lowest concentration of 0.08 mg/ml (Figure 3c). Libraries B4/2 and F4/2 retained capacity to inhibit osteoclast resorption by 36% and 29% respectively when assayed at 0.21 mg/ml (Figure 3b). Thus, a similar inhibitory profile of libraries A4 to G4 was re-confirmed and the osteoclast resorption inhibitory activity was shown to be dose dependent. Hence, libraries A4, B4, D4, E4, F4, and G4 have the potential to regulate of osteoporosis. As already mentioned, library C4 is a lactoferrin hydrolysate that has been previously described to inhibit bone resorption (International patent Publication No. WO 2003/082921). EXAMPLE 3 Osteoclast hioassay of milk proteins and crypteins

[0081] In Example 4, parent milk protein AO to GO and milk cryptein libraries A4 to G4 were assayed in osteoclast assay to determine whether milk cryptein libraries had unique osteoclast inhibitory activity when compared with parent milk proteins. Figures 3(a) and (b) are graphical and photographic representations of the osteoclast bioassay data of milk proteins (AO to GO) and milk cryptein libraries (A4 to G4) assayed at 0.42 mg/ml. Libraries A4 to G4 are 24 hour chymotryptic hydrolysates of proteins α-lactalbumin (A4), β-lactoglobulin (B4), lactoferrin (C4), lactoperoxidase (D4), α-casein (E4), β-casein (F4) and κ-casein (G4) generated as outlined in WO2004/008148 from parent proteins AO to GO α-lactalbumin (AO), β-lactoglobulin (BO), lactoferrin (CO), lactoperoxidase (DO), α-casein (EO), β-casein (FO) and κ-casein (GO). Libraries were assayed in osteoclast assays as outlined in Example 2 and bars represents mean ± SEM (n=6) for control and test libraries. Groups with different superscripts are significantly different (p < 0.0001; one-way ANOVA; Fishers multiple comparison test). The photographic representations reflect the graphical representations of the osteoclast bioassay data of parent milk proteins AO to GO and milk cryptein libraries A4 toG4. The milk proteins AO to FO have no effect on decreasing osteoclast resorption with the only exception being GO reducing osteoclast resorption by 50%. While all the cryptein libraries A4 to G4 decreased osteoclast resorption by 50 to >80%. These data confirm that inhibition of osteoclast resorption can be attributed to the milk crypteins present in the libraries generated and not the intact parent milk proteins from where they were derived, with the exception of GO. Bone formation and bone resorption are closely coupled processes involved in the normal remodelling of bone. Osteoclasts and osteoblasts communicate with other to maintain bone homeostasis. A desirable therapeutic approach is to reduce osteoclast resorption activity without significantly affecting osteoclast survival. Thus, libraries A4, B4, D4, E4, F4 and possibly G4 match these criteria.

[0082] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

Autelitano et al, Drug Discovery Today 77:306-314, 2006

Chatterton et al, J. Diary Journal 16(11):1229-1240, 2006

Hodge et al, J. Bone Miner Res 19(2): 190-199, 2004

Marshall, Altern. Med. Rev. 9(2)ύ36-\56, 2004

Meisel, Curr Med Chem 12(16) :1905-\9\9, 2005

Meisel and Bockelmann, Antonie van Leewenhoek 76(1-4): 207-215, 1999

International Patent Publication No. WO 2003/082921

International Patent Publication No. WO 2005/107491

International Patent Publication No. WO2004/008148

Claims

CLAIMS:

1. A method for inhibiting osteoclasts or a process associated with osteoclast resorption in a subject, said method comprising administering to the subject an effective amount of an osteoclast regulator, the regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

2. The method of Claim 1 for treating osteoporosis or a related condition or a complication arising therefrom.

3. The method of Claim 2 wherein the complication is bone resorption, bone weakness or cancer.

4. The method of Claim 1 or 2 or 3 wherein the osteoclast regulators are derived from milk or a milk product or are non-dairy mimetics or agonists thereof.

5. The method of any one of Claims 1 to 4 wherein the subject is a human.

6. The method of any one of Claims 1 to 4 wherein the subject is a veterinary, livestock or racing industry animal.

7. Use of a regulator of osteoclasts selected from the list consisting of (i) α-casein, β- casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii) in the manufacture of a nutraceutical, or food supplement to control osteoporosis or related condition in a subject.

8. Use of a regulator of osteoclasts selected from the list consisting of (i) α-casein, β- casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii) in the manufacture of a medicament to control osteoporosis in a subject.

9. Use of Claim 7 or 8 wherein the subject is a human.

10. Use of Claim 7 or 8 wherein the subject is a veterinary, livestock or racing industry animal.

11. A nutraceutical formulation comprising a regulator of osteoclasts selected from the list consisting of (i) α-casein, β -casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).

12. A therapeutic composition when used in the treatment of osteoporosis or a related condition or a complication arising therefrom in a subject, the composition comprising a regulator of osteoclast selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii), the composition further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.

13. A food additive or supplement which reduces osteoporosis or a related condition in a subject, the additive or supplement comprising a regulator of osteoclasts selected from the list consisting of (i) α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoperoxidase; (ii) a cryptein or mutein of (i); and (iii) a mimetic or agonist of a regulator from group (i) or (ii).