HK1074635B - Lactoferrin - Google Patents

Description

Lactoferrin

RELATED APPLICATIONS

The present application claims priority from new zealand application No. 518121 filed on 3/4/2002, the contents of which are incorporated herein by reference.

Background

Lactoferrin is an 80KD iron-binding glycoprotein found in most of the external secretions including tears, bile, bronchial mucus, gastrointestinal fluids, cervical-vaginal mucus, semen, and milk. It is the main component of the secondary specific particle of the circulating polymorphonuclear neutrophils. The most abundant sources of lactoferrin are mammalian milk and colostrum.

The lactoferrin content in the circulation is 2-7 μ g/ml. It may have a variety of biological functions including iron metabolism, immune function, and regulation of embryonic development. Lactoferrin has antimicrobial activity against a variety of pathogens, including gram-positive and gram-negative bacteria, yeast, and fungi. The antimicrobial effect of lactoferrin results from its binding ability to iron necessary for pathogen growth. Lactoferrin also inhibits the replication of a variety of viruses and increases the susceptibility of certain bacteria to antibiotics by binding to the liposome a component of lipopolysaccharide on bacterial membranes.

Summary of The Invention

The present invention relates to lactoferrin polypeptides having the effects of stimulating bone growth and inhibiting bone resorption. In particular, the present invention relates to pure lactoferrin polypeptides containing no more than 2 (i.e., 0, 1, or preferably 2) metal ions per molecule. A "pure" polypeptide refers to a polypeptide that is free of other biological macromolecules and that is at least 65% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%) pure in dry weight. The purity of the polypeptide may be determined by any suitable standard method, for example by column chromatography, polyacrylamide gel electrophoresis or HPLC analysis. The lactoferrin polypeptide can be a naturally occurring polypeptide, a recombinant polypeptide, or a synthetic polypeptide. Variants of wild-type lactoferrin polypeptides that retain the biological activity of the wild-type lactoferrin polypeptide, such as wild-type lactoferrin polypeptide fragments that contain at least 2 (e.g., 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700) amino acids or recombinant proteins that contain lactoferrin polypeptide sequences, are also within the scope of the invention. The lactoferrin polypeptides of the invention can be of mammalian origin, e.g., from human or bovine milk. The metal ion bound to the polypeptide may be an iron ion (as in naturally occurring lactoferrin polypeptides), a copper ion, a chromium ion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion.

Lactoferrin polypeptides of the invention can be used to stimulate bone growth (e.g., by promoting the proliferation of osteoblasts and chondrocytes) and to inhibit bone resorption (e.g., by inhibiting the growth of osteoclasts). Lactoferrin polypeptides prepared by the invention (e.g., lactoferrin isolated from milk) can include a single species of polypeptide, e.g., a polypeptide that binds 2 ferric ions per molecule. It may also comprise different kinds of polypeptides, for example where some molecules do not bind ions and others bind 1 or 2 ions per molecule; some molecules bind iron ions and others bind copper ions; some molecules are biologically active lactoferrin polypeptides (full length or shorter than full length) containing 0, 1, or 2 metal ions and others are fragments of the polypeptide (the same or different); or all molecules are fragments (identical or different) of the full-length lactoferrin polypeptide containing 0, 1, or 2 metal ions. For example, a mixture of full-length lactoferrin polypeptides and various fragments of full-length lactoferrin polypeptides can be prepared from a hydrolysate of the full-length lactoferrin polypeptide, e.g., an incomplete digest such as a protease digest. Alternatively, the mixture can be prepared by mixing the full-length lactoferrin polypeptides with various fragments (e.g., synthetic fragments) thereof. Alternatively, mixtures of various fragments of a full-length lactoferrin polypeptide can be prepared, for example, by complete digestion of the full-length lactoferrin polypeptide (i.e., no full-length polypeptide is present after digestion) or by mixtures of different fragments of the full-length lactoferrin polypeptide.

The invention also relates to a nutritional composition, which may be a milk, juice, beverage, snack food or food supplement. The nutritional composition contains a lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments thereof in an amount higher than the natural amount. Lactoferrin has been found to stimulate the proliferation of osteoblasts and chondrocytes and to inhibit the growth of osteoclasts. Therefore, the nutritional composition of the present invention may be used for the prevention and treatment of bone disorders such as osteoporosis, rheumatoid or osteoarthritis. The nutritional composition may also include another bone-strengthening ingredient such as calcium, zinc, magnesium vitamin C, vitamin D, vitamin E, vitamin K2, or mixtures thereof, in appropriate amounts.

In addition, the invention also relates to a pharmaceutical composition containing the lactoferrin polypeptide or the mixture of the polypeptide and fragments thereof and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may further comprise other bone-enhancing agents. The invention also relates to the use of the lactoferrin polypeptide or a mixture of the polypeptide and fragments thereof in the manufacture of a medicament for the prevention and treatment of bone diseases.

The present invention provides methods for preventing and treating bone-related disorders (e.g., by stimulating bone growth and inhibiting bone resorption). The method comprises administering to the individual an effective amount of a lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments thereof in need thereof. The method further comprises concurrently administering to the individual an effective amount of another bone-enhancing agent.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Detailed Description

The present invention is based on the surprising discovery of the property of lactoferrin to stimulate osteoblast and chondrocyte proliferation and inhibit osteoclast growth. Therefore, lactoferrin can be used for the prevention and treatment of bone disorders.

The lactoferrin polypeptides of the invention are pure polypeptides that contain no more than 2 metal ions per molecule. In fact, examination of the ion/lactoferrin ratio of the lactoferrin produced indicates that the ratio can range from 0 to 2.5. It may be isolated from a natural source (e.g., mammalian milk) or produced using genetic engineering or chemical synthesis techniques well known to those skilled in the art. The following is an exemplary procedure for isolating lactoferrin from milk:

fresh skim milk (7L, pH6.5) was passed through a 300ml S Sepharose Fast Flow column equilibrated with ultrapure water (milli Q water) at 4 ℃ at a Flow rate of 5 ml/min. Unbound protein was removed by washing with 2.5 bed volumes of water and bound protein was eluted stepwise with approximately 2.5 bed volumes of 0.1M, 0.35M and 1.0M NaCl, respectively. The pink lactoferrin band eluted with 1M NaCl was collected as a single component and dialyzed against milli Q water followed by lyophilization. The lyophilized powder was dissolved in 25mM, pH6.5 sodium phosphate buffer and re-chromatographed in S Sepharose Fastflow column using the above buffer at a flow rate of 3ml/min with a NaCl concentration gradient to 1M. Fractions containing lactoferrin of sufficient purity were pooled together by gel electrophoresis and reverse phase HPLC analysis, dialyzed and lyophilized. The final lactoferrin pure product was obtained by gel filtration on Sephacryl 300 in 80mM dipotassium phosphate solution containing 0.15M KCl at pH 8.6. The selected fractions were combined, dialyzed against milli Q water and lyophilized. HPLC analysis and the fact that the spectral ratio of the iron ion saturated form of lactoferrin (280nm/465nm) is 19 or less indicate that the lactoferrin produced is more than 95% pure.

Iron ion saturation of lactoferrin was achieved by adding an excess of 5mM ferric nitrilotriacetate (ferrinitrilotriacetate) in a 1: 2 molar ratio to a 50mM Tris, 10mM sodium bicarbonate solution containing 1% purified lactoferrin at pH7.8 (Foley and Bates (1987) Analytical Biochemistry 162, 296-300). The excess of iron nitrilotriacetate was removed by dialysis against 100 volumes of milli Q water (refreshed twice) for 20 hours at 4 ℃. The iron ion-loaded (holo-) lactoferrin was then lyophilized.

An aqueous solution of a 1% sample of highly purified lactoferrin was dialyzed at 4 ℃ for 30 hours against a 30-fold volume of a solution containing 0.1M citric acid and 500mg/L disodium EDTA at pH2.3 to prepare deironized (apo-) lactoferrin (Massons and Heremans (1966) preparations of the Biological fluids 14, 115-. Citrate and EDTA were removed by dialysis against 30 volumes of milli Q water (refreshed) and the resulting colorless solution was lyophilized.

The lactoferrin polypeptides of the invention can contain iron ions (e.g., in naturally occurring lactoferrin polypeptides) or non-ferrous metal ions (e.g., copper ions, chromium ions, cobalt ions, manganese ions, zinc ions, or magnesium ions). For example, iron ions may be removed from lactoferrin isolated from milk, and then loaded with other types of metal ions. For example, the loading of copper ions can be achieved by the same method as the loading of iron ions described above. The method of Ainscough et al ((1979) Inorganica Chimica Acta33, 149-153) can be used to load other metal ions onto lactoferrin.

In the preparation of the lactoferrin polypeptides of the invention, the polypeptides may be of a single species or of different species. For example, each of the polypeptides may contain a different number of metal ions or different types of metal ions; or the polypeptides may be of different lengths, e.g., some are full-length polypeptides and others are fragments, and each of the fragments can represent a particular portion of a full-length polypeptide. Such formulations may be obtained from natural sources or by mixing different classes of lactoferrin polypeptides. For example, a mixture of lactoferrin polypeptides of different lengths can be prepared by protease digestion (in whole or in part) of full-length lactoferrin polypeptides. The degree of digestion can be controlled according to methods known to those skilled in the art, such as controlling the amount of protease used or the incubation time. Complete digestion can produce a mixture of various fragments of full-length lactoferrin polypeptides; partial digestion can produce a mixture of full-length lactoferrin polypeptides and various fragments thereof.

Lactoferrin polypeptides or mixtures of such polypeptides and fragments thereof as described above are used for the preparation of the nutritional compositions of the invention for the prevention and treatment of bone-related disorders. Examples of such disorders include, but are not limited to, osteoporosis, rheumatoid or osteoarthritis, hepatic osteodystrophy, osteomalacia, rickets, cystic fibrosis, renal osteodystrophy, osteopetrosis, osteopenia, osteogenesis imperfecta (fibrositis-osteopetrosis), secondary hyperparathyroidism, hypoparathyroidism, hyperparathyroidism, chronic kidney disease, sarcoidosis (sarcoidosis), glucocorticoid-induced osteoporosis, idiopathic hypercalcemia, Paget's disease, and osteogenesis imperfecta. The nutritional composition can be a food supplement (such as a capsule, sachet or tablet) or a food product (such as milk, juice, beverage, herbal tea bag or candy). The composition may also include other nutritional ingredients such as proteins, carbohydrates, vitamins, trace elements or amino acids. The composition may be in the form of a suitable oral preparation such as a tablet, hard or soft capsule, aqueous or oily suspension or syrup, or in the form of an aqueous solution suitable for parenteral use such as an aqueous propylene glycol solution, or a buffered aqueous solution. The amount of active ingredient in the nutritional composition varies to a large extent depending on the specific needs of the individual. As recognized by those skilled in the art, the amount of the active ingredient may also vary depending on the route of administration and possible co-use with other bone enhancers.

The invention also includes a pharmaceutical composition comprising the lactoferrin polypeptide or a mixture of the polypeptide and fragments thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used for preventing and treating the above bone-related disorders. The pharmaceutical composition may also include an effective amount of other bone-enhancing agents. The pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. An effective dose is the dose required to produce a therapeutic effect. Correlation of animal and human dose (in milligrams per square meter of body surface area) as described by Freireich et al (1966) in Cancer chemither. rep.50: 219, as described in table 219. Body surface area can be estimated from the height and weight of an individual, see, e.g., Scientific Tables, Geigy pharmaceuticals, Ardley, New York, 1970, 537. As recognized by those skilled in the art, the effective dosage may also vary depending on the route of administration, the use of excipients, and the like.

The lactoferrin polypeptide of the present invention or a mixture of the polypeptide and fragments thereof can be formulated into dosage forms for different administration routes by a conventional method. For example, it can be formulated into capsules, capsules or tablets for oral administration. The capsule may contain any conventional pharmaceutically acceptable material such as gelatin or cellulose. Tablets may be made by compressing, in a conventional manner, the lactoferrin polypeptide mixture or the mixture of the polypeptide and fragments thereof in admixture with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite (bentonite). The lactoferrin polypeptides of the invention or the mixture of the polypeptides and fragments thereof can also be administered in the form of hard-shell tablets or capsules containing a binding agent, such as lactose or mannitol, conventional fillers and tablet formers. The pharmaceutical compositions of the present invention may be administered parenterally. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose containing the active ingredient, or other known pharmaceutically acceptable excipients. Cyclodextrins, or other solubilizing agents known to those skilled in the art, may be used as pharmaceutical excipients for the release of the therapeutic ingredient.

The effectiveness of the compositions of the present invention can be evaluated in vitro and in vivo. See, for example, the following examples. In short, the ability of the composition to promote osteoblast and chondrocyte proliferation in vitro can be tested. For in vivo studies, the formulation can be injected into an animal (e.g., a mouse) and then evaluated for its effect on bone tissue. Appropriate dosage ranges and routes of administration can be determined based on the results.

The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further inventive effort, one skilled in the art can, in light of this disclosure, utilize the present invention to its fullest extent. All publications cited herein are incorporated herein by reference in their entirety.

Effect of lactoferrin in promoting proliferation of primary rat osteoblasts

Collagenase digestion of calvaria bone of rat suckling mice from day 20 was used to separate osteogenic bone according to the method described by Lowe and colleagues (Lowe, et al (1991) Journal of Bonean and Mineral Research 6, 1277-A cell. Calvarial bones were aseptically excised, and the frontal and parietal bones were delaminated from their periosteum. Only the central portion without the sutured tissue was collected. The calvaria bone was treated 2 times for 15 minutes with Phosphate Buffered Saline (PBS) containing 3mM EDTA (pH7.4) in a water bath shaken at 37 ℃. After one wash with PBS, the calvaria bone was treated twice with 3ml of 1mg/ml collagenase for 7 minutes at 37 ℃. After discarding the first and second digested suspensions, the calvaria bone was further treated 2 times with 3ml of 2mg/ml collagenase (30 min, 37 ℃). The third and fourth digested suspensions were collected, centrifuged and the cells washed with Dulbecco 'digested Eagle' medium (DME) containing 10% fetal calf serum (FCS or FBS), then suspended in DME/10% FCS medium to 75cm³In a culture flask. The cells were incubated at 5% CO₂The culture was carried out at 37 ℃ in 95% air. After 5-6 days, a confluent monolayer can form, at which point subculture is performed. After trypsinization with trypsin-EDTA (0.05%/0.53 mM), cells were rinsed in Minimal Essential Medium (MEM) containing 5% FCS and resuspended in fresh medium at 5X 10⁴Cells/ml density seeded in 24-well plates (0.5 ml cell suspension per well, i.e. 1.4X 10⁴Cells/cm²). Alkaline phosphatase Activity and high levels of osteocalcin production [ see Groot, et al (1985) Cell Biol Int Res 9, 528]And adenylate cyclase sensitive to parathyroid hormone and prostaglandins [ see Hermann-Erlee, et al (1986) Ninth International Conference on calcium regulating homonas and bone metabolism, p409]The identification result of (a) indicates that these cells have osteoblast-like properties.

Proliferation studies (cell counts and thymidine incorporation) were performed in both actively growing and non-actively growing cell populations. To generate actively growing cells, the sub-confluent monolayer of the cell population (24 h after subculture) was placed in fresh MEM medium containing 1% FCS and a lactoferrin sample. To generate non-actively growing cells, the sub-confluent monolayer population was placed in fresh serum-free medium containing 0.1% Bovine Serum Albumin (BSA) and a lactoferrin sample. Transferring after adding the lactoferrin prepared as described aboveCytometric analysis was performed 6, 24 and 48 hours after the protein samples (i.e., purified lactoferrin, holo-lactoferrin and apo-lactoferrin). Cells were detached from the wells of the plate by exposing the cells to trypsin/EDTA (0.05%/0.53 mM) for about 5 minutes at 37 ℃ and then cell counting was performed. The counting was performed in a haemocytometer chamber. 2 hours before the termination of the culture, the culture solution is prepared³H]Incubating thymidine (1. mu. Ci/well) with the cells, and³H]incorporation experiments of thymidine into actively growing and non-actively growing cells. Cells were washed with cold thymidine containing MEM and then terminated at 6, 24 or 48 hours by the addition of 10% trichloroacetic acid. The precipitate was washed 2 times with ethanol to ether (3: 1) and the pores were dried at room temperature. The residue was re-dissolved with 2M KOH for 30 min at 55 ℃, neutralized with 1M HCl, and then one aliquot was taken for radiolabelling. For cell counts and thymidine incorporation, each experiment was performed 4 different times with at least 6 well panel at each time point.

The significant increase in osteoblast proliferation rate (i.e. increased incorporation of thymidine into the DNA of growing cells) indicates that the purified lactoferrin sample is very effective in mitotic response of the cells. The observed potent osteogenic response was compared to that of insulin-like growth factor 1 (IGF-1), a known osteoblast mitogen. In the same osteoblast culture system

Under these conditions, IGF-1 showed 1.25 times the maximal effect compared to the control group, whereas lactoferrin was 2.26 times more effective at the highest experimental dose (10. mu.g/ml) compared to the control group.

Lactoferrin-promoting effect on chondrocyte proliferation

Chondrocytes were isolated under sterile conditions by removing cartilage (full thickness slices) from the tibial and femoral surfaces of sheep. Bone pieces were placed in DME medium containing 5% FBS (v/v) and antibiotics (50g/L penicillin, 50g/L streptomycin, and 100g/L neomycin) and carefully treated with a scalpel bladeAnd (5) cutting. The tissue was removed and digestion was completed by incubation with pronase (0.8% w/v, 90 min) followed by collagenase (0.1% w/v, 18 h) at 37 ℃. Cells were separated from the digest by centrifugation (10 min, 1300rpm) and resuspended in DME/5% FBS, undigested debris was removed by filtration through a nylon mesh of 90Fm pore size, and centrifuged again. The cells were subsequently washed and resuspended 2 times with the same medium and inoculated in 75cm of DME/10% FBS-containing medium³In a culture flask, at 5% CO₂95% air, and 37 ℃. After 7 days, a confluent monolayer could form, at which point subculture was performed. Following trypsinization with trypsin-EDTA (0.05%/0.53 mM), cells were rinsed in DME/5% FBS medium, resuspended in fresh medium, and then plated in 24-well plates (5X 10)⁴cells/mL, 0.5 mL/well). Thymidine incorporation assays were performed on growth arrested cell populations as described above for osteoblast-like cell cultures. Lactoferrin stimulates the proliferation of chondrocytes at concentrations above 0.1 μ g/ml.

Lactoferrin promotes osteoblast proliferation in organ culture

Organ culture of neonatal mice has been previously reported (Comish, et al (1998) Am J Physiol274, E827-E833). Briefly, 2 day old newborn mice were subcutaneously infused with radiolabeled⁴⁵Ca. After 3 days calvaria bones were excised and placed on a grid in a Petri dish containing 0.1% bovine serum albumin/Media 199. Lactoferrin was added and calvarial bones were cultured for 48 hours. 4 hours before the end of the culture period, add [ 2 ]³H]-thymidine. The experiment was terminated and analyzed⁴⁵Ca release and thymidine incorporation. Lactoferrin was found to stimulate DNA synthesis reflecting the proliferation of osteoblast lineage cells.

Lactoferrin signalling by MAP kinase in osteoblasts

This method has been reported previously (Grey, et al (2001) Endocrinology 142, 1098-1106). In particular, primary rat osteoblasts prepared as described above were cultured at 5X 10⁴Cells/ml initial density were seeded in 6-well tissue culture plates and grown in 5% FCS in MEM as 80-90% confluent monolayers. After serum starvation overnight, cells were treated with lactoferrin-containing MEM/0.1% BSA at room temperature. In an experiment to identify the effect of a signal transduction inhibitor of lactoferrin-induced phosphorylation of p42/44MAP kinase, cells were pretreated with the inhibitor for 30 minutes before lactoferrin was added. After completion of the indicated time of treatment, the culture broth was aspirated, the cells were washed with ice-cold PBS, followed by ice-cold HNTG lysis buffer (50mM HEPES, pH7.5, 150mM NaCl, 1% Triton, 10% glycerol, 1.5mM MgCl. RTM.) containing a combination of protease inhibitors and phosphatase inhibitors (1mM PMSF, 1. mu.g/ml peptin, 10. mu.g/ml leupeptin, 10. mu.g/ml aprotinin, 1mM sodium vanadate, 500mM NaF)₂1mM EDTA) to scrape the cells. The lysate was vortexed briefly, centrifuged at 13,000rpm at 4 ℃ and then stored at-70 ℃ until detection. Protein content in cell lysates was determined by DC protein assay (BioRad, Hercules, CA). The same amount of whole cell lysate (30-50. mu.g) was subjected to 8% SDS-PAGE and transferred to nitrocellulose membrane, which was immunolabeled overnight at 4 ℃ with an antibody against phospho-p 42/44MAP kinase (1: 1000). As a control protein, the same filtrates (filters) were taken and re-labeled with an antibody against all p42/44MAP kinase (1: 400). The nitrocellulose membrane was incubated with HRP-linked secondary antibody at room temperature for 1h and analyzed with ECL. The immunolabeling was repeated at least 3 times. It was found that lactoferrin exhibits a dose and time dependent induction of phosphorylation of p42/44MAP kinase in osteoblasts in the concentration range 1-100. mu.g/ml.

Lactoferrin stimulates bone growth in vivo

The mouse model used in these studies was as described in previous reports (Commish, et al (1993) Endocrinology 132, 1359-. Lactoferrin (0mg, 0.04mg, 0.4mg, and 4mg) was injected daily for 5 days, and the animals were sacrificed after one week. Bone formation was determined by fluorescent labeling of newly formed bone. Bone resorption and bone aggregation indices were determined by conventional light microscopy, supplemented with software image analysis. Local injections of lactoferrin into adult mice increased calvarial bone growth and significantly increased bone area after only 5 injections.

Application example 1

Yogurt blocks containing 14% to 17% solids (with or without fruit) can be prepared by the following method:

medium-heat skimmed milk powder (109-152g) and ALACO stabiliser (100g) were redissolved in about 880ml of water at 50 ℃. Anhydrous milk fat (20g) was then added and mixed for 30 minutes. The mixture was then heated to 60 ℃, ground under 200bar pressure and pasteurized at 90 ℃. After cooling to a temperature of 40-42 ℃, the starting mixture and the lyophilized protein preparation as described above (up to 50mg of lactoferrin at 95% purity or an equivalent amount of material not reaching such a high purity) are added. Fresh fruit can be added at this point if desired. The mixture is then filled into containers and incubated at 40 ℃ until pH4.2-4 and cooled in a hairdryer.

An alternative method of preparing the same yogurt block is to blend a dry specified amount or dose ratio of lactoferrin into dry milk solids before it is used to prepare yogurt.

Application example 2

The dry mixture of skimmed or whole milk powder, calcium and the above lyophilized lactoferrin preparation can be made into milk product preparation or composition, which can be used as functional food or functional food ingredient. Such compositions can be used in reconstituted milk, milk powder ingredients, dairy desserts, functional foods, cheeses or butters or beverages, and nutritional formulations or food supplements. A mixture of powdered milk, calcareous active lactoferrin in a dry weight ratio of between 90: 9.5: 0.5 and 94: 5.95: 0.0001 provides a composition suitable for such use.

Application example 3

The composition of a mixture of milk powder, calcium and a rich lactoferrin composition can be used as a functional bone health food, a component of a bone health food, or as a component of a food that releases bone health nutrients in some health foods.

For such compositions, the calcium and protein content should be adjusted to the required and allowable nutritional limits. Commercially available component milk powders, depending on their origin, typically contain 300 and 900mg calcium per 100g of milk powder. A calcium source may be added to the milk powder to increase the calcium content to 3% by weight of the component milk powder mix. The protein content of the commercially available component milk or dairy protein powder varies depending on the type of the component, the method of manufacture and the requirements of use. The component milk powder usually contains 12 to 92 percent of protein. Examples include commercially available skim and whole milk powders, food grade caseins, metal caseins, milk protein concentrate powders, spray dried ultrafiltration or microfiltration retentate powders, and milk protein isolate products. The lactoferrin enriched preparation can be added to a mixture of protein and calcium to prepare a nutritional milk powder which can be used as an ingredient of health foods and beverages. Such a mixture is suitable for use as an ingredient in the preparation of yoghurt and yoghurt beverages, acid beverages, ingredient milk powder mixtures, pasteurized liquid milk products, UHT milk products, cultured milk products, yoghurt beverages, milk and cereal combinations, malted milk, soy milk combinations. For these uses, the calcium content in the mixture may be 0.001-3.5% (w/w), the protein content may be 2-92%, and lactoferrin as an osteoblast proliferation ingredient may be added at a content of 0.000001-5.5%.

Other embodiments

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the foregoing, it will be apparent to those skilled in the art that the essential features of the present invention can be determined, and that various changes and modifications can be made to the present invention to adapt it to various usages and conditions without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Claims (6)

1. Use of human lactoferrin or bovine lactoferrin containing not more than 2 iron ions per molecule in the manufacture of a nutritional or pharmaceutical composition for stimulating bone growth and inhibiting bone resorption, wherein the lactoferrin is free of other biological macromolecules and has a purity of at least 65% on a dry weight basis.

2. The use of claim 1, wherein the lactoferrin contains 1 iron ion per molecule or 2 iron ions per molecule.

3. The use of claim 1 or 2, wherein the composition is in an oral or parenteral dosage form.

4. Use of human lactoferrin or bovine lactoferrin containing not more than 2 iron ions per molecule in the manufacture of a nutritional or pharmaceutical composition for the treatment of a bone-related disorder, wherein the lactoferrin is free of other biological macromolecules and is at least 65% pure on a dry weight basis, wherein the bone-related disorder is osteoporosis or osteoarthritis, osteomalacia, osteopenia, osteogenesis imperfecta, glucocorticoid-induced osteoporosis.

5. The use according to claim 4, wherein the lactoferrin contains 1 iron ion per molecule or 2 iron ions per molecule.

6. The use of claim 4 or 5, wherein the composition is in an oral or parenteral dosage form.