Lactoferrin

This application claims priority to New Zealand Application Ser. No. 518121, filed Apr. 3, 2002, the content of which is incorporated herein by reference.

Lactoferrin is an 80kD iron-binding glycoprotein present in most exocrine fluids, including tears, bile, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and milk. It is a major constituent of the secondary specific granules of circulating poly-morphonuclear neutrophils. The richest source of lactoferrin is mammalian milk and colostrum.

Lactoferrin circulates at a concentration of 2-7 μg/ml. It has multiple postulated biological roles, including regulation of iron metabolism, immune function, and embryonic development. Lactoferrin has anti-microbial activity against a range of pathogens including Gram positive and Gram negative bacteria, yeasts, and fungi. The anti-microbial effect of lactoferrin is based on its capability of binding iron, which is essential for the growth of the pathogens. Lactoferrin also inhibits the replication of several viruses and increases the susceptibility of some bacteria to antibiotics and lysozyme by binding to lipid A component of lipopolysaccharides on bacterial membranes.

This invention relates to a lactoferrin polypeptide that is capable of stimulating skeletal growth and inhibiting bone resorption.

Specifically, this invention features a pure lactoferrin polypeptide containing no more than two (i.e., 0, 1, or, preferably, 2) metal ions per molecule. A “pure” polypeptide is a polypeptide free from other biological macromolecules and at least 65% (e.g., at least 70, 75, 80, 85, 90, 95, or 99%) pure by dry weight. The purity of a polypeptide can be measured by any appropriate 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 a wild-type lactoferrin polypeptide (e.g., a fragment of the wild-type lactoferrin polypeptide containing at least 2 (e.g., 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700) amino acids, or a recombinant protein containing a lactoferrin polypeptide sequence) that maintain the biological activity of a wild-type lactoferrin polypeptide are within the scope of the invention. A lactoferrin polypeptide of the invention can be of a mammalian origin, e.g., from human or bovine milk. The metal ion bound to the polypeptide can be an iron ion (as in a naturally occurring lactoferrin polypeptide), a 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.g., by promoting proliferation of osteoblasts and chondrocytes) and inhibit bone resorption (e.g., by inhibiting osteoclast development). A preparation of a lactoferrin polypeptide of the invention (e.g., lactoferrin isolated from bovine milk) can contain polypeptides of a single species, e.g., every molecule binding two iron ions. It can also contain polypeptides of different species, e.g., some molecules binding no ion and others each binding one or two ions; some molecules each binding an iron ion and others each binding a copper ion; some molecules each being a biological active lactoferrin polypeptide (full-length or shorter than full-length) that contains 0, 1, or 2 metal ions and others each being a fragment (same or different) of the polypeptide; or all molecules each being a fragment (same or different) of a full-length lactoferrin polypeptide that contains 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, e.g., a partial digest such as a proteinase digest, of full-length lactoferrin polypeptides. Otherwise, it can be obtained by mixing full-length lactoferrin polypeptides with various fragments of full-length lactoferrin polypeptides (e.g., synthetic fragments). A mixture of various fragments of full-length lactoferrin polypeptides, on the other hand, can be prepared, for example, by complete digestion (i.e., no full-length polypeptides remain after digestion) of full-length lactoferrin polypeptides, or by mixing different fragments of full-length lactoferrin polypeptides.

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.g., by stimulating skeletal growth and inhibiting bone resorption). 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.

This invention is based on the unexpected discovery that lactoferrin stimulates osteoblast and chondrocyte proliferation and inhibits osteoclast development. 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. Practically, the measurement of the ion/lactoferrin ratio for a preparation of lactoferrin can be in the range of 0-2.5. It can be isolated from a natural source (e.g., mammalian milk), or produced using genetic engineering or chemical synthesis techniques well-known in the art. The following is an exemplary procedure for isolating lactoferrin from bovine milk:

Fresh skim milk (7 L, pH 6.5) is passed through a 300 ml column of S Sepharose Fast Flow equilibrated in milli Q water, at a flow rate of 5 ml/min and at 4° C. Unbound protein is washed through with 2.5 bed volumes of water and bound protein eluted stepwise with approximately 2.5 bed volumes each of 0.1 M, 0.35 M, and 1.0 M sodium chloride. 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 6.5 and subjected to rechromatography on S Sepharose Fast Flow with a sodium chloride gradient to 1 M in the above buffer and at a flow rate of 3 ml/min. 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 300 in 80 mM dipotassium phosphate, pH 8.6, containing 0.15 M potassium chloride. Selected fractions are combined, dialyzed against milli Q water, and freeze-dried. The purity of this preparation is greater than 95% as indicated by HPLC analysis and by the spectral ratio values (280 nm/465 nm) of ˜19 or less for the iron-saturated form of lactoferrin.

Iron saturation is achieved by addition of a 2:1 molar excess of 5 mM ferric nitrilotriacetate (Foley and Bates (1987) Analytical Biochemistry 162, 296-300) to a 1% solution of the purified lactoferrin in 50 mM Tris, pH 7.8 containing 10 mM sodium bicarbonate. Excess ferric nitrilotriacetate is removed by dialysis against 100 volumes of milli Q water (twice renewed) for a total of 20 hours at 4° C. The iron-loaded (holo-) lactoferrin is then freeze-dried.

Iron-depleted (apo-) lactoferrin is prepared by dialysis of a 1% solution of the highly purified lactoferrin sample in water against 30 volumes of 0.1 M citric acid, pH 2.3, containing 500 mg/L disodium EDTA, for 30 h at 4° C. (Massons and Heremans (1966) Protides of the Biological fluids 14, 115-124). 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.g., a copper ion, a chromium ion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion). 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. ((1979) Inorganica Chimica Acta 33, 149-153) can be used.

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.g., some are full-length polypeptides and some are fragments, and the fragments can each represent a particular portion of a full-length polypeptide. 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.g., by manipulating the amount of proteinase or the time of incubation. 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.