WO2008094493A2 - Compositions containing lactoferrin and calcium, and methods of using same - Google Patents

Abstract

The present invention relates, generally, to compositions containing lactoferrin and calcium, and methods of making the compositions. The compositions may further include additional ingredients useful for promoting and/or maintaining bone health, and/or additional ingredients useful for promoting absorption and/or bioavailability of the lactoferrin and calcium, such as vitamin D. The present invention also relates to methods for promoting and/or maintaining bone health, and treating and/or preventing bone disorders, by administering compositions comprising lactoferrin and calcium.

Description

Compositions Containing Lactoferrin and Calcium, and Methods of Using Same

Background of the Invention:

1. Field of the Invention

[0001] The present invention relates to compositions containing lactoferrin and calcium, and methods of making the compositions. The present invention also relates to methods for promoting and/or maintaining bone health, and treating or preventing bone disorders, by administering compositions comprising lactoferrin and calcium.

2. Description of the Related Art

[0002] The bone of the human body is an organ composed of cortical and trabecular bone, haemopoetic and connective tissues, and cartilage. The primary functions of the bone include the movement of parts of the body, the protection of internal organs, and the provision of a site for haematopoesis. In addition, since the bone is the principal storage site for calcium, phosphorus, sodium, magnesium, and carbonate, the bone plays an important role in mineral homeostasis. [0003] Bones grow in length at the epiphyseal growth plate and involve the cycle of cartilage growth, matrix formation, and calcification of cartilage that serves as the scaffold for bone formation. This process occurs in childhood and throughout the adolescent years until the cartilage growth finally stops, usually in the early twenties. At this point, the epiphyseal plate ossifies completely and the bones can no longer grow in length.

[0004] Another feature of bone growth is the process of bone modeling, where bone is continuously resorbed and replaced by new bone. Active mostly in childhood and adolescence, modeling enables long bones to increase in diameter, to change shape, and to develop a marrow cavity. Modeling continues into the adult life with bone formation balancing bone resorption (the proper term for modeling in adult life is remodeling). Osteoblasts, osteoclasts, and osteocytes are the three cell types involved in the development, growth, and remodeling of bones - osteoblasts are the bone-forming cells, osteoclasts are bone cells that break down and reabsorb bone, and osteocytes are mature bone cells. Bone growth has been shown to be under the influence of many factors, such as genetics, circulating hormones, nutritional intake, mechanical influences, and disease. Any disturbances in the normal cellular activity of the bone will disrupt bone growth, for example, hyperactivity of osteoclasts would lead to bone loss and result in pathologies such as osteoporosis and osteolytic diseases.

[0005] Calcium is recognized as an important nutrient necessary for bone formation and building healthy bones and teeth. One of the problems in supplementing the diet with commercial calcium preparations is that not all calcium sources are equally absorbed or have equal bioavailability. Additionally, many calcium preparations suitable for food and beverage fortification have poor solubility resulting in chalky taste and undesirable aftertaste problems. [0006] Mammalian milk is rich not only in calcium, but also comprises other nutrients and growth factors that are important in the absorption and/or bioavailability of calcium and in the anabolic effects on bone. Human milk is the primary source of nutrition for the developing neonate and provides the essential factors needed during a time of rapid skeletal growth and development. Lactoferrin (LF) is an iron binding glycoprotein found in human breast milk, and has been shown to be a novel anabolic factor in osteoblast formation, and also reduces osteoclast differentiation, leading to the promotion of bone formation and the inhibition of bone resorption. LF is a component of the whey fraction of human milk and is a member of the transferrin family of 80 kDa proteins. It possesses a single polypeptide chain and two iron- binding domains (Baker et al., "Structure, function and flexibility of human lactoferrin," lnt J Biol Macromol 13(3): 122-9 (1991 )). It is secreted by the mammary, salivary, and pancreatic glands and in granules of neutrophils. It is found in high concentrations (average 1-2 g/L) in human milk and circulating levels of LF, predominantly secreted by neutrophils, in normal, healthy subjects range from 2 to 7 μg/mL. LF has been shown to exhibit activities against human pathogens including Gram positive and Gram negative bacteria, yeasts, and fungi and has also demonstrated antiviral activities. Other activities of LF include regulation of iron absorption and immune system modulation. Cellular growth promotion activity has also been ascribed to LF - crypt, intestinal, and epithelial cell proliferation was noted in the presence of LF (Lonnerdal et al., "Lactoferrin: molecular structure and biological function," Annu Rev Nυtr 15: 93-110 (1995)). Therefore, LF has the potential to be highly effective in aiding bone growth, and supporting and maintaining bone health through proliferation of bone cells.

[0007] In vitro studies using bovine LF at physiological concentrations (1-100 μg/mL) have previously been conducted and were shown to stimulate the proliferation and differentiation of both primary and cell-line cultures of human osteoblasts or rat osteoblast-like cells. In addition, bovine LF has been shown to prevent apoptosis in osteoblast cells, suggesting a potent osteoblast survival factor. An in vivo study where bovine LF was subcutaneously injected into mice has been demonstrated to increase bone formation rate as well as mineral apposition rate (Cornish et al., "Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo," Endocrinology 145(9): 4366-74 (2004)). In the same study, bovine LF also has shown to be an inhibitor of osteoclast development in mouse bone marrow cultures. At 100 μg/mL bovine LF, osteoclastogenesis was completely halted, and it was determined that LF acts on both preosteoclasts and more mature osteoclasts. A study by Lorget et al. (Lorget et al., "Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity." Biochem Biophys Res Commun 296(2): 261-6 (2002)) also found that bovine LF reduces bone resorption and confirms the above studies. Because it is the same species of origin, it would be expected that human LF would be a more potent stimulator of human osteoblasts and inhibitor of human osteoclasts.

[0008] The role of lactoferrin in bone health, strength, and repair is further evidenced by the fact that LF exhibits an immune modulatory function. Since the bone and immune cells share the same location and because endogenous synthesis of LF is up-regulated in response to inflammation, there is increasing evidence to suggest that the bone and immune systems are genetically and functionally associated (Baveye et al., "Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process," CHn Chem Lab Med 37(3): 281-6 (1999)). For example, osteoarthritis and osteolytic cancers involve both inflammation and bone loss. Moreover, it has been shown that T-cell activation induces osteoclastic differentiation (Weitzmann et al., "T cell activation induces human osteoclast formation via receptor activator of nuclear factor kappaB ligand-dependent and - independent mechanisms," J Bone Miner Res 16(2): 328-37 (2001)). Thus, it is possible that LF plays an important role in bone remodeling. Oral administration of bovine LF has been demonstrated to decrease the spontaneous production of tumor necrosis factor alpha and interleukin-6 by peripheral blood cells of which have been shown to affect osteoclast differentiation (Zimecki et al., "Lactoferrin increases the output of neutrophil precursors and attenuates the spontaneous production of TNF- alpha and IL-6 by peripheral blood cells," Arch Immunol TherExp 47(2): 113-8 (1999)). In antigen-induced arthritis, interleukin-6 has been shown to be a mediator of bone destruction due to its regulation of T-lymphocyte production of key osteoclastogenic cytokines and inflammation-induced bone marrow osteoclast differentiation (Wong et al., "Interleukin-6 modulates production of T lymphocyte- derived cytokines in antigen-induced arthritis and drives inflammation-induced osteoclastogenesis." Arthritis Rheum 54(1): 158-68 (2006)) and IL-6 has recently been shown to decrease systemic bone loss, as well as local osteoporosis, in inflammatory joint disease.

[0009] In addition to LF, other proteins such as insulin-like growth factors (IGF) (Zapf et al., "Insulin-like growth factors/somatomedins: structure, secretion, biological actions and physiological role." Horm Res 24(2-3): 121-30 (1986)), and transforming growth factors (TGF) (Bonewald et al., "Role of active and latent transforming growth factor beta in bone formation." J Cell Biochem 55(3): 350-7 (1994)) have previously been shown to promote osteoblast proliferation in vitro. The abundant presence of IGF in bone, its mitogenic activity in a variety of cell lines, and its ability to stimulate tissue-specific cellular responses suggests a role in bone cell matrix synthesis (Jia et al., "Insulin-like growth factor-1 and -2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone." Bone 27(6): 785-94 (2000); McQueeney et al., "Roles of insulin-like growth factor-l (IGF-I) and IGF-I binding protein-2 (IGFBP2) and -5 (IGFBP5) in developing chick limbs." Growth Horm IGF Res 11(6): 346-63 (2001). However, unlike these growth factors, LF has been shown to increase new bone formation through proliferative and anti-apoptotic actions in osteoblasts, and at the same time has the capacity to inhibit osteoclastogenesis in vivo (Cornish et al., "Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo." Endocrinology 145(9): 4366-74 (2004)). Therefore, the activity of LF on bone exceeds that of the growth factors. The potential exists for LF to be used in a variety of skeletal disorders, such as osteoporosis, and in the reduction of joint and bone damage and loss caused by inflammatory bone disorders, and in orthopedic practices. [0010] Published PCT Application No. WO 03/082921 A1 describes the use of a pure LF polypeptide containing no more than two metal ions per molecule that can be derived from either human or bovine milk. This LF, or a mixture of the polypeptide and a fragment thereof, can be used to stimulate bone growth and to inhibit bone resorption. This LF can be used as a method of treating bone-related disorders.

[0011] Published PCT Application No. WO 03/095678 A1 describes the use of the low density lipoprotein (LDL) receptor proteins 1 and 2 (LRP-1 and LRP-2) and the interaction between LF and LRP-1 and LRP-2, or p42/44 MAP kinase in the diagnosis and treatment of bone and cartilage disorders. [0012] However, none of the above-mentioned references describes compositions containing lactoferrin and calcium, and none addresses methods for oral delivery of lactoferrin and calcium to promote bone health, maintain bone mineral density, strengthen bones, and repair bone loss. [0013] It is believed that osteoarthritis affects at least 20.7 million Americans. Currently, no pharmacological agents have been shown to retard or prevent this disease. Instead, the focus of treatment has been to relieve pain and maintain quality of life and functional independence. In addition, each year 25 million Americans suffer from fractures as a result of accidents and activity-related injuries. Osteoporosis, which typically affects people over 65 years of age, causes more than 3 million hip, vertebral, forearm, and other fractures each year. Due to the demographic shift towards the aging population, it is possible that the number of people who suffer from osteoarthritis, osteoporosis, and related conditions will escalate in future years.

[0014] Accordingly, there is a great need in the art for compositions including lactoferrin and calcium, and methods of making them. There is also a great need in the art for effective compositions and methods for promoting and/or maintaining bone health, including maintaining bone mineral density, and treating and/or preventing bone disorders, such as osteoarthritis and osteoporosis. Summary of the Invention

[0015] The present invention relates to the promotion and/or maintenance of bone health, including maintaining bone mineral density, and the treatment and/or prevention of bone disorders, and preferably to the treatment of osteoarthritis and osteoporosis. The methods for promoting and/or maintaining bone health, including maintaining bone mineral density, may be accomplished by orally administering lactoferrin and calcium to a healthy person. The methods for treating and/or preventing bone disorders, such as osteoarthritis and osteoporosis, may be accomplished by orally administering lactoferrin and calcium to a patient suffering from any of these conditions. The present invention also relates to pharmaceutical preparations comprising lactoferrin and calcium. There is an unmet need in the art for such compositions and methods.

[0016] The present invention meets the unmet needs in the art, as well as others, by providing methods for promoting and/or maintaining bone health, and treating and/or preventing bone disorders, by oral administration of lactoferrin and calcium. The present invention further provides compositions including lactoferrin and calcium.

[0017] In some embodiments, the present invention is directed to a method of promoting and/or maintaining bone health, including maintaining bone mineral density, or to a method for treating bone disorders, such as osteoarthritis and osteoporosis, by oral administration of lactoferrin, preferably a human lactoferrin, in conjunction with calcium, preferably calcium citrate malate. Further, additional optional ingredients, such as vitamin D, may also be co-administered in accordance with the method. In some embodiments, the ingredients may be administered as a food, a beverage, or a pharmaceutical dosage form such as a tablet or capsule. [0018] In other embodiments, the present invention is directed to a method of promoting and/or maintaining bone health, including maintaining bone mineral density, or to a method of treating bone disorders, such as osteoarthritis and osteoporosis, by oral administration of at least one recombinant human lactoferrin produced in plants, in conjunction with calcium citrate malate. The method may also optionally include providing one or more additional vitamins and minerals, such as vitamin D. [0019] In still further embodiments, the present invention is directed to a composition comprising at least one lactoferrin, preferably a human lactoferrin, and calcium, preferably calcium citrate malate, formulated for oral administration. In some embodiments, the ingredients may be administered as a food, a beverage, or an oral dosage form such as a tablet or capsule. According to additional embodiments, the human lactoferrin is recombinantly produced in plants, preferably monocot plants, and the calcium is calcium citrate malate.

[0020] Other novel features and advantages of the present invention will become apparent to those skilled in the art upon examination of the following or upon learning by practice of the invention. Detailed Description of the Preferred Embodiments

[0021] The present invention relates to methods of promoting and/or maintaining bone health, including maintaining bone mineral density, and to methods of treating bone disorders, such as osteoarthritis and osteoporosis, by orally administering lactoferrin with calcium, and to compositions for oral administration that comprise lactoferrin and calcium. The compositions and methods of the present invention may be beneficially utilized to treat subjects suffering from osteoarthritis and osteoporosis, or to prevent these and other bone disorders in subjects at risk thereof. The compositions and methods of the present invention may also be used to promote continued bone health in healthy subjects.

A. Compositions Comprising Lactoferrin and Calcium

[0022] The lactoferrin (LF) that may be used in connection with the invention may be derived or extracted from natural sources, chemically synthesized, or produced recombinantly using any suitable organism. Natural sources of LF include, but are not limited to, mammalian LF (such as bovine LF), and more particularly human LF. Recombinant LF (rLF), more particularly recombinant human LF (rhLF), may be produced using any suitable host, whether eukaryotic or prokaryotic. Presently preferred recombinant hosts are yeast cells, plant cells, mammalian cells, or bacterial cells. Most preferred are plant cells, especially monocot plant cells, such as rice. Recombinant production of LF in a suitable host would be of routine skill to those in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, CSH Laboratory Press, 3^rd Edition, 2001 , hereby incorporated by reference. [0023] In preferred embodiments, production of recombinant lactoferrin may be accomplished using the teachings of, for example, U.S. Patent Application Nos. 10/077,381 , 10/411 ,395, PCT/US2004/041083, and/or PCT/US2003/39107, each of which is hereby incorporated by reference. The recombinant lactoferrin produced in accordance with these teachings may be utilized with or without further purification. [0024] The calcium that may be used in connection with the invention may be selected from calcium carbonate, coral calcium, calcium formate, calcium citrate, microcrystalline hydroxyapatite (MCHC), amino acid chelates of calcium, and calcium citrate malate (CCM). CCM is the presently preferred form of calcium for inclusion in the compositions of the present invention, as it has shown high bioavailability and effectiveness in the treatment of bone health related disorders. [0025] In preferred embodiments, the inclusion of calcium citrate malate in the compositions of the present invention may be accomplished using the teachings of, for example, U.S. Patent Nos. 5,128,374, 5,314,919, 5,151 ,274, 6,509,326, and/or 5,186,965, each of which is hereby incorporated by reference. [0026] The compositions according to the present invention can optionally include one or more additional ingredients designed to promote bone health, treat or prevent a bone disorder, and/or promote the bioavailability of the lactoferrin and calcium contained in the oral formulation. Such optional additional ingredients may include, but are not limited to, iron, zinc, potassium, magnesium, manganese, copper, vitamin D, vitamin A, vitamin E, vitamin C, vitamin K, and trace minerals. Calcium and iron interactions are known to inhibit absorption of one another. Therefore, having highly bioavailable calcium and a highly bioavailable iron is advantageous for the absorption of both calcium and iron. Lactoferrin itself is a good source of bioavailable iron. It is also envisioned that other nutrient fortifications, for example, dietary fiber, may also be incorporated.

[0027] The compositions according to the present invention can be formulated in any manner suitable to deliver the dose. Examples include a tablet, a caplet, a hard or soft capsule, a lozenge, a cachet, a dispensable powder, granules, a suspension or solution, an elixir, a liquid, a food product, or any other form reasonably adapted for oral administration.

[0028] In accordance with further embodiments of the invention, the LF may be treated such that it is not subject to degradation in the gut. This may be accomplished by providing the LF in a coated oral dosage form that is not broken down in the stomach, or by employing protein stabilization technology to prevent the environment of the stomach from denaturing the LF before it can provide its benefits to the subject. One exemplary protein stabilization technology is set forth in U.S. Patent No. 5,648,112, which is hereby incorporated by reference. 1. Food Products

[0029] Calcium is typically absorbed better when eaten with meals. Accordingly, the present invention relates to compositions containing lactoferrin and calcium that may be provided in the form of liquid or solid food, liquid concentrate or beverage, and/or tablet compositions comprising lactoferrin and a source of calcium. Preferably, these food products include natural and/or recombinant human LF and CCM. The food products may be prepared with or without vitamin D or other microingredients, including vitamins and minerals, and may include excipients and/or formulation agents. Products preferably include, but are not limited to, powders, tablets, liquid supplements, dietary supplements, fruit juice and dairy beverages, carbonated beverages, meal replacement beverages or bars, cultured fruit and dairy products, powdered drink preparations, spoonable puddings, and gelatins. [0030] Food and beverage products comprising calcium and lactoferrin, with or without vitamin D and/or other microingredients, can provide stable, good tasting, highly efficacious delivery of bioactive LF and bioavailable calcium, in order to accomplish any of the various methods of the invention in a more effective manner than each individual ingredient alone.

[0031] An advantage of the invention is providing food, beverage, and supplement compositions with good taste impressions containing a highly bioavailable source of calcium, and optionally vitamin D, in combination with lactoferrin. A food, beverage, or supplement product comprising lactoferrin fortified with highly solubilized calcium can provide for a stable, good tasting, highly efficacious method for building and maintaining bone mineral density in humans and other animals. Compared to nutritional regimens with calcium supplementation known in the art, this method affords higher bioavailability and greater efficacy in building and maintaining bone and in the treatment of osteoarthritis, osteoporosis, and/or related bone disorders.

[0032] A presently preferred aspect of the invention relates to beverages having a pH from 2.5 to 7.5, but most preferably from pH 2.5 to 4.2. The composition containing LF and calcium, per defined dose or serving size, preferably comprises at least 10 mg up to 1,000 mg of lactoferrin. Solubilized calcium is preferably provided in an amount of at least 0.05% by weight, most preferably from about 0.1% to 80% by weight. The compositions may optionally provide at least 10% RDA of vitamin D and/or other trace minerals which can include but are not limited to iron, zinc, potassium, magnesium, manganese, and copper, all of which are important nutrients for bone growth and maintaining bone health. In addition to the lactoferrin, calcium, optional vitamin D and/or trace minerals, other optional ingredients typically present in food, beverages, and supplements can be included, for example, other beneficial vitamins (A, E, C and K) and nutrient fortification, for example dietary fiber and sweeteners, texturizers, emulsifiers, stablilizers and other ingredients. [0033] Products preferably include, but are not limited to, supplements, powders, liquid supplements, fruit juice and dairy beverages, powdered drinks, carbonated beverages, meal replacement beverages, cultured fruit and dairy products, spoonable puddings, and gelatins. One embodiment of the invention is a beverage having a pH from 2.5 to 4.5 that contain a stabilized protein, emulsified lipid component, and fruit juice(s).

[0034] It is also envisioned in accordance with the present invention to provide a tablet supplement product including lactoferrin, calcium, and optionally other micronutrient components.

[0035] Additional oral formulations containing lactoferrin and calcium are also envisioned in accordance with this aspect of the present invention.

2. Pharmaceutical Compositions

[0036] The present invention also relates to compositions containing lactoferrin and calcium that are provided in the form of pharmaceutical/nutraceutical compositions, and may take the form of tablets, capsules, and other oral dosage forms. Preferably, these pharmaceutical compositions comprise natural and/or recombinant human LF and calcium citrate malate (CCM).

[0037] To aid in delivering active lactoferrin to the small intestine, for example, the composition may be tableted or encapsulated, and enteric-coated. Enteric coatings prevent the tablet or capsule from dissolving before it reaches the small intestine. Alternatively the material may be spheronized into microparticles and preferably enterically coated. Spheroids may be produced in the size range of 250 μm to 850μm. Enteric coatings are known to be selectively insoluble substances that do not dissolve in the acidic environment of the stomach, but dissolve in the higher pH of the small intestine, resulting in a specific release of lactoferrin in the small intestine.

[0038] The compositions of the invention including LF and calcium can be further formulated together with one or more pharmaceutically acceptable excipients to produce a pharmaceutical composition. The term "excipient" herein means any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration. Excipients include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, lubricants, glidants, crystallization inhibitors, surface modifying agents, substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition.

[0039] Excipients employed in compositions of the invention can be solids, semisolids, liquids or combinations thereof. Compositions of the invention containing excipients can be prepared by any known technique of pharmacy that comprises admixing an excipient with a drug or therapeutic agent.

[0040] Suitable diluents illustratively include, either individually or in combination, lactose, including anhydrous lactose and lactose monohydrate; starches, including directly compressible starch and hydrolyzed starches (e.g., Celutab™ and Emdex™); mannitol; sorbitol; xylitol; dextrose (e.g., Cerelose™ 2000) and dextrose monohydrate; dibasic calcium phosphate dihydrate; sucrose-based diluents; confectioner's sugar; monobasic calcium sulfate monohydrate; calcium sulfate dihydrate; granular calcium lactate trihydrate; dextrates; inositol; hydrolyzed cereal solids; amylose; celluloses including microcrystalline cellulose, food grade sources of α- and amorphous cellulose (e.g., Rexcel™) and powdered cellulose; calcium carbonate; glycine; bentonite; polyvinylpyrrolidone; and the like. Such diluents, if present, constitute in total about 5% to about 99%, preferably about 10% to about 85%, and more preferably about 20% to about 80%, of the total weight of the oral composition. The diluent or diluents selected preferably exhibit suitable flow properties and, where tablets are desired, compressibility. [0041] Compositions of the invention optionally comprise one or more pharmaceutically acceptable lubricants (including anti-adherents and/or glidants). Suitable lubricants include, either individually or in combination, glyceryl behapate (e.g., Compritol™ 888); stearic acid and salts thereof, including magnesium, calcium and sodium stearates; hydrogenated vegetable oils (e.g., Sterotex™); colloidal silica; talc; waxes; boric acid; sodium benzoate; sodium acetate; sodium fumarate; sodium chloride; DL-leucine; PEG (e.g., Carbowax™ 4000 and Carbowax™ 6000); sodium oleate; sodium lauryl sulfate; and magnesium lauryl sulfate. Such lubricants, if present, constitute in total about 0. 1% to about 10%, preferably about 0.2% to about 8%, and more preferably about 0.25% to about 5%, of the total weight of the composition.

[0042] Suitable anti-adherents include talc, cornstarch, DL-leucine, sodium lauryl sulfate and metallic stearates. Talc is a preferred anti-adherent or glidant used, for example, to reduce formulation sticking to equipment surfaces and also to reduce static in the blend. Talc, if present, constitutes about 0.1% to about 10%, more preferably about 0.25% to about 5%, and still more preferably about 0.5% to about 2%, of the total weight of the composition.

[0043] Glidants can be used to promote powder flow of a solid formulation. Suitable glidants include colloidal silicon dioxide, starch, talc, tribasic calcium phosphate, powdered cellulose and magnesium trisilicate.

[0044] Other excipients such as colorants, flavors and sweeteners are known in the pharmaceutical art and can be used in compositions of the present invention. Compositions of the invention can further comprise, for example, buffering agents. [0045] The compositions containing lactoferrin and calcium according to the present invention, regardless of the particular dosage form utilized, preferably include from about 10 mg to about 1000 mg of lactoferrin per dose, and the calcium is provided in an amount of at least 0.05% by weight of the dosage form, most preferably from about 0.1% to 80% by weight of the dosage form. The dosage form may also optionally include at least 10% RDA of vitamin D and/or other trace minerals.

B. Methods of Making Compositions Comprising Lactoferrin and Calcium [0046] The oral dosage forms of the present invention can be prepared by any suitable process, not limited to processes described herein. [0047] An illustrative process for preparing an oral comprises (a) a step of blending lactoferrin and calcium with one or more excipients to form a blend, and (b) a step of tableting or encapsulating the blend to form tablets or capsules respectively. Preferably, these methods are carried out using natural and/or recombinant human LF and CCM. The tablets or capsules may then be optionally provided with an enteric coating layer.

[0048] In a preferred process, solid dosage forms are prepared by a process comprising (a) a step of blending lactoferrin and calcium with one or more excipients to form a blend, (b) a step of granulating the blend to form a granulate, and (c) a step of tableting or encapsulating the blend to form tablets or capsules respectively. Step (b) can be accomplished by any dry or wet granulation technique known in the art. [0049] One or more diluents, one or more disintegrants, and/or one or more binding agents are preferably added, for example in the blending step, a wetting agent can optionally be added, for example in the granulating step, and one or more disintegrants are preferably added after granulating but before tableting or encapsulating. A lubricant is preferably added before tableting. Blending and granulating can be performed independently under low or high shear. A process is preferably selected that forms a granulate that is uniform in drug content, that readily disintegrates, that flows with sufficient ease so that weight variation can be reliably controlled during capsule filling or tableting, and that is dense enough in bulk so that a batch can be processed in the selected equipment and individual doses fit into the specified capsules or tablet dies.

[0050] When oral formulations such as foods or beverages are provided, the lactoferrin and calcium may be added along with the other ingredients that form the food or beverage, and may be blended therewith. If the food or beverage product is made using a process that requires the application of heat, then the lactoferrin is preferably added to the food or beverage after the heating step, in order to avoid denaturing the lactoferrin. Alternatively, a heat stable form of lactoferrin may also be used in accordance with the present invention.

[0051] In accordance with one embodiment of the present invention, a method of preparing composition including lactoferrin, calcium, and trace minerals is provided. Preferably, the composition has high acidity, i.e., a pH of from about 2.5 to about 4.2. The composition is preferably prepared in a manner that avoids detrimental effects on taste, i.e., chalkiness and sourness, and exhibits good stability. An exemplary preparation method is set forth below.

[0052] The first step during preparation of the composition having high acidity is to provide a highly-solubilized meta-stable complex of calcium citrate malate by adding calcium carbonate, calcium oxide, or calcium hydroxide to an aqueous solution of acids. The level of total acid depends on the product composition involved, for example, the level of fruit juices and the desired level of calcium, but total acids preferably range from 0.4% to 2.6% by weight. Other edible acids, such as phosphoric acid, fumaric acid, and the like, can also be included with fruit juices. Preferably, the acid component comprises a mixture of citric and malic acids, and more preferably, the weight ratio of citric acid to malic acid is from 1 : 0.5 to 1 : 3. According to certain aspects of the embodiment, the level of solubilized calcium is preferably from about 0.05% to 0.42% by weight.

[0053] After the highly-solubilized meta-stable complex of calcium citrate malate is prepared, this mixture is added to fruit juice and other formulation components, for example, sweeteners and flavors, to further stabilize the calcium complex and maintain appropriate acidity.

[0054] The second step during preparation of the composition having high acidity is to add a stabilized protein component containing lactoferrin and other source proteins. While a number of methods are known in the art to stabilize and "protect" proteins in highly acid environments, the preferred method for preparing products of the present invention is to use a pre-acidified protein premix having a pH of from about 3.0 to about 4.0. The total protein component is preferably more than 0.08% by weight, and most preferably from 0.2-1.0% of the total product composition, but the final protein level depends upon the specific product composition desired. [0055] The protein source containing the lactoferrin is preferably a protein fraction of rice or other plant-sourced protein. The protein premix is preferably prepared by hydrating a solution of gum stabilizer, pectin, and carboxymethylcellulose, although other ingredients useful in preparing compositions containing a protein fraction may also be selected, as would be understood by one skilled in the art. Then, the protein source, which is preferably a rice or other plant-sourced protein containing lactoferrin, is added under high shear mixing conditions. While under high shear mixing, the protein and gum mixture is acidified to less than about pH 4.0, preferably to a pH of from about 3.0 to about 3.5, and is maintained at a mix temperature of less than about 70⁰F, preferably less than about 60⁰F, throughout the acidification process. Any typical food-grade acidulant can be used, although a presently preferred acidulant is citric acid. The use of the above-described protein premix preparation process beneficially avoids the further need for a homogenization step to stabilize the final pre-acidified protein premix. However, a homogenization step, which is a processing method typically used to stabilize emulsions, may also be performed if desired.

[0056] This pre-acidified protein premix can be used to prepare the final product formulation, such as a liquid beverage or supplement that preferably contains a fruit juice and/or a food acid, where the final product preferably has a pH of less than about 4.0. The final liquid beverage or supplement can be further treated using typical thermal processing methods without significant loss of protein activity. Optionally, the pre-acidified protein premix can also be concentrated and/or freeze dried for later use as an ingredient for various edible products, including, but not limited to, fruit-based fillings and powdered drinks.

C. Methods of Using Compositions Containing Lactoferrin and Calcium [0057] Oral administration is a preferred method of administering the compositions of the present invention, due to the ease of patient compliance. However, other methods of administration that provide LF and calcium to a subject in a form that is bioavailable are also envisioned in accordance with the present invention.

[0058] One aspect of the present invention is directed to methods of using the compositions described herein in order to promote bone health and adequate bone calcification, as well as to prevent and/or treat bone disorders, such as osteoarthritis and osteoporosis. These goals may be accomplished by orally administering the compositions of the present invention that include lactoferrin and calcium. Preferably, these methods are carried out using natural and/or recombinant human LF and CCM.

[0059] The compositions containing lactoferrin and calcium according to the present invention may be administered as hereinbefore described, or in any dose beneficial for promoting and/or maintaining bone health, including maintaining bone mineral density, or for preventing and/or treating a bone disorder, such as osteoarthritis or osteoporosis.

[0060] It will, of course, be appreciated that the above description has been given by way of example only and that modifications in detail may be made within the scope of the present invention.

[0061] Throughout this application, various patents and publications have been cited. The disclosures of these patents and publications in their entireties are hereby incorporated by reference into this application, in order to more fully describe the state of the art to which this invention pertains.

[0062] The invention is capable of considerable modification, alteration, and equivalents in form and function, as will occur to those ordinarily skilled in the pertinent arts having the benefit of this disclosure. [0063] While the present invention has been described for what are presently considered the preferred embodiments, the invention is not so limited. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the detailed description provided above.

Claims

We claim:

1 , Methods of promoting and/or maintaining bone health, including maintaining bone mineral density, and methods of treating bone disorders, such as osteoarthritis and osteoporosis, by orally administering lactoferrin with calcium.

2. Compositions for oral administration comprising lactoferrin and calcium.