milk identified lactoferrin as a bone-active factor. Lactoferrin is .. Structure/ Function relationship of lactoferrin's bone activity. Bovine .. date there have been no oral efficacy clinical trials targeted specifically at bone. High. X-ray crystal structure of bovine lactoferrin showing the C-lobe closed (iron loaded) Lactoferrin and bone; structure-activity relationships. Osteogenic activity. Signal pathways. Structure. Interaction. It is estimated that As of to date, BMPs are a group of the most well studied functional proteins that can be .. Lactoferrin is currently the most frequently reported bioactive peptide.
In contrast, lactoferrin had no effect on bone resorption by isolated mature osteoclasts. Lactoferrin was administered over calvariae of adult mice for 5 d. New bone formation, assessed using fluorochrome labels, was increased 4-fold by a 4-mg dose of lactoferrin.
Thus, lactoferrin has powerful anabolic, differentiating, and antiapoptotic effects on osteoblasts and inhibits osteoclastogenesis. Lactoferrin is a potential therapeutic target in bone disorders such as osteoporosis and is possibly an important physiological regulator of bone growth. Because of this, it contains many growth regulators in addition to the simple substrates necessary for infant development.
Therefore, we assessed the effects of milk proteins on bone cell growth, and found that a number of fractions of whey protein have growth-stimulatory effects in primary cultures of osteoblasts. With a view to determining the identity of the growth-promoting molecules within whey protein, we used HPLC to identify the major proteins in the active fractions. We found that the glycoprotein, lactoferrin, was present in many of these fractions.
On this basis, we hypothesized that lactoferrin stimulates osteoblast growth. Lactoferrin is an kDa iron-binding glycoprotein that belongs to the transferrin family of proteins 1.
- Materials and Methods
It is produced by many exocrine glands and, consequently, is widely distributed in body fluids including tears, saliva, bile, pancreatic fluid, vaginal secretions, semen, and milk 2. Lactoferrin is also a major constituent of the secondary granules of neutrophilic leukocytes, from which it is released during acute inflammation 3.
It acts as an iron chelator, which may contribute to its antimicrobial activity 5but it also has effects on cell growth and differentiation 6embryonic development 7myelopoiesis 8endothelial cell adhesion 9cytokine 1011 and chemokine 12 production, regulation of the immune system 13and modulation of the inflammatory response Its effects on bone have received little attention. Variants of a wild-type lactoferrin polypeptide e.
A lactoferrin polypeptide of the invention can be of a mammalian origin, e.
The metal ion bound to the polypeptide can be an iron ion as in a naturally occurring lactoferrin polypeptidea copper ion, a chromium ion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion. A lactoferrin polypeptide of the invention can be used to stimulate skeletal growth e.
A preparation of a lactoferrin polypeptide of the invention e.
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It can also contain polypeptides of different species, e. For example, a mixture of full-length lactoferrin polypeptides and various fragments of full-length lactoferrin polypeptides can be prepared from a hydrolysate, e. Otherwise, it can be obtained by mixing full-length lactoferrin polypeptides with various fragments of full-length lactoferrin polypeptides e. A mixture of various fragments of full-length lactoferrin polypeptides, on the other hand, can be prepared, for example, by complete digestion i.
The invention further features a nutraceutical composition, which can be milk, juice, a soft drink, a snack bar, or a dietary supplement. The nutraceutical composition contains a lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments of the polypeptide in an amount higher than the naturally occurring amount. Lactoferrin has been found to stimulate osteoblast and chondrocyte proliferation and inhibit osteoclast development.
Thus, a nutraceutical composition of this invention is useful for preventing and treating bone disorders such as osteoporosis and rheumatoid or osteo-arthritis. The nutraceutical composition can further include an adequate amount of another bone-enhancing agent, such as calcium, zinc, magnesium, vitamin C, vitamin D, vitamin E, vitamin K2, or a mixture thereof.
In addition, this invention features a pharmaceutical composition that contains a lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments of the polypeptide and a pharmaceutically acceptable carrier.
Optionally, the pharmaceutical composition also includes another bone-enhancing agent. The invention also encompasses the use of a lactoferrin polypeptide or a mixture of the polypeptide and fragments of the polypeptide described above for the manufacture of a medicament for preventing and treating bone diseases.
This invention provides a method of preventing and treating bone-related disorders e. The method includes administering to a subject in need thereof an effective amount of a lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments of the polypeptide.
The method can further include concurrently administering to the subject an effective amount of another bone-enhancing agent. The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will be apparent from the detailed description, and from the claims.
Lactoferrin and bone; structure-activity relationships.
Thus, it is useful for preventing and treating bone disorders. A lactoferrin polypeptide of the invention is a pure polypeptide containing no more than two metal ions per molecule. It can be isolated from a natural source e. The following is an exemplary procedure for isolating lactoferrin from bovine milk: Fresh skim milk 7 L, pH 6. Unbound protein is washed through with 2.
Lactoferrin eluting as a discreet pink band in 1 M sodium chloride is collected as a single fraction and dialysed against milli Q water followed by freeze-drying. The freeze-dried powder is dissolved in 25 mM sodium phosphate buffer, pH 6.
Fractions containing lactoferrin of sufficient purity as determined by gel electrophoresis and reversed phase HPLC are combined, dialyzed and freeze-dried. Final purification of lactoferrin is accomplished by gel filtration on Sephacryl in 80 mM dipotassium phosphate, pH 8.
Selected fractions are combined, dialyzed against milli Q water, and freeze-dried. Iron saturation is achieved by addition of a 2: The iron-loaded holo- lactoferrin is then freeze-dried. Massons and Heremans Protides of the Biological fluids 14, Citrate and EDTA are then removed by dialysis against 30 volumes of milli Q water once renewed and the resulting colourless solution freeze-dried.
A lactoferrin polypeptide of the invention can contain an iron ion as in a naturally occurring lasctoferrin polypeptide or a non-iron metal ion e. For instance, lactoferrin isolated from bovine milk can be depleted of iron and then loaded with another type of metal ion. For example, copper loading can be achieved according to the same method for iron loading described above.
For loading lactoferrin with other metal ions, the method of Ainscough, et al. In a preparation of a lactoferrin polypeptide of the invention, the polypeptides can be of a single species, or of different species. For instance, the polypeptides can each contain a different number of metal ions or a different species of metal ions; or the lengths of the polypeptides can vary, e.
Such a preparation can be obtained from a natural source or by mixing different lactoferrin polypeptide species. For example, a mixture of lactoferrin polypeptides of different lengths can be prepared by proteinase digestion complete or partial of full-length lactoferrin polypeptides.
The degree of digestion can be controlled according to methods well known in the art, e. A complete digestion produces a mixture of various fragments of full-length lactoferrin polypeptides; a partial digestion produces a mixture of full-length lactoferrin polypeptides and various fragments. A lactoferrin polypeptide or a mixture of the polypeptide and fragments of the polypeptide described above is used to prepare a nutraceutical composition of this invention for preventing and treating bone-related disorders.
Examples of such disorders include, but are not limited to, osteoporosis, rheumatoid or osteo-arthritis, hepatic osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, chronic renal disease, sarcoidosis, glucocorticoid-induced osteoporosis, idiopathic hypercalcemia, Paget's disease, and osteogenesis imperfecta.
The nutraceutical composition can be a dietary supplement e. The composition can also include other nutrients, such as a protein, a carbohydrate, vitamins, minerals, or amino acids. The composition can be in a form suitable for oral use, such as a tablet, a hard or soft capsule, an aqueous or oil suspension, or a syrup; or in a form suitable for parenteral use, such as an aqueous propylene glycol solution, or a buffered aqueous solution. The amount of the active ingredient in the nutraceutical composition depends to a large extent on a subject's specific need.
The amount also varies, as recognized by those skilled in the art, dependent on administration route, and possible co-usage of other bone-enhancing agents. Also within the scope of this invention is a pharmaceutical composition that contains an effective amount of a lactoferrin polypeptide or a mixture of the polypeptide and fragments of the polypeptide described above, and a pharmaceutically acceptable carrier.
The pharmaceutical composition can be used to prevent and treat bone-related disorders described above. The pharmaceutical composition can further include an effective amount of another bone-enhancing agent. The pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an isotonic and absorption delaying agent.
The interrelationship of dosages for animals and humans based on milligrams per meter squared of body surface is described by Freireich, et al. Body surface area can be approximately determined from height and weight of the subject. Effective doses also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, and the like. A lactoferrin polypeptide of the invention or a mixture of the polypeptide and fragments of the polypeptide can be formulated into dosage forms for different administration routes utilizing conventional methods.
For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration.
Lactoferrin and bone; structure-activity relationships.
Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the lactoferrin polypeptide or a mixture of the polypeptide and fragments of the polypeptide with a solid carrier and a lubricant.
Examples of solid carriers include starch and sugar bentonite. The lactoferrin polypeptide or a mixture of the polypeptide and fragments of the polypeptide can also be administered in a form of a hard shell tablet or a capsule containing a binder, e. The pharmaceutical composition can be administered via the parenteral route.
Cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent. The efficacy of a composition of this invention can be evaluated both in vitro and in vivo.