CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/349,698 filed on May 28, 2010, and U.S. Provisional Application No. 61/423,983 filed on December 16, 2010, both of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is

150108_406PC_SEQUENCE_LISTING.txt. The text file is 80 KB, was created on May 27, 2011 and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

Preparations and compositions are described that comprise a lactoferrin and a nutritional mixture.

Description of the Related Art

Presently, the standard of care for treating hypoferremia and iron deficiency anemia (IDA) comprises administering large quantities of iron from inorganic sources, for example, ferrous sulfate. Toxicity is a major problem with oral ferrous sulfate resulting in many adverse effects, including gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation (see, e.g., Kadiiska et al, J. Clin. Invest. 96: 1653-1657 (1995); Oldenburg, et al, Eur. J. Clin. Invest. 30:505-510 (2000); Reifen et al, Dig. Dis. Sci. 45:394-397 (2000)). In pregnancy, hypoferremia and IDA represent a risk factor for maternal and infant health. In both industrialized and developing countries, hypoferremia and IDA in pregnancy is highly prevalent due to increased iron requirement, enhanced blood volume, and development of the fetal- placenta unit (see, e.g., Umbreit, Am. J. Hematol. 78:225-31 (2005); School, Am. J. Clin. Nutr. 81 : 1218-22 (2005)). In addition to enhanced maternal risks, pregnancy- associated anemia results in preterm delivery, retardation of fetal growth, low birth weight, and inferior neonatal health. A need exists for safe and efficacious therapeutic agents for managing the care of subjects who have hypoferremia and IDA or who are risk of developing hypoferremia and/or IDA.

BRIEF SUMMARY

Provided herein are preparations and compositions comprising lactoferrin that are useful for improving maternal and fetal health. Described herein are the following particular embodiments.

Embodiment 1. A preparation comprising an isolated lactoferrin and a nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation, and wherein the preparation is formulated to supplement the diet of a subject who is pregnant or who is desirous of becoming pregnant or who is postpartum.

Embodiment 2. The preparation according to Embodiment 1, wherein the nutritional mixture comprises one or more vitamins selected from folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E.

Embodiment 3. The preparation according to either Embodiment 1 or Embodiment 2, wherein the nutritional mixture comprises at least the vitamins folic acid and Vitamin B6.

Embodiment 4. The preparation according to either Embodiment 1 or

Embodiment 2, wherein the nutritional mixture comprises at least folic acid, Vitamin

B6, and Vitamin B 12.

Embodiment 5. The preparation according to any one of Embodiments

1-4, wherein the nutritional mixture further comprises one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 6. The preparation according to Embodiment 1, wherein the nutritional mixture comprises (a) folic acid, Vitamin B6, and Vitamin B12; (b) one or more vitamins selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E; and (c) one or more minerals selected from calcium, chromium, copper, magnesium, manganese, molybdenum, selenium, and zinc.

Embodiment 7. The preparation according to any one of Embodiments

1-6, wherein the nutritional mixture comprises folic acid in an amount at least 1.5 times greater than the daily recommended amount for a non-pregnant adult. Embodiment 8. The preparation according to any one of Embodiments 1-6, wherein the nutritional mixture comprises at least folic acid, Vitamin B6, Vitamin B12, biotin, and Vitamin D.

Embodiment 9. The preparation according to Embodiment 8, wherein the nutritional mixture comprises biotin in an amount sufficient to provide between 30- 300 μg biotin per day to the subject.

Embodiment 10. The preparation according to Embodiment 8, wherein the nutritional mixture comprises biotin in an amount sufficient to provide between 290-310 μg biotin per day to the subject.

Embodiment 11. The preparation according to Embodiment 8, wherein the nutritional mixture comprises Vitamin D in an amount sufficient to provide between 1000-2000 International Units per day to the subject.

Embodiment 12. The preparation according to Embodiment 8, wherein the nutritional mixture comprises Vitamin D in an amount sufficient to provide between 1800-2200 International Units per day to the subject.

Embodiment 13. A preparation comprising an isolated lactoferrin and a nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation, and wherein the preparation is formulated to supplement the diet of a child or an infant.

Embodiment 14. The preparation according to Embodiment 13, wherein the nutritional mixture comprises one or more vitamins selected from folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E.

Embodiment 15. The preparation according to either Embodiment 13 or Embodiment 14, wherein the nutritional mixture comprises at least the vitamins folic acid and Vitamin B6.

Embodiment 16. The preparation according to either Embodiment 13 or Embodiment 14, wherein the nutritional mixture comprises at least the vitamins folic acid, Vitamin B6, and Vitamin B12.

Embodiment 17. The preparation according to any one of Embodiments

13-16, wherein the nutritional mixture further comprises one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 18. The preparation according to any one of Embodiments 13-16, wherein the nutritional mixture comprises (a) folic acid, Vitamin B6, and Vitamin B 12; (b) one or more vitamins selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E; and (c) one or more minerals selected from calcium, chromium, copper, magnesium, manganese, molybdenum, selenium, and zinc.

Embodiment 19. The preparation according to any one of Embodiments 1-18, wherein the preparation further comprises one or more additional active nutritional ingredients.

Embodiment 20. The preparation according to any one of Embodiments 1-19, wherein the preparation comprises (a) a first composition comprising the isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition comprising the nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation.

Embodiment 21. The preparation according to Embodiment 20, wherein the first composition and the second composition are formulated together to form the preparation.

Embodiment 22. The preparation according to Embodiments 1-21 , wherein the preparation is formulated for oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, or subcutaneous administration.

Embodiment 23. The preparation according to Embodiment 22, wherein the preparation is formulated for oral administration.

Embodiment 24. The preparation according to Embodiment 20, wherein the first composition and the second composition are each formulated separately and wherein the first composition is formulated for oral, parenteral, lingual, buccal, intranasal, transdermal, vaginal, rectal, intramuscular, subcutaneous, or topical administration, and wherein the second composition is formulated for oral, lingual, buccal, intranasal, intramuscular, subcutaneous, transdermal, or parenteral

administration.

Embodiment 25. The preparation according to Embodiment 24, wherein the second composition is formulated for oral administration.

Embodiment 26. The preparation according to Embodiment 24, wherein the first composition and the second composition are each formulated separately and each is formulated for oral administration.

Embodiment 27. The preparation according to any one of Embodiments 20-26, wherein the first composition or the second composition further comprises one or more additional active nutritional ingredients.

Embodiment 28. The preparation according to any one of Embodiments

1-27, wherein an inorganic source of iron is absent. Embodiment 29. The preparation according to any one of Embodiments 1-28, wherein the daily dose of the preparation provides to a subject the lactoferrin in an amount sufficient to prevent or treat hypoferremia or iron deficiency anemia.

Embodiment 30. The preparation according to any one of Embodiments 1-29, wherein the amount of the lactoferrin is an amount sufficient to provide between 10 mg per daily dose and 1000 mg per daily dose.

Embodiment 31. The preparation according to any one of Embodiments 1-29, wherein the amount of the lactoferrin is an amount sufficient to provide (a) at least 10 mg per daily dose; (b) at least 25 mg per daily dose; (c) at least 50 mg per daily dose; or (d) at least 100 mg per daily dose.

Embodiment 32. The preparation according to any one of Embodiments 1-12 and 19-29, wherein the amount of the lactoferrin is an amount sufficient to provide at least 200 mg per daily dose.

Embodiment 33. The preparation according to any one of Embodiments 1-12 and 19-29, wherein the amount of the lactoferrin is an amount sufficient to provide (a) at least 300 mg per daily dose; (b) at least 500 mg per daily dose; (c) at least 600 mg per daily dose; (d) at least 750 mg per daily dose; or (e) at least 1000 mg per daily dose.

Embodiment 34. The preparation according to any one of Embodiments 1-33 wherein the lactoferrin is human lactoferrin or bovine lactoferrin.

Embodiment 35. The preparation according to Embodiment 34, wherein the lactoferrin is recombinant human lactoferrin or recombinant bovine lactoferrin.

Embodiment 36. The preparation according to Embodiment 34 or Embodiment 35, wherein the lactoferrin is a polypeptide fragment of lactoferrin, and wherein the polypeptide fragment is either lobe N or lobe C of the lactoferrin.

Embodiment 37. The preparation according to any one of Embodiments

1-36, wherein the lactoferrin has any percent saturation with one or more of iron(III), zinc, copper, and manganese.

Embodiment 38. The preparation according to Embodiment 37, wherein the lactoferrin has 10-30% saturation with iron(III).

Embodiment 39. The preparation according to Embodiment 37, wherein the lactoferrin is apolactoferrin.

Embodiment 40. A method of supplementing the daily diet of a subject comprising administering to the subject the preparation according to any one of Embodiments 1-39.

Embodiment 41. A method for preventing or treating hypoferremia or iron deficiency anemia in a subject who is pregnant, who is desirous of becoming pregnant, or who is postpartum, said method comprising administering to the subject the preparation according to any one of Embodiments 1-12 and 19-39.

Embodiment 42. A method for preventing or treating hypoferremia or iron deficiency anemia in a subject who is an infant or child, said method comprising administering to the subject the preparation according to any one of Embodiments 13- 31 and 34-39.

In another embodiment is provided a use of the preparation according to any one of Embodiments 1-12 and 19-39 for preventing or treating hypoferremia or iron deficiency anemia in a subject who is pregnant, who is desirous of becoming pregnant, or who is postpartum. In yet another embodiment is provided a use of the preparation according to any one of Embodiments 1-12 and 19-39 for the manufacture of a medicament for preventing or treating hypoferremia or iron deficiency anemia in a subject who is pregnant, who is desirous of becoming pregnant, or who is postpartum. In still another embodiment is provided the preparation according to any one of Embodiments 1-12 and 19-39 for use in preventing or treating hypoferremia or iron deficiency anemia in a subject who is pregnant, who is desirous of becoming pregnant, or who is postpartum.

In certain other embodiments provided herein is a use of the preparation according to any one of Embodiments 13-31 and 34-39 for preventing or treating hypoferremia or iron deficiency anemia in a subject who is an infant or child. In another embodiment is provided a use of the preparation according to any one of Embodiments 13-31 and 34-39 for the manufacture of a medicament for preventing or treating hypoferremia or iron deficiency anemia in a subject who is an infant or child. In yet another embodiment, a preparation according to any one of Embodiments 13-31 and 34-39 is provided for use in preventing or treating hypoferremia or iron deficiency anemia in a subject who is an infant or child.

In another embodiment, a preparation is provided herein that comprises an isolated lactoferrin and a nutritional mixture comprising vitamins, wherein the nutritional mixture comprises at least folic acid and Vitamin B6, and wherein a biologically effective amount of inorganic iron is excluded from the preparation. In another particular embodiment, the nutritional mixture comprises at least folic acid, Vitamin B6, and Vitamin B12. In an additional embodiment, the nutritional mixture further comprises at least one mineral selected from calcium, chromium, chloride, copper, iodine (iodide), fluorine (fluoride), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In a more particular embodiment, the at least one mineral is calcium. In yet another embodiment, the nutritional mixture further comprises at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In still more specific

embodiments, the nutritional mixture comprises (a) folic acid, Vitamin B6, and Vitamin B12; (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E; (c) calcium; and (d) at least one mineral selected from chromium, copper, magnesium, manganese, molybdenum, selenium, and zinc. In certain embodiments, the preparation is formulated to supplement the diet of a pregnant subject, and in certain particular embodiments, the preparation comprises an amount of folic acid at least 1.5 times greater than the daily recommended amount for a non-pregnant adult. In certain specific embodiments, the preparations described above and herein are formulated to supplement the diet of a child or infant. In another embodiment, the preparation further comprises one or more additional active nutritional ingredients. In certain embodiments, the preparation comprises (a) a first composition comprising the isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition comprising the nutritional mixture, wherein a biologically effective amount of inorganic iron is excluded from the preparation. In still other embodiments, the first composition and the second composition are formulated together to form the preparation. In other certain embodiments, the preparation is formulated for oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, or subcutaneous administration. In a more particular embodiment, the preparation is formulated for oral administration. In another embodiment, the first composition and the second composition are each formulated separately, wherein the first composition is formulated for oral, parenteral, lingual, buccal, intranasal, transdermal, vaginal, rectal,

intramuscular, subcutaneous, or topical administration, and wherein the second composition is formulated for oral, lingual, buccal, intranasal, intramuscular, subcutaneous, transdermal, or parenteral administration. In a more particular embodiment, the second composition is formulated for oral administration. In still another particular embodiment, the first composition and the second composition are each formulated separately and each is formulated for oral administration. In more specific embodiments, when the preparation comprises a first and a second

composition, the first or second composition further comprises one or more additional active nutritional ingredients. In a more specific embodiment, inorganic iron is absent. In certain embodiments, the daily dose of the preparation provides to a subject the lactoferrin in an amount sufficient to prevent or treat hypoferremia or iron deficiency anemia. In more particular embodiments, the amount of the lactoferrin is an amount sufficient to provide at least 10 mg per daily dose, at least 25 mg per daily dose, at least 50 mg per daily dose, at least 100 mg per daily dose, at least 200 mg per daily dose, at least 300 mg per daily dose, at least 500 mg per daily dose, at least 600 mg per daily dose, at least 750 mg per daily dose, or at least 1000 mg per daily dose. In certain embodiments, the lactoferrin is human lactoferrin or bovine lactoferrin. In other particular embodiments, the lactoferrin is recombinant human lactoferrin or

recombinant bovine lactoferrin. In still another particular embodiment, the lactoferrin is a polypeptide fragment of lactoferrin, and wherein the polypeptide fragment is either lobe N or lobe C of the lactoferrin. In additional specific embodiments, the lactoferrin has any degree of saturation with one or more of iron(III), zinc, copper, and manganese. In more particular embodiments, the lactoferrin is 10-30% iron-saturated lactoferrin. In other certain particular embodiments, the lactoferrin is apolactoferrin.

Also provided herein is a method of supplementing the daily diet of a subject comprising administering to the subject the preparation according to any one of the above embodiments described above and herein. In another embodiment, a method is provided for preventing or treating hypoferremia or iron deficiency anemia in a subject, which method comprises administering to the subject the preparation according to any one of the above embodiments described above and herein. In certain other embodiments, is provided a preparation according to any one of the above embodiments described above and herein for use in supplementing the daily diet of a subject. In certain other embodiments, is provided a preparation according to any one of the above embodiments described above and herein for use in preventing or treating hypoferremia or iron deficiency anemia in a subject. In yet other embodiments provided herein is a preparation according to any one of the above embodiments described above and herein for supplementing the daily diet of a subject. In still yet other embodiments provided herein is a preparation according to any one of the above embodiments described above and herein for preventing or treating hypoferremia or iron deficiency anemia in a subject. In another embodiment, a use of a preparation according to any one of the above embodiments described above and herein is provided for the manufacture of a medicament for supplementing the daily diet of a subject. In yet another embodiment, a use of a preparation according to any one of the above embodiments described above and herein is provided for the manufacture of a medicament for preventing or treating hypoferremia or iron deficiency anemia in a subject.

As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes at least one composition, one or more compositions, or a plurality of compositions, respectively. Similarly, reference to "a cell" or "the cell" includes reference to one or more cells and equivalent terms (e.g., plurality of cells) known to those skilled in the art, and so forth. Use of the conjunction "or" is meant to illustrate choice or possibilities and unless stated otherwise, the use of "or" does not mean that the terms or phrases joined by the conjunction are alternatives that are exclusive of each other. When referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 20% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may "consist of or "consist essentially of the described features.

As used herein, any concentration range, percentage range, ratio range, or integer range is understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size, thickness, height, weight, mass, volume, molarity, or pH are to be understood to include any integer or fraction thereof within the recited range, unless otherwise indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 summarizes the arms of a clinical study in which pregnant women (N=296) who were diagnosed with hereditary thrombophilia (HT) and iron deficiency (ID) (i.e., hypoferremia) or iron deficiency anemia (IDA) were randomized into two arms to be treated with either bovine lactoferrin (bLf) (N= 128) or ferrous sulfate (N=124), and self-selected to a third control arm by refusing treatment with iron supplementation (N=44). Also depicted are the number of women who completed the study and the number who were withdrawn and the reasons for withdrawal.

Figure 2 presents the mean values of red blood cells (RBC), hemoglobin (HB), total serum iron (TSI), and serum ferritin in 251 women affected by hereditary thrombophilia (HT), hypoferremia, and iron deficiency anemia (IDA) who completed the clinical study depicted in Figure 1 and described in Example 1. The circle in the "A" column designates 126 HT affected pregnant women in the A arm (treated with bLf). The circle in the "B" column designates 86 HT affected pregnant women in the B arm (treated with ferrous sulfate). The circle in the "C" column designates 39 HT affected pregnant women in the C arm (control group; absence of treatment). Each point represents the mean, and the bars represent 95% confidence intervals.

DETAILED DESCRIPTION

Presently, the standard of care for treating hypoferremia (iron deficiency (ID) in the absence of anemia), and anemia, such as iron deficiency anemia (IDA), comprises administering large quantities of inorganic iron as iron salts. Patients who are at risk of developing hypoferremia or IDA include women who are pregnant or are postpartum. Certain medical conditions, such as thrombophilia, further increase the risk that a pregnant woman will develop hypoferremia or IDA. Accordingly, prenatal vitamins recommended for women who are desirous of becoming pregnant or who are pregnant include iron salts. Infants (including neonates), particularly preterm infants, may also be at risk of developing hypoferremia or IDA, and currently the U.S. Food and Drug Administration requires the addition of iron salts to infant formulas. Iron salts are also often administered to subjects who have acute or chronic anemia, hypoferremia, and/or iron deficiency anemia.

However, administration of inorganic sources of iron, such as ferrous sulfate, often fails to treat these conditions due to poor bioavailability. In addition, because of toxicity associated with inorganic iron salts, administration of these iron salts to a subject frequently causes serious adverse effects (including gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation) (see, e.g., Kadiiska et al, J. Clin. Invest., supra; Oldenburg, et al., Eur. J. Clin. Invest., supra; Paesano et al., Biochem. Cell Biol. 84:377-380 (2006); Reifen et al, Dig. Dis. Sci., supra). Many people who have blood disorders (such as anemia, including chronic anemia and IDA, or hypoferremia) also have other diseases, disorders, or conditions associated with the blood disorder, caused by the blood disorder, or exacerbated by the blood disorder, that further compromise their general health and well-being.

Iron toxicity has been considered a risk factor for a variety of different conditions in non-pregnant individuals and in pregnant women (see, e.g., Weinberg, Oxidative Medicine and Cellular Longevity 2(2): 107-109 (2009)). Toxic effects of iron on mother and fetus relate, at least in part, to catalysis of free radical formation and oxidative stress (see, e.g., Weinberg, Metallomics 2(l l):732-40 (2010); Epub 2010 Sep 24; Favier et al, supra).

The combination of inefficient absorption and poor tolerability of iron salts leads to a significant lack of patient compliance, which is detrimental to the health of the subject intended to be treated, and in the case of a pregnant subject, potentially detrimental to both mother and fetus. The compositions and preparations described in detail herein that comprise lactoferrin but exclude an inorganic source of iron provide benefits of lactoferrin to the subject receiving the preparation in the absence of any undesired effect of iron.

Provided herein are preparations and compositions that comprise an isolated lactoferrin (Lf), such as isolated bovine lactoferrin (bLf), and a dietary nutritional mixture that lacks an inorganic source of a biologically effective amount of iron (i.e., the preparations and compositions lack an inorganic source of iron). In one embodiment, the preparations and compositions are formulated for use by a subject who is pregnant or who is desirous of becoming pregnant or who is postpartum; therefore the preparation and compositions comprise a nutritional mixture formulated appropriately to supplement the nutritional needs of the pregnant or postpartum subject. In other embodiments, the preparations and compositions that comprise Lf and a dietary nutritional mixture and lack an inorganic source of a biologically effective amount of iron are formulated for use by an infant or child. In still other embodiments, the preparations and compositions that comprise Lf and a dietary nutritional mixture and lack an inorganic source of a biologically effective amount of iron are formulated for use by a non-pregnant subject who is at risk of developing an anemia or who has an anemia.

In certain embodiments, the nutritional mixture includes one or more vitamins, for example, folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E. In more specific embodiments, at least two vitamins, folic acid and Vitamin B6 are included in the nutritional mixture. In certain embodiments, the supplementary dietary nutritional mixture comprises at least folic acid, Vitamin B6, and Vitamin B12. The nutritional mixture may further comprise one or more minerals, including for example, calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. These

compositions and preparations are described in greater detail herein. Unless stated otherwise, the nutritional compositions described herein are understood to lack (i.e., exclude, do not include) an inorganic source of a biologically (or pharmacologically) effective amount of iron.

The preparations and compositions may further comprise additional vitamins and/or minerals and/or one or more other active dietary (i.e., nutritional) ingredients. In another particular embodiment, the nutritional mixture may further comprise at least one non-dietary (i.e., non-nutritional) ingredient or component that is beneficial to the health and well-being of the subject recipient (such as an adult, including a pregnant adult, an infant, or a child) and is generally regarded as safe (GRAS) (e.g., stool softener or anti-nausea agent). Optionally, the preparations and compositions may further comprise any one or more of other micronutrients, macronutrients, enzymes, amino acids, herbs and plants and extracts thereof, and other/or specialty ingredients. In certain embodiments, the preparations and

compositions described herein may be prepared and administered to a subject as dietary supplemental preparations and dietary supplemental compositions, respectively.

These preparations and compositions described above and herein lack (i.e., do not include; exclude) an inorganic source of a biologically effective amount of iron. Inclusion of lactoferrin in these compositions and preparations provides a safe, effective, and non-toxic alternative to administration of an inorganic iron source. These preparations and compositions are useful for maintaining and/or restoring iron homeostasis, preventing (i.e., reducing the likelihood of occurrence in a statistically, biologically, or clinically significant manner) or treating hypoferremia or IDA, and supplementing or providing a subject's dietary requirements for various vitamins and minerals.

When iron requirements or the loss of iron exceed the quantity of iron absorbed, a negative iron balance occurs, and iron stores decrease. The deficiency of systemic iron can be divided in two categories: iron deficiency without anemia (ID; also called hypoferremia herein and in the art) and iron deficiency anemia (IDA). In hypoferremia, total serum iron levels decrease but hemoglobin levels remain normal. In iron deficiency anemia (IDA), the lack of iron can be so severe that iron stores are absent or unavailable resulting in abnormally low hemoglobin. IDA may also be characterized by low serum transferrin saturation, low hematocrit, and hypochromic, microcytic red blood cells.

Presently, the standard of care for treating hypoferremia and IDA comprises administering large quantities of iron from an inorganic iron source, for example, ferrous sulfate. As described herein and observed by clinical practitioners, iron salts, such as ferrous sulfate, causes adverse effects, including gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation (see, e.g., Kadiiska et al, J. Clin. Invest. 96: 1653-57 (1995); Paesano et al, Biochem. Cell Biol, supra; et al, Reifen et al, Dig. Dis. Sci. 45:394-97 (2000)). In addition, oral administration of ferrous sulfate also has a negative impact on hematological markers and induces an increase in serum concentration of proinflammatory mediators, such as inflammatory cytokines (e.g., IL-6) (see, e.g., Paesano et al., Biometals, supra; Paesano, Biochimie, supra). Other undesired consequences of iron supplementation are specific to pregnant women; administration of iron salts during pregnancy has been suggested to result in an increased risk that a woman will develop gestational diabetes (see, e.g., Bo et al, Am. J. Obstet. Gynecol. 2009; 201(2): 158.el-6. Epub 2009 Jun 13; Favier et al, Gynecol. Obstet. Fertil. 32(3):245-50 (2004); Weinberg Med. Hypotheses 73(5):714-15 (2009) Epub 2009 May 31), which in turn may predispose the subject to developing metabolic syndrome in later life (see, e.g., Bo et al, Metab. Syndr. Relat. Disord. 4(2): 113-21 (2006)).

As described herein (see Example 1), unexpectedly, administration of ferrous sulfate was less effective in restoring hematological parameters in a population at risk than omitting treatment with any inorganic source of iron. Administration of ferrous sulfate was less effective in restoring RBCs, hemoglobin, and serum ferritin in patients who were at risk of a thrombotic event occurring (pregnant women with thrombophilia) than not administering a source of inorganic iron (untreated).

Unexpectedly, serum ferritin levels of untreated patients were in the normal range but were below normal in patients treated with ferrous sulfate. By contrast, administering lactoferrin to pregnant women with thrombophilia effectively restored hematological parameters to acceptable physiological levels compared with patients treated with ferrous sulfate and compared with patients who were untreated.

Without wishing to be bound by theory, the inability of an organic source of iron to increase total serum iron concentration underscores the poor bioavailability of such compounds, suggesting that sequestration of compounds, such as ferric sulfate iron, in host cells prevents the export of iron to plasma. The poor pharmacodynamics of inorganic sources of iron has the potential of producing dangerous intracellular iron concentrations in enterocytes, macrophages and

hepatocytes.

Infants, particularly infants born from women who have hypoferremia or IDA, may also have or develop hypoferremia or IDA and thus may remain at risk of developing conditions or disorders that can be attributed to or are exacerbated by hypoferremia or IDA (see, e.g., Carter et al, Pediatrics 2010 Aug; 126(2):e427-34.

Epub 2010 July 26; Lukowski et al, Nutr. Neurosci. 13(2):54-70 (2010)). Accordingly, regulatory agencies such as the U.S. Food and Drug Administration require that infant formulas contain an inorganic source of iron. Infant formulas may be formulated as "low iron" formulas containing approximately 2 mg elemental iron per liter or may be formulated as "high iron" formulas containing approximately 12 mg elemental iron per liter. Supplementation with inorganic sources of iron in infant formulas and children's nutritional compositions that result in iron excess, however, can have serious deleterious effects on the health of the infant or child (see, e.g., Rao et al., Clin.

Perinatal. 2009 March:36(l):27-42 doi: 10.1016/j.clp.2008.09.013).

Lactoferrin (Lf) is a cationic, high-affinity iron-binding glycoprotein (see, e.g., Baker et al, Cell Mol. Life Sci. 62:2531-39 (2005)). Lactoferrin is an important regulator of systemic iron homeostasis and is capable of restoring

hematological parameters in hypoferremia and IDA (see, e.g., Paesano et al., Biochem. Cell Biol. 84:377-380 (2006); Paesano et al, Biochimie, supra; International Patent Application Publication No. WO 2007/065482; U.S. Patent Application Publication No. 2009/0156484). Recent reports have described that pregnant women have a significant decrease of total serum iron and serum ferritin concentrations (see, e.g., Paesano et al., Biometals, supra; Paesano et al, Biochimie, supra; Provenzano et al., Clin. J. Am. Soc. Nephrol. 4:386-393 (2009)).

Accordingly, in particular embodiments, the preparations and

compositions described herein are formulated for administration to an infant (including a neonate/newborn) or a child, and comprise an isolated lactoferrin (Lf) in combination with a nutrient mixture formulated with vitamins and/or minerals and/or additional nutrients in the combinations and amounts appropriate for administration to an infant or child. The nutritional mixture may include one or more vitamins, for example, folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5,

Vitamin A, Vitamin C, Vitamin D, and Vitamin E. In certain particular embodiments, the nutrient mixture comprises at least the vitamins Vitamin B6 and folic acid. In certain embodiments, the nutrient mixture further comprises at least Vitamin B 12 and may further include one or more additional vitamins. The nutritional mixture may further comprise one or more minerals, including for example, calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. The nutrient mixture may further comprise at least one additional active dietary ingredient and/or at least one active non-dietary ingredient. The nutrient mixture and preparations and compositions comprising the nutrient mixture described herein lack (i.e., do not include; exclude) a biologically (or pharmacologically) effective amount of inorganic iron (or a source thereof). The compositions and preparations comprising a nutritional mixture, such as infant formula and children's nutritional mixtures and compositions, are described in greater detail herein.

In another embodiment, the compositions and preparations comprising an isolated lactoferrin and a nutritional mixture but that exclude an inorganic source of a biologically effective amount of iron are formulated for a non-pregnant adult who has or who is at risk of developing hypoferremia or an anemia, such as IDA. The nutritional mixture may include one or more vitamins, for example, folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E. In certain particular embodiments, the nutrient mixture comprises at least the vitamins Vitamin B6 and folic acid. In certain embodiments, the nutrient mixture further comprises at least Vitamin B12 and may further include one or more additional vitamins. The lack of sufficient amounts of folic acid, Vitamin B6 and Vitamin B 12 are believed to contribute to development and/or perpetuation of anemic conditions. The nutritional mixture may further comprise one or more minerals, including for example, calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. The nutrient mixture may further comprise at least one additional active dietary ingredient and/or at least one active non-dietary ingredient.

Subjects who would benefit from receiving compositions and

preparations described herein include those whose level of one or more hematological parameters is below the range desired for healthy, non-anemic and non-hypoferremic individuals. For example, subjects who have decreased levels, or levels not within the range considered normal for a particular population, of red blood cells, total serum iron, hemoglobin, and/or serum ferritin (or other hematological parameters) may benefit from receiving the preparations comprising Lf and a nutritional mixture, which may provide statistically, biologically, or clinically significant improvement of one or more hematological parameters (e.g., statistically, biologically, or clinically significant increase in the level of red blood cells, total serum iron, hemoglobin, and/or serum ferritin). The levels and ranges that are considered normal may vary with gender, age, and weight (or mass) of the subject, which levels and ranges are familiar to persons skilled in the clinical and medical arts. In certain instances, other particular parameters, such as hepcidin, that are indicators of hematological status may be above the level or range of levels desired for or healthy, non-anemic and non-hypoferremic individuals.

Subjects who would benefit from receiving the preparations comprising

Lf and a nutritional mixture described herein include subjects with other types of anemia, for example, older and elderly subjects who have anemia, including those with unexplained anemia (i.e., anemia of unknown etiology). Other subjects who are anemic or at risk of developing anemia and who would benefit from the compositions and preparations described herein include subjects who need increased red blood cell production. Subjects who need to increase their ability to produce red blood cells include those who have a blood disorder, such as, but not limited to an anemia that can be categorized generally as one or both of anemia of chronic disease or chronic anemia of the critically ill. Anemia of chronic disease may occur as a result of the underlying chronic disease or occur as an effect of a therapeutic agent or therapeutic procedure (e.g., dialysis) or a combination of both. For example, anemia of chronic disease and/or anemia of the critically ill include anemia associated with a decline or loss of kidney function (chronic kidney disease, also referred to as chronic renal failure) and/or anemia associated with or resulting from kidney dialysis (i.e., hemodialysis or renal dialysis); anemia associated with malignancy (which includes anemia associated with a side effect of one or more chemotherapeutic agents administered (e.g., platinum containing agents)); anemia associated with inflammation and immunological diseases and disorders (e.g., psoriasis, rheumatoid arthritis; inflammatory bowel disease (including Crohn's disease and ulcerative colitis)); anemia associated with chronic infection including chronic viral infection (e.g., HIV, hepatitis B, hepatitis C); chronic heart failure; and chronic obstructive pulmonary disease (COPD). Other anemias of chronic disease and anemias of the critically ill include, but are not limited to, anemia associated with myelosuppressive therapy, such as anemia associated with

administration of a chemotherapeutic drug (e.g., a platinum based drug), or associated with administration of a biological, or associated with administration of an anti-viral drug (e.g., zidovudine for treatment of HIV/ AIDS; ribavarin for treatment of hepatitis C), or administration of radiation therapy; anemia associated with the progression of non-myeloid cancers (see, e.g., Weiss, The Oncologist 8(suppl): 18-29 (2003)); anemia associated with erythropoietin deficiency (such as occurs with kidney damage and/or disease); anemia associated with congestive heart failure; anemia associated with excessive blood loss; and anemia associated with blood loss during surgery or post- surgery. An anemia can become severe such that the subject is in need of treatment with an erythropoiesis stimulating agent (ESA). Blood disorders also include a hematopoiesis disorder (i.e., formation of blood or blood cells); hemachromatosis; and deficiencies or disorders in iron metabolism.

Other subjects who may benefit from administration of the preparations comprising Lf and a nutritional mixture described herein include subjects who are at risk of a thrombotic event occurring. In the United States, approximately 250,000 cases of venous thromboembolism (VTE) (also called venous thromboembolic disease (VTD)), which includes deep venous thrombosis (DVT) and pulmonary embolism (PE), are diagnosed annually and at least 50,000 cases are fatal. Venous thrombosis occurs when red blood cells and fibrin, and to a minor degree, platelets and leukocytes, form a mass within an intact vein. Typically, a pulmonary embolism occurs when a thrombus or a portion of the thrombus detaches from a vein wall and lodges within a pulmonary artery. Subjects at risk for occurrence of a thrombotic event include, for example, subjects who have at least one risk factor for VTE and/or who have one or more certain underlying medical conditions, diseases, or disorders. Exemplary risk factors that a subject may have include by way of nonlimiting example an autoimmune disease, hypertension, chronic microbial infection, decreased pulmonary function,

thrombophilia, an endothelial injury, malignancy, cardiovascular disease, obesity, inflammatory bowel disease, hormone use (for example, birth control pills, replacement hormone therapy for amelioration of menopausal symptoms), chemotherapy, and radiation therapy, pregnancy, and postpartum.

Normal pregnancy represents a hypercoagulable state; therefore, during pregnancy and postpartum, women have an increased risk of a thrombotic event occurring. The risk of occurrence of a thrombotic event increases when the woman has one or more other risk factors for thrombosis. Women with recurring adverse pregnancy outcomes, such as recurrent miscarriages, or who are at risk of an adverse pregnancy outcome, include women with thrombophilia. These women are in need of therapies to reduce the risk of pregnancy-associated complications. As discussed herein, clinical trials have demonstrated greater efficacy and safety of orally

administered bovine lactoferrin (bLf) compared to ferrous sulfate in treating

hypoferremia and IDA in uncomplicated pregnancies (see, e.g., Paesano et al.,

Biometals, supra; Paesano et al, Biochimie 91 :44-51 (2009); Epub 2008 June 14;

Paesano et al., Biochem. Cell Biol. 84:377-380 (2006)). In a human clinical trial described herein (see Example 1), pregnant women who had a history of problematic pregnancy and who had a diagnosis of hereditary thrombophilia (HT) and,

hypoferremia or IDA, and who were treated with bLf had a decreased incidence of miscarriage than women who were not treated with lactoferrin (i.e., women who were either treated with ferrous sulfate and women who did not receive either lactoferrin or ferrous sulfate).

Other subjects who may benefit from receiving the preparations comprising Lf and a nutritional mixture described herein include subjects who may have or who are at risk of developing inflammation (which includes chronic

inflammation), characterized, at least in part, by increased production of

proinflammatory cytokines, such as interleukin (IL)-ip, IL-6, IL-8, tumor necrosis factor (TNF)-a. Any mucous membrane may be affected, including oral (including buccal), pharyngeal, bronchial, uterine, endometrial, esophageal, gastric, intestinal, rectal, penile, vaginal, ocular, nasal, and/or olfactory mucous membranes, and mucous membranes of the ear or auditory canal.

Without wishing to be bound by any particular theory, when inflammation occurs in any one of the mucous membranes in a subject, the subject is at risk for developing, or concomitantly has, hypoferremia or iron deficiency anemia. During inflammation, a gradual transfer of free iron from the circulation to the tissues increases the physiological concentration of free iron at the mucous membrane to approximately 10 ^~18 M. This increased concentration of free iron at the mucous membrane can lead to additional undesired consequences including an overproduction (i.e., increased production) of reactive oxygen species, such as superoxides, at the tissue, which occurs, for example, by the iron-induced Fenton reaction. The increase in free iron at the mucous membranes also induces an increased production of

proinflammatory cytokines. In addition, the increase in free or available iron can facilitate microbial growth (including viruses and bacteria) resulting in microbial infections of the mucous membranes. Infections of the mucous membranes,

particularly of oral, intestinal, and vaginal epithelium can lead to tissue damage extensive enough to cause bleeding, which results in a further reduction of iron from circulation. Lactoferrin is capable of providing therapeutic benefit to a subject who has any one, two or more of inflammation, microbial infection, and hypoferremia or anemia. (See, e.g., International Patent Application Publication No. WO 2007/065482; U.S. Patent Application Publication No. 2009/0156484).

Preparations and Compositions

In one embodiment, a preparation is provided that comprises an isolated lactoferrin and a nutritional mixture (also called herein a nutrient mixture), which preparation does not include (i.e., excludes) an inorganic source of a biologically (or pharmacologically) effective amount of iron. The nutritional mixture may include one or more vitamins, including but not limited to, folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E. In certain particular embodiments, the nutritional mixture comprises at least two (i.e., two or more) vitamins, folic acid and Vitamin B6, and in another embodiment, the nutritional mixture may further comprise Vitamin B12 to provide at least sufficient amounts of folic acid, Vitamin B6, and Vitamin B 12 to supplement the diet of the subject. In another specific embodiment, the nutritional mixture comprising vitamin B6 and folic acid may further comprise Vitamin B12 and/or at least one other additional vitamin.

In other specific embodiments, the nutritional mixture may further comprise at least one (i.e., one or more) mineral. The one or more minerals in the nutrient mixture may include, for example, one or more of calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In certain specific embodiments, the preparations described above and herein may further comprise at least one (i.e., one or more) other dietary (i.e., nutritional) ingredient and/or at least one (i.e., one or more) non-dietary ingredient (e.g., a stool softener or anti-nausea agent or other

ingredient/agent generally regarded as safe (GRAS)). Other nutrients may include bioflavonoids referred to as Vitamin P.

In another embodiment, the preparation comprises an isolated lactoferrin (or a composition comprising the isolated lactoferrin and a physiologically suitable excipient) and a separate (or second) composition comprising the nutritional mixture, which comprises vitamins (e.g., folic acid, Vitamin B6, Vitamin B12), and/or at least one (i.e., one or more) minerals, and/or at least one (i.e., one or more) other dietary (i.e., nutritional) ingredient. The preparation may further comprise at least one (i.e., one or more) non-dietary ingredient. An inorganic source of a biologically (or

pharmacologically) effective amount of iron is excluded from (i.e., not included in) each composition and, thus, is also excluded from the preparation. Thus, the preparation and each of the any one or more compositions lack inorganic iron (i.e., lack an inorganic source of iron) in an amount sufficient to provide a detectable inorganic iron-associated biological (or pharmacological) activity or effect, particularly an undesired biological activity or effect.

As described herein, the nutrient mixtures may include any one or more vitamins, including but not limited to vitamins A, C, D, E, K, and B vitamins, which include folic acid, Vitamin B6, thiamine (also called Vitamin Bl), riboflavin (also called Vitamin B2), niacin, pantothenic acid (also called Vitamin B5), biotin, and vitamin B12, which are important to growth, metabolism, and function of the body. Folic acid, which is the synthetic form of folate (also called B9), is involved in RBC maturation and in the synthesis of purines and pyrimidines. Accordingly, adequate intake of folate is especially important during pregnancy and infancy. Vitamin B6 is required for hemoglobin synthesis. Vitamin B12 (also called cobalamin) is also required for RBC formation. Accordingly, in certain embodiments the nutrient mixture and the preparations described herein comprise at least the vitamins, folic acid, Vitamin B6, and Vitamin B 12. As understood in the art, certain vitamins if taken in excess may induce or contribute to a toxic or undesired effect. By way of example, Vitamin A, particularly when included in a nutrient mixture for prenatal use is included in an amount insufficient to cause birth defects.

An optimal dose of lactoferrin that is included in the preparations and compositions described herein may generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the subject (i.e., patient). Results of animal studies with lactoferrin have indicated that lactoferrin is well tolerated with minimum toxic effects at doses of 2000 mg/kg/day (see, e.g., Yamauchi et al, Food Chem. Toxicol. 38:503-12 (2000)). In certain embodiments of the methods provided herein, lactoferrin may be administered from about 5 to 50 mg lactoferrin per day, from about 10 to 1000 mg lactoferrin per day, from about 50 to 400 mg lactoferrin per day, from about 100 to 400 mg lactoferrin per day, from about 100 to 200 mg lactoferrin per day, from about 200 to 400 mg lactoferrin per day, from about 400 to 1000 mg per day, from about 1 gram to 5 grams per day, or from about 5 grams to 10 grams per day, or from about 10 to about 15 grams per day, which may be administered in one or in multiple doses. In certain

embodiments, the range of lactoferrin in the compositions and preparations described herein is formulated to provide lactoferrin in an amount from between about 10-25, 10- 50, 10-100, 10-200, 50-100, 50-200, 50-400, 100-200, 100-400, 200-400, 200-600, 400- 600, 400-800, 400-1000, 400-1500, 500-2000, 600-800, 1000-2000, 1000-1500, 1500- 2000, 2000-2500, 2500-3000, or 2000-5000 mg per day. The minimum dose is the amount effective for use in any of the methods described herein. By way of example, in certain embodiments, the minimum dose (which is the minimum daily dose) of lactoferrin is an amount sufficient to maintain iron homeostasis, and/or prevent (i.e., reduce the likelihood of occurrence) and/or treat hypoferremia or iron deficiency anemia or other anemia. In particular embodiments, about 10 mg, 25 mg, 50 mg, 100 mg, about 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 mg or more per day may be administered in one dose per day or in multiple doses (e.g., 2, 3, 4, 5) per day to provide the desired daily amount. In particular embodiments, compositions and preparations comprising lactoferrin are formulated for administration 2 times per day to provide at least about 10 mg, 25 mg, 50 mg, 100 mg, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 mg or more mg per day. In one particular embodiment, the total daily dose of lactoferrin that may be administered by the compositions and preparations described herein may be 50, 100, or 200 mg per day, which may be delivered in a single dose or in two separate doses. By way of additional example, a preparation formulated for administration to a subject who is pregnant, desiring to become pregnant, or postpartum may be formulated to provide between about 200-600 mg lactoferrin per day, which may be provided in a single dose per day or in two or more doses per day. In a particular embodiment, a lactoferrin is formulated at a dose to provide about 200 mg per day, and may be prepared and administered in two individual doses of 100 mg each. In another certain embodiment lactoferrin may be formulated for administration to provide about 100 mg lactoferrin per day, which in a more specific embodiment is administered in two individual doses of 50 mg each per day. By way of an additional example, when a preparation or composition described herein that comprises lactoferrin is delivered to a child or to an infant, such as in an infant formula or in oral drops that may be administered multiple times per day, the total dose of lactoferrin per day may be at least 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, at least 50 mg, 100 mg, or 200 mg/day. The use of the minimum dosage of a therapeutic or prophylactic agent that is sufficient to provide effective therapy (i.e., clinical or biological benefit) is usually preferred.

Infants and children may also receive an isolated lactoferrin in amounts greater than 50 mg/day as discussed above (e.g., 50, 100, 200 or more mg/ day).

As described in great detail herein, lactoferrin is not merely a molecule that is used for delivery and/or stability of a vitamin such as B12 and/or folic acid, unlike use of lactoferrin in folic acid/lactoferrin or Vitamin B 12/lactoferrin complexes that are formed by complexing multiple molecules of the vitamin(s) to a molecule of lactoferrin by ultrafiltration (see, e.g., U.S. Patent No. 6,500,472). As described herein, lactoferrin is a biologically and physiologically active ingredient of the preparations that comprise a nutrient mixture and that may also comprise folic acid, Vitamin B6, and may further comprise Vitamin B 12.

The amounts of each of the vitamins that can be included in the nutrient mixture can be readily determined given the extensive knowledge in the nutritional, metabolic, clinical, and related arts, with which a person skilled in these arts is familiar. Reference values have been developed by the U.S. Food and Drug Administration to aid consumers and clinicians determine the content of a nutrient in a food, drug, or in a dietary supplement. Dietary Reference Intake (DRI) is a general term for a set of reference values of a nutrient. DRIs include at least three reference values:

Recommended Dietary Allowance (RDA), Adequate Intake (AI), and Tolerable Upper Intake Level (UL). The RDA value provides the recommend average daily intake that is sufficient to meet nutrient requirements of the vast majority of healthy individuals, which may be different depending on the individual's age (e.g., infant (which includes newborn/neonate), toddler, child, pre-teen, teenager, young adult, middle aged adult, senior adult) and gender and whether the individual is pregnant, postpartum, or lactating. Nutritional mixtures are formulated using the appropriate amounts of each ingredient (or source of each ingredient) that will provide the desired amount to the subject or that will provide the desired amount when metabolized by the subject, which formulation procedures are accomplished according to methods and techniques with which the person skilled in the medical and nutritional arts is familiar. The RDA, AI, and UL of a particular vitamin, mineral, or other nutritional ingredient may increase or decrease as medical and nutritional practitioners respond to research study data.

Accordingly, a nutritional ingredient that may be included in the preparations and compositions described herein may be adjusted to provide the amount of the nutrient that appropriately supplements the diet of a subject and/or reduces or eliminates any toxic effect if the dose of a particular nutrient is too high. By way of example, as discussed herein, certain health organizations have increased the UL of Vitamin D. In addition, studies have indicated that pregnant women not only tolerate doses of 4000 IU per day but may also need these higher amounts to maintain maternal and fetal health. Administration of the RDA of a vitamin, mineral, or other micronutrient or

macronutrient or other active dietary ingredient may be administered as a single dose or may be administered in multiple (e.g., two, three, or four) doses per day.

Persons skilled in the art also appreciate that certain vitamins in excess

(such as vitamin A) may be toxic and care is taken not to exceed a particular daily value that is based on the recommended dietary allowance. Beta carotene, alpha-carotene and beta-cryptoxanthin are provitamin A carotenoids that can be converted into retinol (a useable, active form of Vitamin A) in the body. Beta carotene is more efficiently converted into retinol compared with alpha-carotene and beta-cryptoxanthin; therefore, beta carotene is often added to vitamin mixtures as the provitamin A carotenoid.

Also known in the art is that a subject who is at risk of a thrombotic event occurring and who is receiving warfarin (e.g., COUMADIN®) must have carefully regulated vitamin K intake, a vitamin that is involved in clot formation.

Studies have also suggested that vitamin E has blood-thinning effects and may increase risk of excess bleeding. Accordingly, a nutrient mixture that may be administered to a person at risk of a thrombotic event should be formulated appropriately such that the nutrient mixture and compositions comprising the nutrients do not place the subject at greater risk of occurrence of a thrombotic event.

One or more minerals that may be included in the preparations and nutrient mixtures described herein include as non-limiting examples, calcium, chromium, chloride, copper, iodine (iodide), fluorine (fluoride), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In a particular embodiment, the preparation and nutritional mixtures may comprise at least one of calcium, chromium, copper, iodine (iodide), magnesium, manganese, molybdenum, selenium, and zinc. In certain specific embodiments, the mineral included in the nutrient mixture is calcium. The source of a mineral may be one or more mineral salts of the mineral, which are typically more water soluble. By way of non-limiting example, calcium may be included in the nutrient mixture as calcium carbonate and/or calcium citrate, and magnesium may be included as magnesium oxide and/ or magnesium citrate .

The vitamins and minerals included in the nutrient mixtures and the preparations may be chemically synthesized or isolated from a natural source according to methods known and routinely practiced in the art. Sources of vitamins and minerals for inclusion in the nutrient mixtures described herein may also be commercially available.

The process by which the ingredients of the preparation are combined and mixed is performed in a manner appropriate for maintaining (i.e., retaining) the desired biological activity of the lactoferrin and of each of the vitamins and minerals. The preparations described herein may be formulated by combining individual ingredients and/or by combining compositions comprising one or more individual ingredients. In one embodiment, the isolated lactoferrin and the nutritional mixture are combined or mixed together (i.e., formulated) to provide the preparation. In another embodiment, a composition comprising an isolated lactoferrin and at least one physiologically suitable excipient is combined with or formulated with a composition that comprises the nutritional mixture. Optionally, at least one other active dietary (i.e., nutritional) ingredient and/or at least one non-dietary ingredient is formulated together with the lactoferrin (or composition comprising the lactoferrin) and the nutritional mixture (or composition comprising the nutritional mixture) to provide the preparation. At least one other active dietary (i.e., nutritional) ingredient includes, for example, any one or more of other micronutrients, macronutrients, enzymes, amino acids, herbs and plants and extracts thereof, and other specialty ingredients, which are described in greater detail herein. Any active dietary ingredient and/or any non-dietary ingredient may optionally be included in (added to, combined with) (a) a composition comprising the isolated lactoferrin; (b) a composition comprising the nutritional mixture, or (c) separately added to the preparation. In other embodiments of the preparation, the composition comprising the isolated lactoferrin (which can be referred to as a first composition) and the

composition comprising the nutritional mixture (which can be referred to as a second composition) are formulated separately and administered to the subject separately. One composition may be administered prior to, concurrent with, or subsequent to

administration of the second composition (also referred to as coordinate

administration). Administration of the preparations is described in greater detail herein.

The specific vitamins, minerals, and other active dietary {i.e., nutritional) ingredients and amounts or ranges of amounts of each that may be used in the nutritional mixtures described herein can be formulated using the extensive knowledge in the nutritional and medical arts {see, e.g., Office of Dietary Supplements, National Institutes of Health; Internet web site, dietarysupplements.nlm.nih.gov). The lactoferrin and the specific mixture of nutritional vitamins, and which may include minerals, may be formulated dependent on whether the intended user is an infant, a child, or an adult. Formulations for an adult may further be prepared dependent on whether the adult is a young adult, middle-aged, or a senior adult. The lactoferrin and the specific nutritional mixture may be formulated dependent on the gender and/or general health status of the intended user. The lactoferrin and the specific mixture of nutritional vitamins and minerals may be formulated to treat or prevent a particular disease, disorder, or condition as described herein. In certain embodiments, the preparations are formulated appropriately for a subject who is pregnant (or desiring to become pregnant), or postpartum, or lactating.

A nutritional mixture described herein may be formulated for a subject who has a disease, disorder, or condition and who cannot obtain or who has difficulty obtaining all essential and necessary vitamins and minerals, and other dietary nutrients from food consumption {i.e., from daily diet or food intake). By way of non-limiting examples, preparations may be formulated for (1) a pregnant or lactating subject; (2) an infant or child who has or who is at risk of developing iron deficiency (hypoferremia) or iron deficiency anemia; (3) an elite athlete; (4) an elderly subject; (5) a subject with one or more diseases, disorders or conditions that results in poor uptake of a vitamin or mineral or other nutrient from dietary sources; or (6) a subject with one or more diseases, disorders or conditions that requires greater intake of a vitamin, mineral, and/or other nutrient than compared to a subject who does not have the one or more diseases, disorders or conditions.

The one or more other active nutritional ingredients {i.e., components) that may be included in the preparations and compositions described herein include one or more amino acids, one or more fatty acids, fiber, one or more antioxidants, one or more enzymes, one or more herbs and/or plants (or extracts thereof) (also known as botanicals), and/or other dietary ingredients. An "active" nutritional ingredient means that the nutritional ingredient is present in nutritional mixture in an amount sufficient to have the desired biological or pharmacological effect in the host. In certain particular embodiments, when an antioxidant is at least one dietary ingredient included in the nutritional mixture, the nutrient mixture is formulated for administration to a subject with the proviso that the subject is a human and not a non-human animal. Non-limiting examples of herbs and plants that may be included in the preparations include but are not limited to ginger root, chamomile flower extract, and raspberry leaves. Examples of active nutritional ingredients also include enzymes, for example, amylases, proteases, various pancreatic and digestive enzymes, lipase, among others.

Other additional active nutritional ingredients include micronutrients and macronutrients that may also be called specialty ingredients in the art. Exemplary categories of specialty ingredients include fatty acids. The preparations and

compositions described herein may thus comprise one or more fatty acids, for example, DHA (docosahexaenoic acid), an omega-3 fatty acid, eicosapentaenoic acid (EPA) a- linolenic acid (ALA), and linoleic acid. The preparations and compositions may also further comprise one or more of choline; inositol; bioflavonoid complex; PABA; and coenzyme Q10.

In one embodiment, the preparations comprising Lf and a nutritional mixture are formulated for administration to a subject who is pregnant or who is desirous of becoming pregnant or planning to become pregnant or who is postpartum. These preparations comprising Lf and a nutritional mixture (which may also be called herein prenatal preparations and prenatal compositions) also lack an inorganic source of a biologically (or pharmacologically) effective amount of iron. A prenatal preparation comprising Lf and a nutritional mixture (as described herein) comprises the

combination and amounts of each vitamin and optionally at least one mineral, and/or at least one active nutritional ingredient, that support the dietary needs of a pregnant, postpartum, or lactating subject. The amount of each ingredient, which may typically, but not necessarily, be included in the preparation, is an amount that provides the RDA for the subject. The amounts can be readily determined by a person skilled in the art, given the extensive knowledge in the art regarding nutritional needs and requirements of pregnant, postpartum, and lactating women. The preparations comprising Lf and a nutritional mixture for use by a pregnant subject (or a subject who is planning to become pregnant) or postpartum subject may be useful for preventing (i.e., reducing the likelihood of occurrence) and/or treating iron deficiency and/or iron deficiency anemia.

Prenatal mixtures may be distinguished from nutrient supplements administered to non-pregnant adults with respect to daily dose requirements of certain vitamins and minerals. In particular, the recommended dietary allowance (RDA) of folic acid for a pregnant subject, or a non-pregnant female who is desirous of becoming pregnant, is at least 600 μg folic acid, whereas the RDA for a non-pregnant adult (not including a non-pregnant female who is desirous of becoming pregnant) is 400 μg (see, e.g., Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes:

Thiamin, riboflavin, niacin, vitamin B 6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academy Press, Washington, DC (1998)). The range of folic acid recommended for a pregnant subject is between 600-1000 μg. Greater amounts of folic acid may be recommended by a caregiver and can be formulated in the

compositions and preparations described herein. For example, a caregiver typically recommends that a woman who has previously given birth to a child with a neural tube defect receive greater than 1000 μg folic acid per day. Compositions comprising greater than 1.0 mg folic acid are typically obtained by prescription. Thus, in certain embodiments, prenatal preparations and prenatal compositions described herein that exclude an inorganic source of a biologically effective amount of iron differ, at least in part, from nutrient supplements for non-pregnant adults by inclusion of a sufficient amount of folic acid to provide a daily dose (i.e., recommended dietary allowance) at least 1.5 times the folic acid requirement (e.g., at least 600 μg). Vitamin supplements for non-pregnant adults (males and females greater than 19 years of age) and for teenagers from about 14-18 years of age, typically include approximately 400 μg folate. Accordingly, in certain embodiments when a preparation and/or composition comprising a nutrient mixture is administered to a subject, particularly a non-pregnant adult, the folic acid ingredient is included in the composition in an amount sufficient to provide approximately 400 μg folate or between about 300-500 μg per day to the subject.

By way of non-limiting example, in one embodiment, a prenatal preparation comprising Lf and a prenatal nutritional mixture of vitamins comprises folic acid in an amount sufficient to provide at least the recommended daily allowance of 600 μg (i.e., 1.5 times the RDA). In certain other embodiments, folic acid is present in an amount sufficient to provide a daily value of about between 600-980 μg, 600-650 μg, 650-700 μg, 700-750 μg, 750-800 μg, 800-850 μg, 850-900 μg, 900-950 μg, 900-975 μg, 950-975 μg, 975-980 μg, 980-990 μg, or 975-995 μg. In certain embodiments folic acid is present in an amount sufficient to provide a daily value of about 600 μg, 650 μg, 700 μ§, 750 μ§, 800 μ§, 850 μ§, 900 μ§, 950 μ§, 960 μ§, 980 μ§, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, or 4.0 mg. When the preparation comprising the nutritional mixture is administered to a postpartum subject, the nutritional mixture may be either a nutritional mixture formulated for a non-pregnant adult (i.e., to provide about 400 μg folic acid per day or about between 300-500 μg per day) or a nutritional mixture formulated for a pregnant adult as described in detail above (e.g., a nutritional mixture that comprises at least 600 μg folic acid, which is 1.5 times the recommended RDA for a non-pregnant adult).

In other certain embodiments, the nutritional mixture is formulated for use by an infant or child. By way of example, the National Institutes of Health's Office of Dietary Supplements suggests that the RDA for folate in infants between 0 to 6 months of age is about 65 μg per day and for infants between about 7 to 12 months is about 80 μg per day. The RDA for children between 1 and 3 years of age is about 150 μg per day, for children 4-8 years old is about 200 μg per day, and for children 9-13 years of age is about 300 μg per day.

As described herein, the amount of each vitamin (or source thereof) that may be formulated in the nutritional mixture can be readily determined by persons skilled in the art. By way of non-limiting example, a source of vitamin B6 is formulated in a nutrient mixture to provide the range of vitamin B6 amounts between about 2-25, 1.5-3 mg, 3-5 mg, 5-7 mg, 6-8 mg, 8-12 mg, 7-10 mg, 9-11 mg, 10-12, mg, 10-15 mg, 15-20 mg, 20-30 mg, 3-35 mg, 35-40 mg, 40-45 mg, or 40-45 mg per day to the subject. In more specific embodiments, the amount of vitamin B6 provided may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 22, 25, 30, 33, 35, 37, 40, 42, 43, 44, or 45 mg per day. A nutrient mixture comprising folic acid and vitamin B6 and that further comprises vitamin B 12 may be formulated with a source of vitamin B12 in the nutrient mixtures to provide between about 2-15 μg, 2.0-2.5 μg, 2.5-3 μg, 3 -5 μg, 5-10 μg, 10- 20 μg, or 20-30, 30-40, 40-50, 50-75, 75-100, 100-200, 200-400, 400-600, 600-800, 300-1200, 1200-1500 μg, or 2-20 mg, 20-50 mg, 50-75 mg, 75-100 mg, 100-120 mg 120-140 mg, 140-144 mg, or 144-150 mg per day. In more specific embodiments, a source of vitamin B 12 is formulated in the nutrient mixture to provide to the subject an amount of vitamin B12 about 2.0, 2.2, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 15.0, 20.0, or 40.0 μg per day. In other more specific embodiments, when large doses of vitamin B 12 are indicated for treating a subject, the amount of B12 that may be provided is about 1, 5, 10, 15, 20, 40, 60, 75, 100, 110, 120, 130, 140, 144, 145, 150, or 155 mg per day. In another particular embodiment, when the subject is in need thereof, the nutrient mixture may be formulated to provide significantly greater amounts of vitamins B6 and B12 than RDAs and than typically present in a dietary supplement. By way of non- limiting example, a subject may receive such a nutrient mixture and lactoferrin when the subject has thrombophilia (including hereditary thrombophilia). A person skilled in the medical arts can readily determine whether a subject is in need of a nutritional mixture comprising the greater amounts of vitamins B6 and B12. The subject may be a pregnant or a non-pregnant subject. By way of example, the nutrient mixture may comprise folic acid formulated to provide approximately 400 μg folate to the subject. The mixture may comprise a source of vitamin B6 in the nutrient mixture to provide between about 40-44 mg vitamin B6, which is to be delivered daily to the subject (i.e., to provide approximately 300 mg per week). The amount of vitamin B 12 to be provided by the nutrient mixture is between about 140-144 mg, which is to be delivered daily to the subject (i.e., to provide approximately one gram vitamin B12 per week). The amounts of each of folic acid, vitamin B6, and vitamin B12 per dose may be determined according to whether the desired daily amount is provided in one, two, or more doses per day.

In certain embodiments, the nutrient mixture comprises at least biotin, and in other embodiments, comprises at least biotin, Vitamin B6, folic acid, and Vitamin B 12. In humans, biotin acts as a coenzyme in the synthesis of fats, glycogen, and certain amino acids. Certain studies indicate that many pregnant women may have insufficient levels of biotin, which may increase the risks of birth defects (see, e.g., Mock et al, Am. J. Clin. Nutr. 75:295-99 (2002); Watanabe et al, Congenit. Anom. (Kyoto) 50(l):21-28 (2010)). In certain embodiments, nutritional mixtures comprising biotin are formulated to provide a daily amount of about 30 μg per day, such as for a non-pregnant adult. For nutritional mixtures formulated for use by a subject who is pregnant or who is desirous of becoming pregnant (i.e., prenatal formulations of the nutritional mixture), biotin may be included in an amount to provide between about 30 μg to about 300 μg per day. In particular embodiments of a prenatal formulation, biotin may be included in the nutritional mixture to provide a level of biotin between about 50 μg to about 300 μg per day, about 100 μg to about 300 μg per day, about 125 μg to about 300 μg per day, about 150 μg to about 300 μg per day, about 175 μg to about 300 μg per day, about 200 μg to about 300 μg per day, about 225 μg to about 300 μg per day, about 250 μg to about 300 μg per day, about 275 μg to about 300 μg per day, or about 290-310 μg per day. Biotin may be included at a level to provide about 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or about 300 μg per day. In nutritional compositions formulated for use by an infant, biotin may be included at a level to provide between about 1.5 to about 8 μg per day. In more particular embodiments, a nutrient mixture formulated for infants from 0 to about 6 months comprises biotin in an amount to provide about 5 μg per day and for infants from about 7 to about 12 months, the nutritional formula may provide about 6 μg per day. Nutritional mixtures formulated for use by children may include biotin at a level to provide about between 8 to 30 μ per day. For example, a nutritional mixture for administration to children from about 1-3 years may be formulated to provide biotin at about 7-9 μg per day (e.g., about 8 μg per day). A nutritional mixture for children from about 4-8 years may be formulated to provide biotin at about 10-14 μ per day (e.g., about 12 μ per day); for children about 9-13 years, the nutritional mixture may be formulated to provide about 15-25 μg biotin per day (e.g., about 18, 20, or 22 μg per day); and for children of about 14-18 years, the nutritional mixture maybe formulated to provide about 20-30 μg per day of biotin (e.g., about 20, 22, 25, 26, 28, or 30 μg biotin per day).

The preparations and nutritional mixtures described herein may include a source of Vitamin D (also called calciferol). Vitamin D is an essential vitamin required for bone health, including health and development of fetal bone. Vitamin D

insufficiency in pregnant women is thought to increase the risk of preeclampsia and may increase the risk of impaired fetal and infant growth and increase risk of developing an autoimmune diseases during infancy (see, e.g., Mulligan, Am. J. Obstet. Gynecol. 202:429.el-9 (2010), 2009 Oct 19 (Epub ahead of print)). Recent research indicates that humans may need and can tolerate without adverse effect at least about 4000 International Units (IU) Vitamin D per day. Previously, the upper limit recommended was approximately 2000 IU per day. The nutritional mixtures described herein may include a source of Vitamin D sufficient to provide between about 200-300, 300-400, 400-600, 600-800, 600-1000, 800-1000, 900-1100, 1000-2000, 1100-1400, 1250-1500, 1500-1600, 1600-1800, 1800-2100, 1800-2200, 2000-2200, 2200-2400, 2400-2600, 2600-2800, 2800-3000, 2900-3100, 3000-3200, 3200-3400, 3400-3600, 3600-3800, or about 3800-4000 IU per day. In more particular embodiments, the nutritional mixture may comprise at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, or 4000 IU Vitamin D per day. In yet another specific embodiment, a nutritional mixture formulated for use by a pregnant subject or a subject who desires to become pregnant may comprise a source of Vitamin D to provide about 600, 1000, 1500, 2000, 2500, or 3000 IU Vitamin D per day. The nutritional mixtures as described herein, and thereby the

preparation, may further comprise at least one mineral, and/or at least one other nutritional (i.e., dietary) ingredient. The preparation may further comprise at least one non-dietary ingredient. As with other preparations comprising Lf and a nutritional mixture described herein, an inorganic source of a biologically (or pharmacologically) effective amount of iron is excluded from the preparation. In another embodiment, a nutritional mixture, including a nutritional mixture formulated in a prenatal preparation, comprises one or more (i.e., at least one) minerals, which may include but is not limited to calcium, chromium, chloride, copper, iodine (iodide), fluorine (fluoride), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In another embodiment, the nutritional mixture, such as a prenatal nutritional mixture, comprises one or more of calcium, chromium, copper, iodine (iodide), magnesium, manganese, molybdenum, selenium, and zinc. In a more specific embodiment, the nutritional mixture includes the mineral, calcium.

Other active nutritional ingredients that may be included in the nutritional mixtures described herein, including a prenatal nutritional mixture, include but are not limited to fatty acids (e.g., DHA (docosahexaenoic acid), omega-3 fatty acids, eicosapentaenoic acid (EPA) a-linolenic acid (ALA), linoleic acid); choline; inositol; bioflavonoid complex (a source of antioxidants); PABA; and coenzyme Q10. Also included may be herbal or plant sources of vitamins, minerals, and other ingredients, such as ginger root, chamomile flower extract, and raspberry leaves. In addition, certain prenatal preparations and compositions may further comprise non- dietary (i.e., non-nutritional) ingredients, such as, an anti-nausea agent and/or a stool softener.

A person skilled in the art will also be familiar with recommendations provided by regulatory agencies, health agencies, or medical organizations with respect to content of nutrients to be included in a nutrient mixture. By way of example, the American Academy of Pediatrics Committee on Nutrition has published

recommendations for nutrient content and recommended amounts of each nutrient in infant formulas.

In accordance with U.S. Food and Drug Administration requirements and guidelines, infant formulas may be formulated as "low iron" formulas containing approximately 2 mg elemental iron per liter or may be formulated as "high iron" formulas containing approximately 12 mg elemental iron per liter. Persons skilled in the clinical arts may recommend use of "high iron" formulas or other "high iron" iron supplements for infants (e.g., pre-term neonates) who are at increased risk of developing hypoferremia or IDA or who have been diagnosed with hypoferremia or IDA. Because of the potential toxic effects of iron salts and the benefits of lactoferrin, alternative compositions and preparations are provided herein whereby the infant formula or other nutrient mixture that is administered to an infant in need thereof to reduce the likelihood of occurrence of hypoferremia or IDA or treat hypoferremia and IDA comprises lactoferrin and lacks an inorganic source of iron.

Non-dietary ingredients that are included in the preparations comprising Lf and a nutritional mixture described herein are ingredients that provide a clinical or therapeutic benefit to a subject. A non-dietary ingredient included in a preparation comprising Lf and a nutritional mixture may have a designation by a regulatory agency as an ingredient that is generally regarded as safe (GRAS). In other particular embodiments, non-dietary {i.e., non-nutritional) ingredients are excluded from the preparations and compositions described herein. By way of example, the compositions and preparations described herein do not include a non-dietary ingredient referred to as a mucin complex (which as described in U.S. Patent No. 7,638,142 is included in an ingested composition for treatment of dry eye).

As described herein, each vitamin, mineral, and additional nutritional ingredient {i.e., or source for each vitamin, mineral, and nutritional ingredient) that may be formulated in the nutritional mixture can be readily determined by persons skilled in the art. By way of non-limiting example, the source(s) of calcium is formulated to provide to the subject a range of amounts between about 125-300, 100-125, 125-300, 125-150, 150-175, 175-200, 200-225, 225-250, 250-300 mg, or 300-350 mg per day. In more specific embodiments, the amount of calcium provided to the subject may be about 100, 125, 150, 175, 200, 250, 275, 290, 300, or 350 mg per day. By way of non- limiting example, in a particular embodiment, a nutrient mixture comprises about between 2-25 or 9-11 mg vitamin B6 and 400-980 or 600-980 μg folic acid. In a more specific embodiment, when the nutrient mixture is intended for use in a prenatal nutrient mixture, the mixture comprises between 2-25 mg vitamin B6 and 600-980 μg folic acid. In other embodiments, the nutrient mixture further comprises 2-15 μg vitamin B 12. In more specific embodiments, the nutrient mixture comprising vitamin B6 and folic acid, which may further comprise vitamin B 12, comprises about between 2-25 or 9-11 mg vitamin B6 and 400-980 or 600-980 μg folic acid, and which may further comprise between 2-15 μg vitamin B12, may also further comprise between about 125-300 mg calcium. In more specific embodiments as noted above, when the nutrient mixture is intended for use in a prenatal nutrient mixture, vitamin B6 and folic acid are between about 2-25 mg vitamin B6 and 600-980 μg folic acid, respectively. In still other specific embodiments, the nutrient mixture formulated as a prenatal nutrient mixture that comprises one or more or all of folic acid, Vitamin B6, and Vitamin B12 may also comprise biotin, for example, in an amount sufficient to provide about 300 μg per day. In yet other specific embodiments, these nutrient compositions may further comprise a source of Vitamin D sufficient to provide about 2000 IU per day.

With respect to methods for reducing the likelihood of occurrence of iron deficiency or iron deficiency anemia or treating iron deficiency or iron deficiency anemia in an infant or child, the preparations and nutritional mixtures that comprise a lactoferrin and a physiologically suitable excipient, and the preparations and nutritional mixtures that further comprise at least folic acid and Vitamin B6 or at least folic acid, Vitamin B6, and Vitamin B12, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation and compositions, may further comprise additional active nutritional ingredients that provide the minimum daily nutritional requirements of the infant or child. In certain embodiments, the preparation and/or nutritional mixture is in the form of an infant formula and further comprises the additional vitamins, A, C, D, E, K, thiamin (Bi), biotin, and riboflavin (B ₂ ). The infant formulation preparation may also comprise one or more minerals, for example, but not limited to calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In more specific embodiments, the infant formulation comprises calcium, magnesium, zinc, manganese, and copper. Additional nutrients recommended by pediatricians and regulatory bodies include a protein source (e.g., bovine milk proteins, goat milk proteins, soy proteins); fat, linoleic acid, niacin, pantothenic acid, calcium,

phosphorous, iodine, sodium chloride, potassium chloride, carbohydrates (e.g., lactose in a milk based formula, or added carbohydrates such as sucrose, glucose, dextrins, natural or modified starches), and nucleotides. The amounts of each nutritional component may be readily determined by persons skilled in the nutritional arts.

The nutritional mixtures for use in the methods described herein may also readily be formulated for infants and children according to art-determined reference values of a nutrient as described herein (e.g., RDA, AI, and UL). The RDA values provide the recommend average daily intake that is sufficient to meet nutrient requirements of the vast majority of healthy infants and children and are readily available to persons skilled in the nutritional and medical arts. The RDA values are determined according to the infant or child's age. For older children, (9-18 years) the RDA values are determined for the child depending on age and gender. Recommended daily allowances of nutrients are typically set forth as requirements for four age groups: between 0-6 months, 7-12 months, 1-3 years, and 4-8 years. Recommended daily allowances of nutrients for older children are typically set forth as requirements for males from 9-13 years, males from 14-18 years, females from 9-13 years, and females from 14-18 years. The amounts of nutritional ingredients included in a nutritional mixture may also be adjusted appropriately to meet the needs of an infant or child who has one or more conditions, diseases, or disorders, which may dictate that the infant or child has a lower or higher daily requirement of a nutritional ingredient.

As understood in the chemistry arts, inorganic iron refers to iron or a source of iron that is not associated with a carbon-containing molecule, such as for example, transferrin, lactoferrin, hemoglobin (or other heme containing molecule), or other iron containing polypeptide or peptide (e.g., an amino acid-iron chelate).

Inorganic iron may be in the form of an iron salt, which includes for example ferrous sulfate, ferrous gluconate, and ferrous fumarate. The preparations comprising Lf and a nutritional mixture described herein lack an inorganic source of a biologically effective amount of iron; that is, an insufficient amount of inorganic iron is present in the compositions and preparations to have a detectable, biological effect or activity, particularly an undesired biological or pharmacological effect or activity (e.g., the capability to increase the level of a proinflammatory cytokine, and/or the capability to effect gastrointestinal discomfort, nausea, vomiting, diarrhea, and/or constipation). As described herein, an amount of inorganic iron that exhibits or has a detectable biological activity or effect (i.e., biologically effective amount) is excluded from the preparations and from each of the compositions described herein, including compositions comprising an isolated lactoferrin and from compositions comprising a nutrient mixture. An insufficient amount of iron from an inorganic source to effect a biological activity is also understood to include a total lack or absence of an inorganic source of iron.

Exemplary biological activities of iron from an inorganic source include any one of the biological activities observed in the studies described herein (see

Example 1) and in the art (see, e.g., Paesano et al, Biometals, supra; Paesano et al, Biochimie, supra; Paesano et al, Biochem. Cell Biol, supra) and include, but are not limited to, the undesired capability to cause an increase in production of inflammatory immune modulators (including for example, inflammatory cytokines, C-reactive protein) and to induce an inflammatory state; to cause a decrease in one or more hematological parameters (e.g., decrease in total serum iron, which may result in failure to restore iron homeostasis); and to cause at least one toxicity in a subject (including but not limited to inducing an inflammatory status of the subject, gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation). Stated another way, the amount of iron from an inorganic source that may be present in a preparation or composition described herein is insufficient to cause, induce, mediate, or facilitate an undesired biological activity that results in an undesired effect, including an adverse effect or toxicity.

An amount of iron that is insufficient to have at least one detectable biological activity (including, for example, any one or more of the undesired biological activities described herein) may be detectable or may be undetectable by physical methods used in the art for detecting iron or detecting an inorganic iron salt (e.g., inductively coupled plasma - mass spectrometry (ICP-MS)). Lactoferrin

The preparations and compositions described herein comprise an isolated lactoferrin. Lactoferrin is a globular, cationic, non-heme iron-binding protein, which is a glycoprotein in milk, other secretory fluids, and white blood cells, and is synthesized by exocrine glands and by neutrophils at infection and inflammation sites. Lactoferrin has antibacterial, antiviral, and anti-fungal, and anti-inflammatory properties (see, e.g., Conneely, J. Amer. Coll. Nutr. 20(5):389S-395S (2001); van der Strate et al, Antiviral Res. 52: 225-39 (2002); Valenti et al, Cell. Mol. Life Sci. 62:2576-87 (2005); Ward et al, Cell. Mol. Life Sci. 62: 2540-48 (2005); Bellamy et al, Biochim. Biophys. Acta 1121 : 130-36 (1992)). Without wishing to be bound by theory, lactoferrin may reduce inflammation by reducing and/or maintaining the production of proinflammatory factors such as IL-Ιβ, IL-6, IL-8, and TNF-a, for example, to a level that reduces, abrogates, prevents, minimizes destructive inflammatory effects (see, e.g., International Application Publication No. WO 2007/065482, which is incorporated herein by reference in its entirety).

Lactoferrin belongs to the family of transferrin proteins, which also includes serum transferrin (see, e.g., Baker et al, Biochem. Cell Biol. 80:27-34 (2002) and references cited therein). Lactoferrins between species share approximately 70% sequence identity (see, e.g., Baker, Adv. Inorg. Chem. 41 :389-463 (1994)). The amino acid sequence of lactoferrin contains a two-fold internal repeat, and the N-terminal half has approximately 40% sequence identity with the C-terminal half, which results in the protein folding into two homologous halves. Compared with serum transferrin, lactoferrin has a more potent iron-withholding activity: lactoferrin retains iron at a ph as low as pH 3.5, whereas, serum transferrin begins to lose iron at pH 6 (see, e.g.,

Mazurier et al, Biochim. Biophys. Acta 629:399-408 (1986); Peterson et al,

Biochemistry 39:6625-33 (2000)). The protein surface of the lactoferrin molecule has regions with high concentrations of positive charge that result in a high isoelectric point (~ pi 9) for the polypeptide. For example, in human lactoferrin, one region of positive charge includes the N-terminus portion of the mature lactoferrin that has an amino acid sequence of GR R S (SEQ ID NO: 15) (see, for example, amino acid residues 1-6 of SEQ ID

NOS:8, 9, and 10), which projects from the protein surface-terminus of the polypeptide chain, together with the adjacent carboxy terminal portion of helix 1, which includes a positively charged region, for example, the amino acid sequence, RKVR (SEQ ID NO: 16) or RRVR (SEQ ID NO: 17). This region provides a site for binding heparin (see, e.g., Van Berkel et al, Biochem. J. 328:145-151 (1997)) and glycosaminoglycans (see, e.g., Mann et al, J. Biol. Chem. 269: 2366-23667 (1994)) and may be the site that binds to DNA. The N-terminal portion is contiguous with helix 1 of the N-lobe, which forms the main part of the bactericidal domain (see, e.g., Bellamy et al, Biochim.

Biophys. Acta 1121 : 130-136 (1992)), which is characterized by the presence of surface arginine residues. Despite the virtually identical fold of lactoferrin and other members of the transferrin family, other transferrins do not share this bactericidal activity, presumably because they lack the necessary surface features (see, e .g, Baker, Biochem. Cell Biol., supra).

While bovine lactoferrin (bLf) does not share with serum transferrin the same N-terminal repeat of arginine residues, bLf has a highly positively charged region at its N terminus. In bovine lactoferrin, the domain responsible for bactericidal activity, which is also the heparin binding domain, includes residues 17-42 of the mature bLf polypeptide (Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala -Pro- Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala (SEQ ID NO: 18)) (see, for example, SEQ ID NO: 11-14) (see, e.g., Bellamy Biochim. Biophys. Acta, supra; Shimazaki et al., J. Dairy Sci. 81 :2841-49 (1998)). Motifs, KCRR (SEQ ID NO: 19) at positions 18-21 and RMKK (SEQ ID NO:20) at positions 25-28 (see SEQ ID NOS: l 1-14), are believed to be particularly important for heparin binding (Shimazaki et al., supra).

Lactoferrin has the capability to tightly but reversibly bind two Fe ³ ions (also referred to as ferric ions, iron III, or Felll) together with two carbonate ions (C0 ₃ ^2~ ), requisite to stabilize ferric ion binding. The presence of lactoferrin in tissues and secretions and transferrin in blood assures that iron is tightly complexed. The high affinity for ferric ions (approximately 10 ²² M) ensures that the concentration of free iron does not exceed 10 ^"18 M, at which point ferric hydroxides would precipitate (see, e.g., Aisen et al., "Physical biochemistry of the transferrins" in Iron carriers and iron proteins, vol. 5, Loehr, ed. VCH Publishers, New York, pp. 241-351 (1989); Baker et al, Cell. Mol. Life Sci. 62:2531-2539 (2005)). The high affinity of lactoferrin for iron also limits the availability of iron for microbial growth and inhibits iron-catalyzed formation of reactive oxygen species (see, e.g., Weinberg, Biochim. Biophys. Acta 1790:600-605 (2009)). Lactoferrin is an important regulator of systemic iron homeostasis and is capable of restoring hematological parameters in hypoferremia and IDA (see, e.g., Paesano et al., Biometals, supra; Paesano et al., Biochimie, supra;

Paesano et al, Biochem. Cell Biol. 84:377-380 (2006)). As discussed herein, several functions, dependent and independent of iron binding ability, have been attributed to lactoferrin (see, e.g., Valenti et al. Cell Mol. Life Sci. 62: 2576-87 (2005)).

Recent clinical trials suggest use of bovine lactoferrin for treating pregnancy-associated anemia (see, e.g., Paesano et al, Biometals, supra; Paesano et al, Biochimie, supra; Paesano et al, Biochem. Cell Biol., supra; Valenti et al., in Riv. It. Ost. Gin. Vol. 17 (2007); Paesano et al, Ginecologo Rivista di Ostetricia e

Ginecologia, Vol. 3, Suppl. al No. 1 Marzo 2008). In subjects with hypoferremia and IDA, a lactoferrin, such as bLf, can restore the physiological transport of iron from tissues to circulation, thereby normalizing iron homeostasis in patients with anemia (see, e.g., Paesano et al., Biometals, supra; Paesano et al., Biochimie, supra; Paesano et al., Biochem. Cell Biol., supra; Valenti et al., in Riv. It. Ost. Gin. supra; Paesano et al., Ginecologo Rivista di Ostetricia e Ginecologia, supra). Moreover, in direct contrast to ferrous sulfate, oral bLf is capable of exerting an anti-inflammatory effect by effecting a decrease in serum levels of inflammatory cytokines (such as IL-6, IL-Ιβ, IL-8, and TNF-a) in uncomplicated pregnancies (see, e.g., Paesano et al, Biometals, supra;

Paesano et al., Biochimie, supra).

The capability of lactoferrin to effect a decrease or reduction in the level of an inflammatory mediator or effector, such as a proinflammatory cytokine, means that the level of the inflammatory cytokine is reduced in a statistically, clinically, or biologically significant manner to a level that is within range of the amount of the inflammatory mediator or effector that is produced in the absence of an inflammatory response to an inflammatory stimulus. Stated another way, lactoferrin is capable of inhibiting, reducing, or preventing, the production of an inflammatory mediator, such as an inflammatory cytokine, to maintain, retain, or re-establish the level of the mediator to a level that is typically produced in a cell or in a subject in the absence of an inflammatory response to an inflammatory stimulus.

The biological effects of a lactoferrin may be achieved when the lactoferrin has any degree (i.e., percent) of saturation of the binding sites for iron (III), wherein "any degree" is understood not to exceed 100%. Even when no iron or an amount of iron substantially equivalent to no iron (i.e., in an amount less than about 1%) is bound by the lactoferrin (referred to as the "apo" form (i.e., apolactoferrin) wherein the degree of saturation of iron sites is equal to 0% or equal to an amount substantially equivalent to 0%), desired biological effects may be observed. The biological effects of a lactoferrin may also be achieved when the lactoferrin is partially saturated, or when the lactoferrin is completely saturated (referred to as the "holo" form of the lactoferrin (i.e., hololactoferrin) when the degree of saturation of iron sites is equal to about 100%). A lactoferrin used in the methods described herein may have any degree of saturation of the iron binding sites ranging from 0% saturation to and including 100% saturation, including but not limited to saturation from about 0%-20%, 0-40%, 10-30%, 10-15%, 10-18%, 13-16%, 10-35%, 10-40%, 10-50%, 10-60%, 15- 30%, 15-40%, 15-50%, 15-60%, 10-70%, or 10-80%. In a specific embodiment, a lactoferrin has a degree of saturation of the iron binding sites ranging from about 15- 30%. In another specific embodiment, a lactoferrin has a degree of saturation of the iron binding sites ranging from about 10-15%), 13-16%), 10-30%), or from about 10%>- 35%. The level of saturation may be achieved by using a mixture of lactoferrin molecules that have differing percent saturation to achieve a desired level of saturation.

The binding sites for iron can be occupied at any degree (i.e., percent) of saturation by Fe (III) and/or optionally, with different kinetics and affinities, by one or more other transition metals that have similar chemical and physical properties. These metals can be, for example, one or more of zinc (Zn), copper (Cu), aluminum (Al), gallium (Ga), chromium (Cr) and manganese (Mn). Accordingly, in one embodiment, lactoferrin used in the methods and compositions described herein has any degree of saturation by Fe (III) and one or more of Zn, Cu, and Mn. In another certain embodiment, lactoferrin has any degree of saturation with one or more of Zn, Cu, and Mn.

The isolated lactoferrin included in the compositions and preparations described herein and used in the methods described herein may be an isolated human lactoferrin, bovine lactoferrin, murine lactoferrin, or buffalo lactoferrin. In certain embodiments, the compositions described herein comprise bovine lactoferrin; in other embodiments, the compositions comprise human lactoferrin. Isolating bovine lactoferrin can be produced in large quantities by isolating the polypeptide from cow's milk. Isolated lactoferrin may also be obtained from commercial sources. Any lactoferrin, including bovine lactoferrin, described herein also may be produced recombinantly according to methods routinely practiced in the molecular biology, protein expression arts, and protein isolation arts. Full-length lactofemn has a molecular weight of approximately 80 kDa. The molecular weight of lactoferrin has also been reported to be 78 kDa. The difference in reported molecular size may represent the presence or absence of one N- linked oligosaccharide modification.

The amino acid sequences of human lactoferrin and bovine lactoferrin have been long known in the art and are readily available from any one of several public protein databases or commercially available protein databases. The majority of full-length human lactoferrin polypeptide species that have been sequenced are 711 amino acids in length, which includes a 19-amino acid signal peptide. Accordingly, an exemplary mature (i.e., without the signal peptide) lactoferrin polypeptide has 692 amino acids. Exemplary amino acid sequences for human lactoferrin are located in the GenBank database (National Center for Biotechnology Information (NCBI)) and include but are not in any way limited to Accession Nos. AAA59511.1 (SEQ ID NO: 1), ACF19793.1 (SEQ ID NO:2), and AAW71443.1 (SEQ ID NO:3). The amino acid sequences of mature human lactoferrin (i.e., without the 19-amino acid signal peptide) as represented by SEQ ID NOS: l, 2, and 3, are provided in SEQ ID NOS:8, 9, and 10, respectively.

The full-length bovine lactoferrin polypeptide species that have been sequenced are 708 amino acids, and the bovine lactoferrin polypeptides also include a 19-amino acid signal peptide. Accordingly, an exemplary mature (i.e., without the signal peptide) lactoferrin polypeptide has 689 amino acids. Exemplary amino acid sequences available in the art for bovine lactoferrin include, but are not limited to, GenBank Accession Nos. AAA30610.1 (SEQ ID NO:4), AAA30617.1 (SEQ ID NO:5), AAA30609.1 (SEQ ID NO:6), and AAA21722.1 (SEQ ID NO:7). The amino acid sequences of mature bovine lactoferrin (i.e., without the 19-amino acid signal peptide) as represented by SEQ ID NOS:4-7, are provided in SEQ ID NOS: 11-14, respectively. The encoding polynucleotide sequences for human and bovine lactoferrin (and other lactoferrin species) can be readily obtained in a similar manner from publicly available and privately (i.e., for a fee or supporting membership) available databases or by deducing an encoding polynucleotide sequence from the amino acid sequence.

In certain embodiments, the compositions and preparations lactoferrin that is a lactoferrin polypeptide fragment (see, e.g., U.S. Patent No. 7,420,033). As described herein, certain lactoferrin polypeptide fragments retain antimicrobial activity such as a cationic domain at the amino terminal end of lactoferrin (see, e.g., Bellamy, et al, supra; Conneely, supra; Nakamura et al, Protein Exp. Purif. 21;424-31 (2001); Tanaka et al., Biochem. Cell Biol. 81 : 349-354 (2003)). The antimicrobial activity of lactoferrin is structurally distinct and separate from its iron binding activity. Other fragments described in the art include fragments called lobe N and lobe C, and smaller fragments within each of lobe N and lobe C (see, e.g., International Application Publication No. WO 2007/065482). The amino terminal half of lactoferrin is referred to as the N-lobe (or Lobe N) and the carboxy terminal half is referred to as the C-lobe (or Lobe C). Both lobes have the same fold, which is consistent with the high percent sequence identity between the lobes. Each lobe is subdivided into two domains that are separated by an interdomain cleft that includes an iron binding site (see, e.g., Baker et al., Biochem. Cell Biol, supra).

Exemplary human lactoferrin fragments of the amino terminal region of lactoferrin (i.e., Lobe N) include but are not limited to a lactoferrin fragment from amino acid at position 1 to about position 280 of the mature human polypeptide (see, e.g., SEQ ID NOS:8-10). Lactoferrin fragments of lobe C include but are not limited to a lactoferrin fragment from about amino acid at position 285 to amino acid 692 of the mature human lactoferrin polypeptide. Certain exemplary N lobe fragments of bovine lactoferrin include amino acids at positions 1-333 (see, e.g., SEQ ID NOS: l 1-14), which may in certain embodiments, also include the inter-lobe region (typically residues at positions 334-344) (see, e.g., Bai et al, Biometals 2010 Feb 10, epub ahead of print). The N lobe and/or the C lobe may be obtained by recombinant expression of the lobe polypeptide using molecular biology and protein expression methods known and routinely practiced in the art. Alternatively, the lobe of interest may be obtained by proteolytic digest of the lactoferrin. (See also, e.g., U.S. Patent No. 7,420,033.)

The lactoferrin included in the compositions and preparations and used in the methods described herein may be full length lactoferrin, or truncated lactoferrin, or fragments of lactoferrin. Truncated lactoferrin is a lactoferrin polypeptide that comprises less than the full-length amino acid sequence of the polypeptide. As used herein "deletion" has its common meaning as understood by those familiar with the art, and may refer to molecules that lack one or more amino acids of a given sequence from either terminus or from a non-terminal region, relative to a corresponding full length or mature molecule. A truncated lactoferrin polypeptide may have one or more amino acids deleted from either the amino terminus and/or carboxy terminus of the polypeptide. In particular embodiments, a truncated lactoferrin retains at least one iron binding site. Also provided herein are methods for using compositions that comprise a polypeptide that comprises a fragment of lactoferrin as described herein. A lactoferrin fragment may comprise any number of contiguous (i.e., adjacent) amino acids between at least 10 and 700 amino acids (including but not limited to at least 10, 20, 40, 60, 80, 100, 120, 150, 200, 300, 400, and 500 amino acids and any whole number of amino acids between 10 and 690).

In certain embodiments, a lactoferrin polypeptide also includes lactoferrin species that have one or more amino acid substitutions, insertions, or deletions (also called herein a lactoferrin variant). Conservative substitutions of amino acids are well known and may occur naturally in the polypeptide or may be introduced when the polypeptide is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a polypeptide using well-known and routinely practiced mutagenesis methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY 2001)).

Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Deletion or truncation variants of proteins may also be constructed by using convenient restriction

endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in and the DNA re-ligated. Alternatively, random mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare lactoferrin polypeptide variants and fragment variants (see, e.g., Sambrook et al., supra). A bovine lactoferrin variant includes a polypeptide that has at least 85%, 90%, 95%, or 99% amino acid sequence identity to any of the exemplary bovine lactoferrin amino acid sequences known in the art and/or provided in the sequence listing.

Similarly, a human lactoferrin variant includes a polypeptide that has at least 85%, 90%, 95%, or 99% amino acid sequence identity to any of the exemplary human lactoferrin amino acid sequences known in the art and/or provided in the sequence listing.

Given the description in the art regarding regions of lactoferrin that exhibit particular activities (e.g., the N-terminal region comprising anti-microbial activity) and the amino acids that are the ligands for binding to Felll (see, e.g., Baker et al., Biochem. Cell Biol., 2002, supra; see also, e.g., Chappie, Antimicrob. Agents Chemother. 48:2190-98 (2004); Shimazaki et al., J. Dairy Sci. 81 :2841-49 (1998); Tanaka et al., Biochem. Cell Biol. 81 :349-54 (2003); Nakamura et al., Protein

Expression and Purification 21 :424-31 (2001); Chappie et al., Adv. Exp. Med. Biol. 443:215-20 (1998)), persons skilled in the art can readily determine which regions of lactoferrin may be more amenable to alteration (i.e., substitution, deletion, or addition of one or more amino acids) and which regions may not be amenable to change. Also given the description herein and given the many molecular biology, protein expression, and protein isolation techniques and methods routinely practiced in the art for introducing mutations in a polypeptide, preparing polypeptide fragments, isolating the fragments and variants, and analyzing same, lactoferrin variants and fragments having the desired biological activities can be made readily and without undue

experimentation.

Assays for assessing whether the lactoferrin variant folds into a conformation comparable to the non-variant polypeptide or fragment include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of ligand-binding functions, and the sensitivity or resistance of the mutant protein to digestion with proteases (see Sambrook et al., supra). Lactoferrin variants as described herein can be identified, characterized, and/or made according to these methods described herein or other methods known in the art, which are routinely practiced by persons skilled in the art.

Lactoferrin polypeptides, variants and fragments thereof, can be prepared without altering a biological activity of the resulting protein molecule {i.e., without altering one or more functional activities in a statistically significant, clinically significant, or biologically significant manner). For example, such substitutions are generally made by interchanging an amino acid with another amino acid that is included within the same group, such as the group of polar residues, charged residues, hydrophobic residues, and/or small residues, and the like. The effect of any amino acid substitution may be determined empirically merely by testing the resulting modified protein for the ability to function in a biological assay, or to bind to a cognate ligand or target molecule. By way of example, the capability of the lactoferrin variant or fragment to bind iron can be determined according to methods practiced by a person skilled in the art. In particular embodiments, a lactoferrin polypeptide, variant or fragment thereof, retains antimicrobial activity and/or retains the capability to effect a reduction or decrease in the production of at least one inflammation mediator, such as an inflammatory cytokine.

The isolated lactoferrin polypeptide, variant or fragment thereof, retains the capability to maintain and retain, improve, or restore hematological status of a subject as indicated by maintaining, improving, or restoring the level of one or more hematological parameters {e.g., red blood cell count, hemoglobin, total serum iron, serum ferritin, hematocrit). The capability of lactoferrin to improve or restore hematological status includes the capability to improve, increase, re-establish, or restore the level of a hematological parameter that is reduced in a statistically, clinically, or biologically significant manner to a level that is within range of the level of the hematological parameter in the absence of hypoferremia or anemia (including iron deficiency anemia). Other hematological parameters may be increased or elevated when a subject has an anemia. Accordingly, improvement in hematological status would also be indicated by a decrease or reduction of an elevated hematological parameter in a statistically, clinically, or biologically significant manner to a level that is typically measured in a subject in the absence of hypoferremia or an anemia. The isolated lactoferrin polypeptide, variant or fragment thereof, also retains the capability to reduce serum concentrations of proinflammatory modulators, such as inflammatory cytokines, that are elevated during an inflammatory immune response.

Lactoferrin can be prepared by isolating the protein from a source of milk or colostrum. Isolated lactoferrin means that the lactoferrin protein is removed (i.e., partially purified, or totally purified such that other components present in the source of lactoferrin are not detectable) from its original environment (e.g., the natural environment if it is naturally occurring). For example, when the polypeptide is present in a living animal, it is not considered to be isolated; however, the same polypeptide, separated from some or all or most of the co-existing materials in the natural system, is considered isolated. Alternatively, lactoferrin may be produced recombinantly according to methods routinely practiced by a person skilled in the molecular biology art, particularly given that the polypeptide sequence of lactoferrin and encoding nucleotide sequence have been long known in the art. An isolated lactoferrin is typically at least about 90% pure, at least about 95% pure, or at least about 99% pure.

By way of illustration, an isolated lactoferrin may be prepared using any of a variety of well known techniques. Recombinant polypeptides, encoded by nucleotide sequences as described herein and available in the art, may be readily prepared using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed, transduced, or transfected with an expression vector containing a polynucleotide that encodes a recombinant lactoferrin (or fragment or variant thereof). Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Lactoferrin forms that differ in glycosylation may be generated by varying the host cell or by post- isolation processing. Cell culture supernatants or cell extracts that comprise the lactoferrin may then be isolated from the host cellular components using methods and techniques routinely practiced in the protein purification art. Lactoferrin from natural sources, such as bovine milk, may also be isolated using methods and techniques routinely practiced in the protein purification art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al, Current Protocols in Molecular Biology, 2003.

Methods of Using Preparations and Compositions Comprising Lactoferrin and Nutrient Mixtures

In one embodiment, methods are provided for supplementing the diet of a subject by administering to the subject any one of the preparations described herein that comprise a lactoferrin and a nutritional mixture and which lack an inorganic source of a biologically effective amount of iron. The preparations and nutritional

compositions comprise one or more vitamins, for example, folic acid, Vitamin B6, Vitamin B12, biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin C, Vitamin D, and Vitamin E. In certain particular embodiments, the nutritional mixture comprises at least two (i.e., two or more) vitamins, folic acid and Vitamin B6, and in another embodiment, the nutritional mixture may further comprise Vitamin B12 to provide at least sufficient amounts of folic acid, Vitamin B6, and Vitamin B12 to supplement the diet of the subject. In another specific embodiment, the nutritional mixture comprising vitamin B6 and folic acid may further comprise Vitamin B 12 and/or at least one other additional vitamin, such as biotin and/or Vitamin D.

In other specific embodiments, the nutritional mixture may further comprise at least one (i.e., one or more) mineral. The one or more minerals in the nutrient mixture may include, for example, one or more of calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. In certain specific embodiments, the preparations described above and herein may further comprise at least one (i.e., one or more) other dietary (i.e., nutritional) ingredient and/or at least one (i.e., one or more) non-dietary ingredient (e.g., a stool softener or anti-nausea agent or other

ingredient/agent generally regarded as safe (GRAS)). Also as described herein, the preparations (and compositions) lack (i.e., exclude) an inorganic source of a

biologically (i.e., pharmacologically) effective amount of iron. Even though a subject theoretically could ingest the required amounts of vitamins and minerals (and other micro- and macro-nutrients) from food intake, to ensure that sufficient amounts of vitamins and minerals are available in the tissues and cells of the subject for optimum and proper function, many caregivers and nutritionists recommend supplementation of food intake (i.e., diet) with compositions of vitamins and minerals and, optionally, additional active nutritional ingredients. The preparations and compositions described herein may provide adequate amounts of essential nutrients especially for subjects who do not consume a sufficient amount of food or a sufficient variety of foods that provide essential vitamins and minerals. The subject may be a human or non-human mammal. The human may be an infant (including a neonate or newborn), child, teenager, adult (e.g., non-pregnant adult), may be a pregnant (or desiring to become pregnant), postpartum, or lactating.

In another embodiment, the preparations described herein that comprise at least one lactoferrin and a nutritional mixture from which an inorganic source of a biologically (pharmacologically) effective amount of iron is excluded, may be used in methods for preventing (i.e., decreasing the likelihood of occurrence) or treating hypoferremia or iron deficiency anemia in a subject. The preparations may be administered to subjects who have or who are at risk of developing acute or chronic anemia, hypoferremia, and/or iron deficiency anemia.

Anemia, generally, is characterized by an insufficient number of healthy red blood cells to carry adequate oxygen to the body's tissues. In iron deficiency anemia, typically, the red blood cells are smaller than normal; IDA is, therefore, an example of microcytic anemia. In chronic anemia, including anemia related to kidney disease, the red blood cell size is normal (normocytic anemia). In certain anemias, such as pernicious anemia (related to vitamin B 12 deficiency) and anemia related to alcoholism, the red blood cells are larger than normal; these anemias are sometimes called macrocytic anemias.

For a subject who has hypoferremia, (which is typically characterized, at least in part, by total serum iron levels that are decreased compared with the level in a subject (or the same subject) who does not have hypoferremia), administration of the preparations described herein may increase serum iron levels to a level that is normal (i.e., the level of serum iron is restored to a level that is improved or that falls within the normal range). In a subject who has iron deficiency anemia (IDA), the lack of iron can be so severe that iron stores are absent or unavailable resulting in abnormally low hemoglobin. IDA may also be characterized by low serum transferrin saturation, low hematocrit, decreased serum ferritin, and hypochromic, microcytic red blood cells. Accordingly, the preparations described herein may improve, increase, or restore the level of serum iron, hemoglobin, serum ferritin, and hematocrit to within near normal or normal range and/or improve (e.g., increase size) or restore the number, size, shape, and color of the red blood cells.

Maintaining, retaining, improving, or restoring hematological status of a subject is indicated by maintaining, improving, or restoring the level of one or more hematological parameters (e.g., red blood cell count and morphology, hemoglobin, total serum iron, serum ferritin, hematocrit) to the level of the respective parameter observed when the red blood cells are normal, healthy cells, and thereby abrogating or lessening the severity of hypoferremia or anemia. The capability of the preparations described herein that comprise lactoferrin to improve or restore hematological status includes the capability to improve, increase, or restore the level of a reduced hematological parameter in a statistically, clinically, or biologically significant manner to a level that is within range of the level of the hematological parameter in the absence of hypoferremia or anemia (including iron deficiency anemia). Stated another way, lactoferrin is capable of effecting an increase in hemoglobin, total serum iron, hematocrit, red blood cell count, and/or serum ferritin, for example, or other hematological parameters described herein to re-establish or restore the level of the hematological parameter to a level that is typically measured in a subject in the absence of hypoferremia or an anemia.

Other hematological parameters may be increased or elevated, such as the level of hepcidin, for example, when a subject has an anemia associated with inflammation as may be observed when the subject has hypoferremia or IDA.

Accordingly, improvement in hematological status would also be indicated by a decrease or reduction of elevated hepcidin in a statistically, clinically, or biologically significant manner to a level that is typically measured in a subject in the absence of hypoferremia or IDA.

Levels of each hematological parameter discussed herein that are considered normal can be readily ascertained by persons skilled in the art given the relevant characteristics (e.g., age, weight, gender, general health status, etc.) of the subject.

The capability of lactoferrin to effect a decrease or reduction in the level of an inflammatory mediator or effector, such as a proinflammatory cytokine, means that the level of the mediator or effector is reduced in a statistically, clinically, or biologically significant manner to a level that is within range of the amount of the inflammatory mediator or effector that is produced in the absence of an inflammatory response. Stated another way, lactoferrin is capable of inhibiting, reducing, or preventing, the production of an inflammatory mediator, such as an inflammatory cytokine, to maintain, retain, or re-establish the level of the mediator to a level that is typically produced in a cell or in a subject in the absence of an inflammatory stimulus or response.

Subjects who may be in need of prevention or treatment of hypoferremia or iron deficiency anemia include, but are not limited to, pregnant subjects or subjects who are desirous of becoming pregnant or planning to become pregnant. Subjects who are desirous of becoming pregnant or planning to become pregnant include subjects who are planning and intending to undergo in vitro fertilization.

The compositions and preparations described herein may also be administered to a subject for reducing the likelihood that a pregnancy-associated complication will occur (i.e., reducing or decreasing the likelihood in a statistically, biologically, or clinically significant manner). Pregnancy-associated complications include, but are not limited to, preeclampsia, preterm labor, gestational diabetes, miscarriage of the fetus, delivery of a premature neonate, intrauterine fetal death, delivery of a low birth weight neonate, placental abruption, and intrauterine growth restriction. All women who become pregnant have a statistical risk of miscarriage of the fetus due to a fetal chromosomal abnormality, which risk increases with maternal age. The compositions and preparations described herein that comprise a lactoferrin are not expected to and are not intended for use in preventing miscarriages or intrauterine fetal death that may occur due to a fetal chromosomal abnormality.

The hypercoagulable state represents one of the physiological changes in problem pregnancies. The compositions/preparations described herein may be administered to the subject who is pregnant or who is at risk or desirous of becoming pregnant, thereby reducing the likelihood of occurrence of a pregnancy-associated complication. A subject who has an increased risk that a pregnancy associated complication will occur include pregnant subjects who also have thrombophilia. A subject who has thrombophilia may have hereditary thrombophilia, acquired

thrombophilia, or have aspects of both acquired and hereditary thrombophilia.

Hereditary thrombophilia is a genetic predisposition resulting in the formation of thrombosis due to coagulation abnormalities (see, e.g., Rosendaal, Lancet 353: 1167-73 (1999); Kan et al., Thrombosis J. 4: 15-18 (2006)) and may result from one or more of several genetic alterations described herein and in the art. Hereditary thrombophilia is a significant risk for both maternal and infant health due to adverse outcomes including, for example, recurrent miscarriages, intrauterine fetal death, growth retardation, preeclampsia, and placental abruption (see, e.g., Stella et al, Clin. Obstet. Gynecol. 49:850-860 (2006); Patnaik et al, Expert. Rev. Cardiovas. Ther. 5:753-65 (2006)).

In other embodiments, the preparations and compositions described herein may be used in methods for preventing (i.e., reducing the likelihood of occurrence in a statistically, clinically, and/or biologically significant manner) a thrombotic event in a subject who is at risk of a thrombotic event occurring. Subjects at risk for occurrence of a thrombotic event include, for example, subjects who have at least one risk factor for venous thromboembolic disease (VTD) (also called venous thromboembolism (VTE)) and/or who have one or more certain underlying medical conditions, diseases, or disorders.

Venous thrombosis occurs when red blood cells and fibrin, and to a lesser degree, platelets and leukocytes, form a mass within an intact vein. Venous thrombi usually develop within a deep vein, and where blood flow may be sluggish or impeded and/or where a vascular trauma has occurred. Typically, a pulmonary embolism occurs when a thrombus or a portion of the thrombus detaches from a vein wall and lodges within a pulmonary artery, most often within the lobar arteries or distal main pulmonary artery.

Risk factors include the presence of one or more underlying medical conditions, diseases, or disorders. Exemplary risk factors that a subject may have include by way of nonlimiting example, prior history of VTE or an arterial embolism, an autoimmune disease, hypertension, a microbial infection (either acute or chronic), decreased pulmonary function, thrombophilia (which may be acquired or hereditary or a combination of acquired and hereditary), surgery, an endothelial injury, malignancy (cancer), cardiovascular disease, obesity, inflammatory bowel disease (ulcerative colitis or Crohn's disease), hormone use (for example, birth control pills, replacement hormone therapy for amelioration of menopausal symptoms), chemotherapy, and radiation therapy, pregnancy, and postpartum. In a certain embodiment, the method further comprises identifying a subject who has one or more of the aforementioned risk factors, which are described in greater detail herein. Occurrence of thrombotic events and the associated diseases and conditions (i.e., VTE and aortic embolisms) are multifactorial and a subject may have more than one risk factor that contributes to an increased risk of a thrombotic event occurring.

Prior history of a thrombotic event (which includes a thrombosis such as VTE including deep venous thrombosis (DVT) and pulmonary embolism (PE), and arterial thrombosis) is considered a risk factor for recurrence of thrombosis. However, because signs and symptoms of thrombotic diseases can be non-specific and insensitive, accurately establishing a subject's prior history may be problematic. Typical symptoms of DVT include leg pain, edema, erythema, and warmth in the affected limb.

Symptoms of PE are also nonspecific and when the subject does not have preexisting cardiovascular or pulmonary disease (which could also have the same or similar symptoms) include dyspnea, pleuritic chest pain, cough, leg edema, leg pain, hemoptysis, and palpitations (see, e.g., Stein et al, Chest 100:598-603 (1991)). A physical examination often identifies at least one of tachypnea, rales (crackles), tachycardia, a fourth heart sound, closure of the pulmonic valve indicated by accentuation of the second heart sound, DVT, and diaphoresis (see, e.g., Stein et al, Chest (1991), supra). A diagnosis of PE must be differentiated from various cardiovascular diseases, pulmonary diseases, malignancy, anxiety, and muscoskeletal conditions.

Objective testing may be performed to determine the presence of a thrombosis. For example, clinical prediction models, such as the Wells model, assess and assign weight to particular risk factors, signs and symptoms of deep vein thrombosis (see, e.g., Wells et al, Lancet 350: 1795-98 (1997)). Analysis using the Wells model may also be combined with other predictive analyses used in the art such as D-dimer testing (see, e.g., Wells et al, N. Engl. J. Med. 349: 1227-35 (2003)). D- dimers are formed when plasmin degrades cross-linked fibrin. The presence of D- dimers may be determined using tests, such as enzyme-linked immunosorbant assays (ELISA), whole-blood agglutination test, and/or a latex agglutination test. Because D- dimers may be present in patients with cardiopulmonary disease, malignancy, or who have had recent surgery or trauma, (who may be at risk for a thrombotic event but who may not necessarily have a thrombosis), the test is used more frequently for excluding thrombosis as a diagnosis (see, e.g., Bounameaux et al, Thromb. Haemost. 71 :1-6 (1994)). Other tests for determining the presence of deep vein thrombosis include, for example, duplex ultrasonography (see, e.g., Lensing et al, N. Engl. J. Med. 320:342-45 (1989)); contrast venography, impedance plethysmography, magnetic resonance venograph (MRV) (see, e.g., Carpenter et al, J. Vase. Surg. 18:734-41 (1993); Fraser et al, Ann. Intern. Med. 136:89-98 (2002)); computed tomography venography (CTV) (Baldt et al, Radiology 200:423-28 (1996)); CT pulmonary angiography, and combined CTV-CT angiography (CTA) (see, e.g., Stein et al, N. Engl. J. Med. 354:2317-27 (2006)).

A scoring system, such as the Wells model, is also used to predict probability of the presence of PE, which is a more predictive indicator when combined with ventilation perfusion scanning (see, e.g., Wells et al, Ann. Intern. Med. 129:997- 1005 (1998)). The results of Wells model may also be combined with a negative D- dimer test to exclude PE as a cause of a patient's symptoms (see, e.g., Kearon et al, Ann. Intern. Med. 144:812-21 (2006)). Electrocardiography is usually included with testing for PE because in certain patients, especially those with a large PE, show right heart strain. Other tests include chest radiography, arterial blood gas determination, lung scintigraphy (see, e.g., PIOPED Investigators, JAMA 263:2753-59 (1990)), computed tomography angiography (CTA) (see, e.g., Stein et al. (2006), supra; Segal et al, Ann. Fam. Med. 5:63-73 (2007)); echocardiography (see, e.g., Ribeiro et al, Am. Heart J. 134:479-87 (1997); Goldhaber et al. Lancet 341 :507-10 (1993)); and pulmonary angiography.

Other methods and tests include determining levels of fibrinopeptides, which are released at rapid rates during formation of fibrin clots, and blood/urine fibrinopeptide levels may, therefore, be useful measures of thrombus propagation in thrombembolic disease (see, e.g., U.S. Patent No. 7,763,561). The diagnosis and predictive value of each test and method available in the art in combination with a clinical history can readily be determined by persons skilled in the medical and clinical arts (see also, e.g., Qaseem et al, Ann. Fam. Med. 5:57-62 (2007)).

A person skilled in the medical and clinical art may also use one or any combination of diagnostic methods, including physical examination, clinical symptoms, and analytical tests and methods described above, for monitoring the health status of the subject and for monitoring whether the likelihood of a thrombotic event occurring is increasing, decreasing, or remains unchanged.

A subject who is at risk of a thrombotic event occurring may have thrombophilia as a risk factor. Thrombophilia (also called hypercoagulability) is the propensity to develop thrombosis and may be inherited or acquired or both inherited and acquired. Thrombophilia may be hereditary and may result from any one of several genetic mutations. Types of inherited thrombophilia include the factorV Leiden mutation (homozygous or heterozygous), the prothrombin gene mutation (homozygous or heterozygous) (e.g., G20210A; 5'UTR); methylene tetrahydrofolate reductase (MTHFR) C677T (homozygous); dysfibrinogenemia (an inherited thrombophilia in which a mutation in the fibrin molecule results in defective fibrin clot formation); and dysplasminogenemia (inherited plasminogen abnormality); and deficiencies of protein C, protein S, and antithrombin (see, e.g., Robertson et al, Br. J. Haematol. 132: 171-196 (2006)). Protein C deficiency may also be acquired, such as development of autoantibodies specific for protein C.

Other types of thrombophilia include or are related to the presence of anti-phospholipid antibodies (e.g., a lupus anticoagulant and anticardiolipin antibodies), and elevated factor VIII levels. The presence of anti-phospholipid antibodies, heparin- induced thrombocytopenia, and paroxysmal nocturnal hemoglobinuria are types of acquired thrombophilia that are suggested to pose a high risk for thrombosis. Elevated levels of homocysteine due to MTHFR mutation or vitamin deficiency (e.g., Vitamin B6, B12, and folic acid) also considered risk factors for thrombosis. Analytical techniques and tests are available in the art for determining thrombophilia, including genetic testing to determine if a subject has inherited thrombophilia and which gene (or genes) is affected.

Pregnancy and/or the postpartum period is a risk factor that increases the risk of a thrombotic event occurring in a subject. In other certain embodiments, the pregnant or postpartum subject also has thrombophilia (hereditary or acquired or both). Normal pregnancy and the postpartum period (up to at least 8 weeks and in some instances longer) are hypercoagulable states. In normal pregnancy, concentrations of factors VII, VIII, X, and von Willebrand factor, and fibrinogen are increased; free protein S (active, unbound form) is decreased; and plasminogen activator inhibitor type 1 (PAI-1) levels are increased. In addition, levels of PAI-2, which is produced by the placenta, increases dramatically during the third trimester. Other markers of thrombin generation, such as prothrombin Fl + 2 and thrombin-antithrombin (TAT) complexes, also increase (see, e.g., James, Hematology Am. Soc. Hematol. Educ. Program 277-85 (2009), supra). As discussed in greater detail herein, a woman, either pregnant or during the postpartum period, who also has thrombophilia is at even greater risk of thrombosis (James, Crit. Care Med. 38 (2 Suppl):S57-63 (2010)). Moreover, pregnancy-associated complications, such as recurrent miscarriage and intrauterine fetal death occur with greater frequency in women with thrombophilia (see, e.g., Kovac et al, Gynecol. Obstet. Invest. 69:233-38 (2010)); Grandone et al, Semin. Thromb.

Hemost. 35:630-43 (2009); Mantha et al, J. Thromb. Haemost. 8:263-68 (2010; Epub 2009 Nov 13).

Another risk factor for VTE is malignancy, and patients with cancers, such as brain, pancreatic, gastric, acute myelogenous leukemia, and renal cell carcinoma have the highest rates of VTE. Other malignancies with high rates of thrombosis include hematological malignancies, including multiple myeloma, myleoproliferative disorders, and lymphomas (see, e.g., Chew et al, Arch. Intern. Med. 166:458-64 (2006)). The risk of a thrombotic event occurring in a patient who has a malignancy can be increased by other risk factors including, for example, surgery, chemotherapy (see, e.g., Heit et al., Arch. Intern. Med. 160:809-15 (2000); Khorana et al, Cancer 104:2822-29 (2005); Cantwell et al, BMJ (Clinical research ed.) 297: 179- 80 (1988); Clark et al, Cancer 66:2027-30 (1990)), radiotherapy, use of erythroid stimulating agents, and presence of an indwelling catheter.

Cardiovascular disease, including myocardial infarction (also called coronary thrombosis), congestive heart failure, stroke, and hypertension are risk factors for VTE. Other risk factors such as obesity, type 1 or type 2 diabetes, chronic obstructive pulmonary disease (COPD) and other respiratory diseases or conditions, which may each be risk factors for VTE alone, pose increased risk if one or more occur in a patient who also has cardiovascular disease. In addition to COPD, other conditions, diseases, and disorders that result in decreased pulmonary function and therefore pose a greater risk of occurrence of a thrombotic event include asthma, chronic bronchitis, cystic fibrosis, and bronchiectasis.

Autoimmune diseases represent other medical conditions that are considered risk factors for VTE. Autoimmune diseases that are inflammatory diseases, such as inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) present an increased risk of VTE (see, e.g., Irving et al, Clin. Gastroenterol. Hepatol. 3:617-28 (2005)). Another such autoimmune disease is rheumatoid arthritis (see, e.g., Mameli et al, Clin. Exp. Rheumatol. 27:846-55 (2009)). One study reported that patients with rheumatoid arthritis and history of thrombotic events also had increased plasma levels of homocysteine and anti-phospholipid antibodies (see, e.g., Seriolo et al, Clin. Exp. Rheumatol. 19:597-615 (2001)). The presence of lupus anticoagulants is believed to be a significant indicator of an increased risk of thrombosis in patients with lupus erythematosus (see, e.g., Galli et al, Blood 101 : 1827-32 (2001)). Another autoimmune disease that is a risk factor for VTE is psoriasis.

A chronic or acute microbial infection, including an infection that is associated with inflammation, is a risk factor that identifies a person who is at risk of a thrombotic event occurring. Exemplary microbial infections includes, but are not limited to, Epstein-Barr Virus infection, a chronic bacterial infection, a chronic viral infection (e.g., hepatitis B, hepatitis C, human immunodeficiency virus and AIDS), gingivitis or periodontitis, a vaginal microbial infection (which may be an acute or chronic fungal, bacterial, or viral infection), and a urogenital microbial infection (which may be an acute or chronic fungal, bacterial, or viral infection).

A person at risk of a thrombotic event may present with diseases, disorders, or conditions of the kidney such as nephritic syndrome and renal disease, including chronic renal failure. Thrombotic events may occur among patients with renal disease, particularly end-stage renal disease, which includes patients who are receiving dialysis. Hemodialysis vascular access thrombosis, ischemic heart disease, and renal allograft thrombosis are complications of subjects with renal disease (see, e.g., Casserly et al, Semin. Dial. 16:245-56 (2003)). In addition, therapies that treat renal disease, or treat conditions associated with renal disease, such as administration of recombinant erythropoietin, dialyzer bioincompatability, and calcineurin inhibitor administration may contribute to increase risk of the subject to a thrombotic event. Another risk factor for a person at risk of a thrombotic event is endothelial injury. The presence of an indwelling venous device, surgery, major bodily trauma (such as a fracture), immobilization (such as occurs during prolonged travel) and paralysis (which includes anesthetization for greater than about 30 minutes), and varicose veins are exemplary endothelial injury risk factors. Additional risk factors include increasing age, oral contraceptive use, hormone replacement therapy, heparin- induced thrombocytopenia, paroxysmal nocturnal hemoglobinuria, microcythemia, von Willebrand disease, schizophrenia, and hyperthyroidism.

In certain embodiments, the subject at risk for a thrombotic event occurring and who has one or more of the risk factors discussed herein and known in the art may also be presenting hypoferremia and/or iron deficiency anemia. In particular embodiments, the subject is pregnant and also has hypoferremia and/or iron deficiency anemia; in more specific embodiments, the subject may also have thrombophilia (hereditary or acquired or both hereditary and acquired).

The compositions and preparations described herein (which comprise an isolated lactoferrin and a nutritional mixture of vitamins and minerals and exclude an inorganic source of a biologically effective amount of iron) may be administered to a subject who has any one or more of the risk factors at any time, including prior to, concurrent with, or subsequent to any episode of exacerbated symptoms. In a certain embodiment, the patient may be treated periodically or throughout the patient's lifetime with the compositions/preparations described herein according to a dosing regimen determined by a person skilled in the medical art, including the dosing regimens described herein. For a subject who is pregnant, or at risk or desirous of becoming pregnant, the compositions/ preparations may be administered prior to conception, during pregnancy (i.e., during one or more of the first, second, and third trimesters), and/or during the postpartum period.

The compositions and preparations described herein may also be useful for preventing a pregnancy-associated complication (i.e., decreasing or reducing the likelihood of occurrence of a pregnancy-associated complication in a statistically, clinically, or biologically significant manner) in a subject who is pregnant, who is at risk of becoming pregnant, or desirous of becoming pregnant. The subject may also have one or more conditions, disorders, diseases, or risk factors that increase the likelihood that the subject will experience one or more pregnancy-associated complications. The condition or risk factor may be thrombophilia (tendency to develop thrombosis and/or hypoferremia and/or iron deficiency anemia. One or more conditions, disorders, or diseases, (i.e., risk factors) that increase the risk of a pregnancy-associated complication occurring include, but are not limited to, infertility, recurrent pregnancy loss (e.g., recurrent miscarriage or recurrent intrauterine fetal death), inflammatory bowel disease (which includes Crohn's disease and ulcerative colitis), type 1 or type 2 diabetes, hypertension, renal disease (including renal disease for which the subject receives dialysis as well as renal disease for which dialysis is not yet required), microcythemia, an autoimmune disease or disorder (e.g., lupus erythematosus, rheumatoid arthritis, psoriasis). Additional risk factors include von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy (cancer), and microbial infection. A microbial infection includes but is not limited to Epstein-Barr Virus infection; a chronic or acute bacterial infection; gingivitis or periodontitis; a vaginal or urogenital microbial infection (including an acute or chronic bacterial, yeast, or fungal infection); and a chronic viral infection (e.g., HIV/ AIDS, hepatitis B, hepatitis C). Respiratory disorders, diseases, or conditions (e.g., asthma, cystic fibrosis, chronic bronchitis, and chronic obstructive pulmonary disease) may also increase risk of occurrence of a pregnancy-associated complication.

Another risk factor is the development or presence of maternal immune intolerance to a conceptus or fetus. The risk of a pregnancy-associated complication (in particular a miscarriage or intrauterine fetal death) occurring may increase when at least one paternal histocompatability antigen induces an immune response in the pregnant subject, thereby inducing maternal immune intolerance to a conceptus or fetus. Risk may also be increased when at least one fetal antigen or at least one embryonic antigen induces an immune response in the mother, resulting in maternal immune intolerance to the conceptus or fetus. The preparations/compositions described herein may be administered to a subject, who is pregnant or desiring to become pregnant, to induce immune tolerance in the subject to the conceptus or fetus. The preparations described herein may be useful for women who are planning to undergo or are undergoing in vitro fertilization procedures because of infertility and/or recurrent miscarriage.

Administration of these preparations may decrease the risk of a pregnancy associated complication occurring and improve or increase the probability that the subject will conceive and will carry the fetus to term.

A state of inflammation (for example, as characterized by an increase in production of proinflammatory cytokines including but not necessarily limited to IL-6, IL-Ιβ, IL-8, and TNF-a), hypoferremia, and IDA, each can increase the likelihood of a pregnancy-associated complication occurring during pregnancy and/or during the postpartum period. By way of background, in pregnancy, hypoferremia and IDA represent risk factors that can adversely affect maternal and/or infant health (see, e.g., Siega-Riz et al, Am. J. Obstet. Gynecol. 194:512-19 (2006)). In both industrialized and developing countries, hypoferremia and IDA in pregnancy are highly prevalent due to increased iron requirement, enhanced blood volume, and development of the fetal- placenta unit (see, e.g., Umbreit, Am. J. Hematol. 78:225-31 (2005); School, Am. J. Clin. Nutr. 81 : 1218-22 (2005)). In addition to enhanced maternal risks, pregnancy- associated anemia results in preterm delivery, retardation of fetal growth, low birth weight and inferior neonatal health. The degree of fetal hypoferremia is often less severe than observed in the mother because iron transfer from mother to fetus is regulated by the placenta (see, e.g., Bradley et al, Am. J. Physiol. Regul. Integr. Comp. Physiol. 287:R894-R901 (2004); Bastin et al, Br. J. Haematol. 134: 532-543 (2006)). Mechanistically, the placental syncytiotrophoblast acquires ferric iron bound to maternal transferrin at the apical membrane through transferrin receptors (TfR-1) (see, e.g., Cheng et al, Cell 116:565-76 (2004); Mullner et al, Cell 53:815-25 (1988)), which increases noticeably in pregnant women with hypoferremia or IDA (Bastin et al., supra).

As described herein, when iron requirements or the loss of iron exceed the quantity of iron absorbed, a negative iron balance occurs and iron stores decrease. Deficiency of systemic iron may result in hypoferremia, wherein total serum iron levels decrease but hemoglobin levels remain normal. When a subject has iron deficiency anemia (IDA), the lack of iron can be so severe that iron stores are absent or unavailable resulting in abnormally low hemoglobin. IDA may also be characterized by low serum transferrin saturation, elevated serum transferrin, low hematocrit, and hypochromic, microcytic red blood cells.

In industrialized and developing countries, hypoferremia and IDA are caused by increased iron requirements during pregnancy and lactation, as well as physiological losses of iron in menses. Due to enhanced blood volume and

development of the fetal-placenta unit during pregnancy (see, e.g., School, Am. J. Clin. Nutr. 81 : 1218-22 (2005)), hypoferremia and IDA can also represent a high risk for preterm delivery, fetal growth retardation, low birth weight, and inferior neonatal health.

Also by way of background, hepcidin and ferroportin (see, e.g., Bastin et al, Br. J. Haematol. 134:532-543 (2006)) are two proteins known to modulate systemic iron homeostasis through iron absorption, storage, and transport in adults (see, e.g., Ganz, Hematology Am. Soc. Hematol. Educ. Program :29-35, 507 (2006); Domenico et al., Nat. Rev. Mol. Cell Biol. 9:72-81 (2008)). Iron absorption takes place in the proximal duodenum and includes the following steps: (i) reduction of iron from the ferric state (III) to the ferrous state (II) by a ferrireductase (duodenal cytochrome B); (ii) apical uptake by enterocytes followed by trans-cellular trafficking via divalent metal transporter 1 ; (iii) storage into ferritin; and (iv) basolateral efflux by the iron transporter ferroportin (see, e.g., Domenico et al., supra). Ferroportin, the only known cellular iron exporter from tissues into blood, has been found in all cell types involved in iron export, including enterocytes, hepatocytes, placental cells (see, e.g., Donovan et al, Cell Metab. 1 : 191 -200 (2005)) and macrophages, which require ferroportin to recycle 20 mg of iron from lysed erythrocytes for erythropoiesis daily (see, e.g., Nemeth et al, Hematology J. 91 :727-732 (2006)).

Another component of systemic iron homeostasis is hepcidin, a circulating peptide hormone synthesized by hepatocytes in iron loading conditions and secreted in plasma (see, e.g., Krause et al, FEBS Lett. 480: 147-150 (2000)) and urine (Park et al, J. Biol. Chem. 276:7806-7810 (2001)), Epub 2000 Dec 11). Hepcidin regulates the entry of iron into plasma through ferroportin (see, e.g., Loreal et al., Curr. Protein Pept. Sci. 6:279-291 (2005)). By binding to ferroportin, hepcidin causes ferroportin phosphorylation, internalization, and degradation in lysosomes (see, e.g., Nemeth et al, Science 306:2090-2093 (2004); Domenico et al, Mol. Biol. Cell

18:2569-2578 (2007)), thus hindering iron export and enhancing cytosolic iron storage in ferritin. Without wishing to be bound by theory, iron homeostasis disorders appear to arise from hepcidin and/or ferroportin dysregulation (see, e.g., Domenico, et al, Nat. Rev. Mol. Cell Biol, supra; Nemeth et al, supra; Domenico et al, Mol. Biol. Cell, supra).

In pregnant women, absorption of nearly all dietary iron (1-2 mg/day) increases significantly in the second and third trimester of pregnancy to about 4 and 8 mg/day, respectively (see, e.g., Bothwell, Am. J. Clin. Nutr. 72(suppl):257-264 (2000)). Similar to the regulation of maternal systemic iron homeostasis, fetal hepcidin controls the transfer of maternal iron across the placenta to the fetus. Not wishing to be bound by any particular theory, iron transfer to the fetus that occurs after the 30th week of gestation may also involve placental expression of hepcidin and ferroportin (see, e.g., Bastin et al., supra; see also, e.g., Ganz, supra; Domenico et al., Nat. Rev. Mol. Cell Biol, supra). Enhanced placental-fetal iron transport is related to increased expression of ferroportin on the placental basal fetal-facing membrane, which is consistent with unidirectional mother-fetus iron transport (see, e.g., Bradley et al, Regul. Integr Am. J. Physiol. Comp. Physiol., supra; Bastin et al, supra). Studies regarding the mechanisms and effects of iron homeostasis during development of neonates are ongoing (see, e.g., Collard, Pediatrics 123: 1208-16 (2009)).

Regulation of hepcidin expression appears to occur at the transcriptional level. Hepcidin production is increased by iron loading and inflammation and decreased by anemia and hypoxia (see, e.g., Nicolas et al, J. Clin. Invest. 110 1037- 1044 ((2002)). Although the mechanisms surrounding hepcidin regulation by iron, oxygen and anemia are still unclear (see, e.g., Nemeth et al, supra), studies have indicated that IL-6 upregulates the hepcidin gene in hepatocytes (see, e.g., Nemethet al, J. Clin. Invest. 113 :(2004) 1271-1276 (2004)). In infection and in inflammatory disorders, IL-6-induced hepcidin, in combination with ferroportin, results in anemia of inflammation characterized by hypoferremia and IDA despite adequate iron stores (see, e.g., Nemeth et al, Annu. Rev. Nutr. 26:323-342 ((2006)). When iron export is hindered, it is stored in host cells. Inflammation may also contribute to hypoferremia and IDA by hepcidin-independent mechanism(s) through the down-regulation of ferroportin (see, e.g., Weinstein et al, Blood 100:3776-81 (2002)). Independent of IL- 6 induced hepcidin, high levels of serum IL-6 seem to down-regulate ferroportin mRNA expression, thus sequestering iron inside cells and blocking iron flow into plasma (see, e.g., Weinstein et al, supra; Ludwiczek et al, Blood 101 :4148-54 (2003)). This inability to export cellular iron stores leads to hypoferremia, decreased serum transferrin-Fe(III) pool, and iron-limited erythropoiesis.

Recent reports have described that a significant decrease of total serum iron and serum ferritin concentrations (see, e.g., Paesano et al, Biometals, supra;

Paesano et al., Biochimie supra; Provenzano et al., Clin. J. Am. Soc. Nephrol. 4:386- 393 (2009)) related to an increase of serum IL-6 concentration have been observed in pregnant women (see, e.g., Paesano et al, Biometals, supra; Paesano et al, Biochimie supra) and in hemodialysis patients orally treated with ferrous sulfate (see, e.g., Provenzano et al, Clin. J. Am. Soc. Nephrol, supra). These studies suggest that supplemented iron was not exported from cells to circulation, but was accumulated inside the cells resulting in their increased inflammatory status, similar to that reported in animals treated with ferrous sulfate (see, e.g., Kadiiska et al, J. Clin. Invest.

96: 1653-1657 (1995); Oldenburg, et al, Eur. J. Clin. Invest. 30:505-510 (2000); Reifen et al, Dig. Dis. Sci. 45:394-397 (2000)).

Presently, the standard of care for treating hypoferremia and IDA comprises administering large quantities of iron salts, for example, ferrous sulfate. However, oral ferrous sulfate often fails to improve hypoferremia and IDA due, at least in part, to poor bioavailability. As described herein, toxicity is a major problem with oral ferrous sulfate resulting in many adverse effects, including gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation (see, e.g., Kadiiska et al, J. Clin .Invest, supra; Oldenburg, et al, Eur. J. Clin. Invest, supra; Reifen et al., Dig. Dis. Sci. supra). In one study, approximately three-quarters of women receiving ferrous sulfate in a prenatal supplement experienced abdominal pain, dyspepsia, and constipation (see, e.g., Patterson et al, J. Am. Coll. Nutr. 20:337-42 (2001)).

Approximately one-third of the women who received the oral iron containing supplement were unable to receive the inorganic source of iron as prescribed (see, e.g., Patterson et al., supra). The combination of inefficient absorption and poor tolerability results in a significant lack of patient compliance, detrimental to the health of the subject intended to be treated, and in the case of a pregnant subject, potentially detrimental to both mother and fetus (see, e.g., Siega-Riz et al, Am. J. Obstet. Gynecol. 194:512-19 (2006); Patterson et al, supra; Wulff et al, Acta Obstet. Gynecol. Scand. 82:628-35 (2003); Bonar et al, Lancet 1 :457-78 (1969)).

Without wishing to be bound by any particular theory, hereditary thrombophilia (HT) and inflammation may be associated. Inflammation promotes coagulation (see, e.g., Fox et al, Thromb. Haemost. 94:362-75 (2005)) and high plasma levels of IL-6 have been found in HT diagnosed pregnant women with severe preeclampsia (see, e.g., Reitsma et al, J. Thromb. Haemost. 2:619-622 (2004)).

Moreover, elevated intra-amniotic IL-6 and IL-8 levels have been correlated with the occurrence of preterm birth (see, e.g., Hagberg et al, BJOG 112 Suppl 1 : 16-18).

Increases in proinflammatory mediators, including inflammatory cytokines, such as IL- 6, may enhance inflammation, iron overload in tissues, and cell damage. Thus increased levels of inflammatory cytokines, including IL-6, present additional maternal and fetal/neonate or infant risks during pregnancy. As described herein, by contrast to treatment of pregnant women with ferrous sulfate, which increases the level of IL-6 production, administration of bovine lactoferrin to pregnant women, including women who have hereditary thrombophilia, resulted in therapeutic effective treatment of hypoferremia and IDA, decreased IL-6 production, and restoration iron homeostasis and hematological markers.

Prior history of at least one (i.e., one or more) pregnancy-associated complication (which may also be called a pregnancy-associated pathology) described herein is also a contributing risk factor for subjects who may receive the compositions and preparations described herein. A pregnancy-associated complication may affect the health of the mother, the fetus or newborn (neonate), or both the mother and fetus or mother and neonate. Certain pregnancy-associated complications affect the health status of the mother but can, in turn, affect the health of the neonate (e.g., preeclampsia, which may result in premature delivery of the neonate or, more adversely, intrauterine fetal death). A pregnancy-associated complication includes, for example, preeclampsia, preterm labor, gestational diabetes, miscarriage of the fetus, intrauterine fetal death, delivery of a premature neonate, delivery of a low birth weight neonate, placental abruption, maternal intolerance, and intrauterine growth restriction. Accordingly, in certain embodiments, the compositions/preparations described herein may be administered to a subject for preventing or treating (i.e., reducing the likelihood of occurrence of) at least one of preeclampsia, preterm labor, gestational diabetes, miscarriage of the fetus, intrauterine fetal death, delivery of a premature neonate, delivery of a low birth weight neonate, placental abruption, maternal immune intolerance, and intrauterine growth restriction. In a particular embodiment, the compositions and preparations may be administered to the subject for reducing the likelihood of occurrence of a miscarriage or intrauterine fetal death.

The preparations comprising Lf and a nutritional mixture described herein may be administered to a subject who has disruption of placental cells and is at risk of detachment of the placental cells (and consequent detachment of the placenta), and to a subject who is at risk of having the amniotic membrane rupture prematurely (i.e., any time prior to when the fetus is considered full-term or prior to the time the fetus is considered to be of appropriate birth weight). The preparations may also be administered to a subject who is at risk of developing premature contractions, preeclampsia, who is at risk of preterm delivery of the fetus or neonate or delivery of a low birth weight neonate, who is at risk of intrauterine growth retardation of the fetus, and/or who is at risk of intrauterine death of the fetus.

Determining that the reduction in occurrence of a pregnancy-associated complication, for example in occurrence of miscarriage, spontaneous abortion, or preterm delivery, respectively, is statistically, biologically, and/or clinically significant can be determined by performing controlled clinical studies and/or review and analysis of historical data. Such studies and analyses are familiar to persons skilled in the art who determine clinical efficacy of drugs and biologicals. The subject may receive one or more doses of the preparation prior to conception and/or subsequent to conception (including for any length of time during the pregnancy). For example, the subject may receive the preparation during at least the first trimester, which is the time during which most miscarriages occur. In other embodiments, the preparation may be administered to the subject during the first two trimesters or during the entire pregnancy.

Administration of the preparations described herein to the subject may also be initiated after birth of the neonate or continued during the postpartum period, which may reduce the likelihood of a postpartum complication (e.g., a thrombotic event, a postpartum- related psychological condition (e.g., postpartum depression or postpartum psychosis), postpartum fatigue, hypoferremia, IDA) occurring in the mother.

Terms that are used to define fetal death vary in the medical art and vary among different countries and can vary within a country. By way of example, within the United States, even though a fetal death is reported to national and state government agencies, state governments within the United States vary in the definitions related to fetal death. In general, the loss of a fetus at any stage is a fetal demise. Yet, for statistical purposes, fetal losses may be classified according to gestational stage.

Typically, and for statistical purposes, the loss of a fetus prior to 20 weeks of gestation is called a spontaneous abortion (when not an induced termination of pregnancy), which is more commonly called a miscarriage. After approximately 20 weeks of gestation, a fetal death in utero is also known as stillbirth and is also referred to as intrauterine fetal demise, which is also called herein intrauterine fetal death. The preparations and compositions described herein may be administered to a subject who is pregnant, who is at risk of becoming pregnant or who desires to become pregnant and thus is at risk of becoming pregnant, which may prevent (i.e., reducing or decreasing the likelihood of occurrence) miscarriage or intrauterine fetal death (i.e., fetal death or demise at any stage of gestation).

Preeclampsia is a condition in pregnancy that is characterized by abrupt hypertension, which is a sharp rise in blood pressure; proteinuria (excess protein in the urine); high levels of vaginal and serum IL-6 (i.e., the levels of IL-6 are elevated above the levels observed in subjects in the absence of preeclampsia); sometimes edema (typically of the hands, feet, and face), changes in vision, abdominal pain, headaches, and dizziness. Preeclampsia is a common complication of pregnancy that occurs after 20 weeks gestation. Preeclempsia occurs more commonly in women who also have diabetes, are carrying twins, and/or whose mothers also had the condition. Serious complications that may result from preeclampsia include lack of blood flow to the placenta, placental abruption (total or partial separation of the placenta from the uterine wall), HEELP syndrome (hemolysis (the destruction of red blood cells), elevated liver enzymes and low platelet count), and eclempsia (which includes seizures and can lead to more serious adverse outcomes for mother and fetus). The preparations comprising Lf and a nutritional mixture described herein may be administered to a pregnant subject, thereby reducing the likelihood of occurrence (i.e., preventing) preeclampsia or reducing or decreasing the severity of one or more symptoms or consequences of preeclampsia or eclempsia.

The preparations described herein may also be used in methods for reducing or decreasing the likelihood of occurrence of placental abruption. Placental abruption as noted above is the total or partial separation of the placenta from the uterine wall. Consequences of placental abruption include maternal shock, premature birth of the baby, deprivation of the fetus of oxygen and nutrients, later identified neurological problems of the baby, and intrauterine fetal death. Another pregnancy- associated complication that may be prevented (i.e., for which the likelihood of occurrence is reduced or decreased) by administering the preparations described herein includes preterm labor.

The compositions and preparations described herein may be used for reducing or decreasing the likelihood that the pregnancy-associated complication, intrauterine growth restriction of the fetus, will occur. Fetal intrauterine growth restriction (IUGR) has also been referred to in the art as intrauterine growth retardation. The perinatal mortality for infants with IUGR has been reported to be 6 to 10 times greater than that for a normal growth infant population. Thirty percent of all stillborn infants are growth restricted and 50% suffer intrapartum asphyxia. Therefore, IUGR is a clinically significant prenatal problem. Perinatal and neonatal morbidity include intrapartum fetal distress, intrapartum asphyxia, hypoglycemia, hypocalcemia, meconium aspiration, and intrauterine death.

IUGR fetuses are typically characterized as one of two types:

symmetrically impaired or asymmetrically impaired. Symmetrically growth restricted fetuses tend to have impaired head growth at an earlier point in gestation than asymmetrically impaired fetuses, and has been observed more often in fetuses with infection or genetic and anatomic defects. The mortality risk and intrapartum fetal distress risks of symmetrically impaired IUGR fetuses are higher, in the range of 40- 50%. Asymmetric IUGR fetuses tend to have head growth that increases appropriately until late pregnancy and then lags behind growth that is considered normal.

Approximately two thirds of all cases of IUGR are asymmetric and involve many etiologies.

Persons skilled in the art distinguish the features of fetuses with IUGR from the features of fetuses with low birth weight. By way of example, the World Health Organization defines low birth weight as birth weight less than 2500 grams. These neonates may be small but uncompromised. The term, IUGR, may be used synonymously with small for gestational age (SGA) and implies a pathologic condition. Often the term SGA is applied to the neonate and IUGR is applied to the fetus. A widely accepted definition of IUGR is fetal weight below the 10th percentile for gestational age; however, the definition would encompass both constitutionally small and pathologically small fetuses.

As noted above, low birth weight infants with a birth weight of less than

2500 grams are small but not further compromised by a pathological condition as found with an IUGR infant. Low birth weight infants may be further subcategorized as very low birth weight, weighing less than 1500 g, or extremely low birth weight, weighing less than 1000 g. The lower the birth weight of the neonate, the greater increase in observed morbidity and mortality of the neonate. Extremely low birth weight neonates are also generally the youngest of premature newborns, born at approximately 27 weeks gestation or earlier and have the lowest first-year survival rate of low birth weight infants. The preparations and methods described herein may be useful for decreasing the risk or reducing the risk that the pregnant subject will deliver a low birth weight, very low birth weight, or extremely low birth weight neonate by administering a lactoferrin to the subject.

As described herein and understood in the art, a low birth weight infant may or may not have been delivered prematurely. Most human pregnancies last approximately 40 weeks, and as defined in the medical art, a premature birth occurs when an infant is born more than three weeks before the due date. Prematurely born infants are at greater risk for numerous medical and developmental problems, including respiratory problems, intracranial hemorrhage, hydrocephalus, cerebral palsy or other neurological conditions, vision and/or hearing problems, problems of the digestive tract, development delays, learning disabilities, jaundice, anemia, and low blood pressure.

The term, preterm labor, refers to when contractions begin to open the cervix before the 37 ^th week of gestation. Managing preterm labor to delay the time to birth is important for reducing the likelihood of occurrence of one or more

physiological and neurological conditions that affect the health, development, and survival of the infant. Subjects at greater risk that preterm labor will occur and/or delivering a premature infant include those who have had recurrent problematic pregnancies, such as miscarriage or prior incidence of preterm labor/and or preterm delivery, chronic health conditions (e.g., diabetes, high blood pressure, chronic infections), stress, multiple births, poor nutrition, smoking, and alcohol or drug use.

As described herein, during pregnancy, hypoferremia and IDA represent risk factors that can adversely affect maternal and/or infant health. In addition to enhanced maternal risks, pregnancy-associated anemia results in preterm delivery, retardation of fetal growth, low birth weight and inferior neonatal health. The preparations comprising Lf and a nutritional mixture described herein are useful for treating women with recurring adverse pregnancy outcomes, such as recurrent miscarriages, or who are at risk of an adverse pregnancy outcome, which includes women with thrombophilia.

The preparations comprising Lf and a nutritional mixture described herein may also be used in methods for reducing the likelihood of occurrence of at least one pregnancy-associated complication comprising administering these

compositions/preparations to a subject who has or who is at risk of developing immune intolerance to one or more antigens of the fetus or embryo (called herein fetal antigen or embryonic antigen, respectively), and/or one or more paternal histocompatability antigens. Without wishing to be bound by theory, administration of a composition comprising an isolated lactoferrin induces and/or maintains maternal tolerance of the embryo, fetus, and/or conceptus and thus reduces the likelihood of occurrence of a pregnancy-associated complication. Histocompatibility antigens, major and minor, are described in great detail in immunology textbooks with which a person skilled in the art is quite familiar. The conceptus refers to the embryo and adnexa (appendages or adjunct parts) or associated membranes (i.e. the products of conception). The conceptus includes all structures that develop from the zygote, both embryonic and extraembryonic. It includes the embryo as well as the embryonic part of the placenta and its associated membranes: amnion, chorion (gestational sac), and yolk sac.

The preparations comprising Lf and a nutritional mixture described herein and methods for administering these preparations may reduce miscarriage, spontaneous abortion (including recurrent spontaneous abortion), or preterm delivery, in a statistically, biologically, or clinically significant manner. The subject may receive the preparations comprising Lf and a nutritional mixture described herein prior to conception and/or may receive the preparations subsequent to conception (including for any length of time during the pregnancy). For example, the subject may receive the preparations during at least the first trimester, which is the time during which most miscarriages occur. In other embodiments, these preparations may be administered to the subject during the first two trimesters or during the entire pregnancy.

In another embodiment, the preparations comprising Lf and a nutritional mixture described herein may be useful for preventing (i.e., reducing the likelihood of occurrence) or treating colic (e.g., reducing or decreasing the severity, duration, and/or frequency of colic episodes) in a neonate or infant. The method comprises

administering to the infant or neonate breast milk from the birth mother of the neonate/infant who had received the compositions/preparations prenatally, postpartum, (i.e., postnatally), or both prenatally and postpartum. In still another embodiment, the neonate/infant receives a combination of the breast milk from the birth mother and receives an infant formula supplemented with lactoferrin. Components (i.e., ingredients) of infant formulas (both milk based and plant based) are well known and described in the art and sold commercially. When the infant/neonate receives both breast milk and infant formula supplemented with lactoferrin, the breast milk and infant formula may be given to the infant at different times (e.g., two-four hours apart, or a time interval typically observed between feedings of an infant/neonate or that is typical for the particular neonate/infant) or may be administered sequentially within a short time frame. Exogenous isolated lactoferrin that is present in an infant formula may be administered to the neonate/infant because the breast milk of the mother may contain an inadequate level of endogenous lactoferrin. Accordingly, the neonate or infant may be breast fed and concurrently receive exogenous lactoferrin or may receive exogenous lactoferrin subsequent to termination of breast feeding.

Preparations comprising Lf and a nutritional mixture as described in detail herein may be used for treating or preventing postpartum complications (e.g., occurrence of a thrombotic event, a postpartum-related psychological condition (e.g., postpartum depression or postpartum psychosis), postpartum fatigue, hypoferremia, IDA) in the mother. Administration of lactoferrin and a nutritional mixture may be continued if received during pregnancy or may be initiated after birth of the neonate and continued during the postpartum period to reduce the likelihood of occurrence, severity, or duration of a postpartum complication of the mother. Accordingly, the compositions and preparations described herein are useful in methods for reducing the likelihood of a thrombotic event occurring in the mother during the postpartum period by

administering a lactoferrin and a nutritional mixture. In particular embodiments, the nutritional mixture comprises at least folic acid and Vitamin B6 (as described in greater detail herein). In particular embodiments, when the subject has thrombophilia, the nutritional mixture may comprise at least folic acid, Vitamin B6, and Vitamin B12. The likelihood of occurrence, severity, or duration of a postpartum-related

psychological condition (e.g., postpartum psychosis, postpartum-related depression), postpartum related fatigue, hypoferremia, and/or IDA may also be reduced (i.e., decreased in a statistically, biologically, or clinically significant manner) by

administering a preparation comprising Lf and a nutritional mixture, during pregnancy, and/or during the postpartum period. Accordingly, in another embodiment, the preparations comprising Lf and a nutritional mixture described herein may be administered to a subject postpartum to prevent (i.e., reduce the risk of occurrence) or treat hypoferremia or IDA. Dosing and delivery and lactoferrin or the preparation comprising lactoferrin and the nutritional mixture may be performed as described in greater detail herein. For example, lactoferrin and/or the preparation may be administered daily to a subject postpartum for at least one, two, three months, or four months, or longer, including up to one year.

By way of background, postpartum fatigue appears related to anemia, and anemia seems to reduce immune function and increase the risk of infection (see, e.g., Weiss, Best Pract. Res. Clin. Haematol. 18: 183-201 (2005); Weyermann et al, Am. J. Obstet. Gynecol. 192:548-53 (2005)). Anemia has also been linked to postpartum depression (see, e.g., Troy, MCN Am. J. Matern. Child Nurs. 28:252-57 (2003)), and, if severe, can be related to cardiovascular symptoms, dizziness, and need for prolonged hospitalization (see, e.g., Breymann et al, Eur. J. Clin. Invest. 26: 123- 130 (1996)). Cognitive function and emotional distress have also been noted in women with anemia (see, e.g., Beard et al, Am Soc. For Nutritional Sciences 5:267-71 (2005)) while symptoms of depression such as "maternity blues" or "postpartum blues" and a reduced sense of well being has been demonstrated compared to non-anemic women (see, e.g., Meyer et al, J. Perinat. Med. 23:99-109 (1995)).

The compositions and methods described herein may be useful for treating , preventing (i.e., reducing or decreasing the likelihood of occurrence in a statistically, biologically, or clinically significant manner) postpartum psychological conditions include mild depressive conditions referred to in general as "maternity blues" or "postpartum blues," which may be experienced by a woman after birth of a child. Approximately ten percent of postpartum women develop more serious postpartum depression that is characterized by symptoms of depression that can occur in non-postpartum adults (e.g., feelings of guilt, inadequacy, worthlessness, fatigue and exhaustion, mood swings, lack of interest in work, family, and friends). A smaller, but significant percent (approximately 0.1-0.2%) develop postpartum psychosis, such as severe manic-depression, which often requires hospitalization. See, for example, (see, e.g., Weiss, Best Pract. Res. Clin. Haematol. 18: 183-201 (2005); Weyermann et al, Am. J. Obstet. Gynecol. 192:548-53 (2005); Silverman et al, Arch. Womens Ment. Health 13(5)411-15 (2010) (Epub 2010 Apr 13).

The Center for Disease Control (CDC), the American College of Obstetrician and Gynaecologists, and the Institute of Medicine only recommend anemia screening at 4-6 weeks postpartum and only for patients considered to be at "high risk" of developing anemia (see, e.g., Iron deficiency anemia: recommended guidelines for the prevention, detection, and management among U.S. children and women of childbearing age. Institute of Med (IOM) and The National Academy of Sciences, 2000). In the United States, one in eight women have iron deficiency up to twelve months after delivery, and even when extended to the second year of postpartum, one in twelve women have IDA. These statistics suggest more clinicians should screen postpartum women for anemia up to 1-2 years after birth (see, e.g., Bodnar et al., Am. J. Epidemiol. 156:903-912 (2002)). Thus, the preparations comprising Lf and a nutritional mixture described herein may be useful for preventing or treating postpartum fatigue and/or postpartum-related psychological conditions (including postpartum depression and postpartum psychosis) in a postpartum subject. In addition to performing haematological and immunological tests and assays, persons skilled in the medical art can determine and identify subjects who exhibit signs of symptoms of postpartum fatigue and/or postpartum depression by clinical observation, psychological analysis, and physical examination.

Because IDA in the postpartum period may impair a woman's ability to participate in child care, household tasks, and social activities, it may also diminish the productivity in physical and intellectual work (see, e.g., Baker et al, Hematol. Oncol. Clin. North Am. 51 :s2-s9 (2000)). Without wishing to be bound by any particular theory, these changes may also lead to disturbed maternal-infant interactions as shown by one study comparing infants of IDA women to non-anemic parturient controls (see, e.g., Perez et al, J. Nutr. 135(4):850-55 (2005)). These infants when examined at 10 weeks of age were developmentally delayed, and these delays persisted long after correction of the maternal iron status, which, at least in this study, raises the possibility that postpartum maternal IDA may irreversibly impair childhood development (see, e.g., Perez et al, J. Nutr. 135(4):850-55 (2005)).

Compositions and preparations described herein (which comprise an isolated lactoferrin and a nutritional mixture of vitamins and minerals and exclude an inorganic source of a biologically effective amount of iron) may be administered to an infant (including a neonate/newborn) or child to reduce the likelihood of occurrence (i.e., reduce in a statistically, biologically, or clinically significant manner) of iron deficiency (ID) (i.e., hypoferremia) or iron deficiency anemia (IDA). Infants that are at risk of developing hypoferremia or IDA include infants whose birth mothers had ID or IDA. Infants at risk of developing hypoferremia or IDA also include pre-term infants, who also may be susceptible to iron overload (see, e g., Rao et al, Clin. Perinatol. 36:27-42 (2009)). The compositions and preparations described herein that comprise lactofemn and lack an inorganic source of iron may therefore provide particular benefit to pre-term infants.

The compositions and preparations for administration to infants and children include infant formula and nutrient mixtures (liquid, semi-solid, gel, or solid) that are formulated and prepared appropriately by a person skilled in the art and which formulations may depend, at least in part, on the age and health status of the infant or child. As described herein, the compositions and preparations comprising lactoferrin comprise a nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation or composition. In certain

embodiments, the nutritional mixture further comprises at least folic acid and Vitamin B6 and in other certain embodiments, the nutritional mixture further comprises at least Vitamin B 12.

The health and well-being of the subject, including a pregnant subject, and the health and development of the fetus and neonate are monitored by physical examination, assessment of vital signs, recording and analyzing of any adverse events, and clinical evaluations. The health and status of a fetus is assessed by monitoring fetal movement, fetal heart tones, growth, and which include physical examination and may also include ultrasonography. Monitoring the subject, fetus, and neonate includes any one of several methods and techniques that are routinely practiced in the medical art and described herein.

Methods and Techniques for Monitoring Hematological and Immunological Status

Monitoring hematological status and immunological status of a subject described herein may be performed using any one of several methods and techniques routinely practiced in the medical art. As described herein and practiced by a person skilled in the art in the medical art, a dosing regimen for the preparations described herein, which comprise lactoferrin and a nutrient mixture and that exclude an inorganic source of a biologically effective amount of iron, may be adjusted depending on the hematological status and/or immunological status of the subject being treated.

To assess the hematological status of the subject, at least one

hematological parameter (such as by way of non-limiting example red blood cells (including red blood cell count, morphology), hemoglobin level, hematocrit, serum ferritin, total serum iron) is analyzed and evaluated using methods routinely practiced by clinical technicians and scientists and which are described in greater detail herein. Accordingly, before, during, and after treatment of a subject with the preparation described herein, the hematological status of the subject may be monitored. The levels and ranges that are considered normal may vary with gender, age, and weight (or mass) of the subject, and which are familiar to persons skilled in the clinical and medical arts. In certain instances, other particular hematological parameters, such as the level of hepcidin, may be above the level or range of levels desired for or healthy, non-anemic and non-hypoferremic individuals.

Hematological status may be monitored by performing tests that indicate the level of one or more of hemoglobin, hematocrit, MCV (mean corpuscular volume), absolute reticulocyte count, white blood cell count with differential and platelet count, ferritin, total iron binding capacity, transferrin saturation, serum iron, transferrin receptor levels, and hepcidin. The level of prohepcidin may also be determined. A subset of hematologic indicators to be determined includes red blood cell count, hemoglobin, total serum iron, serum ferritin, and hematocrit (%). Techniques for determining each of these hematological parameters, including iron analyses, are well known and routinely practiced in the clinical art and/or described in the literature. For example, methods for determining hepcidin and prohepcidin levels are described in the literature. For certain assays and techniques, assay kits are commercially available. The level of each hematological parameter (or indicator) in a sample from a subject may then be compared with predetermined levels and ranges of each parameter that set forth the normal level and/or range for the particular parameter. As described herein, determining the appropriate level of hemoglobin and other parameters of hematologic or immunologic status are well within the skill of a person skilled in the medical art, which person also considers the subject's overall health status, condition to be treated, and any one or more underlying diseases associated or not associated with the condition to be treated.

Typically and by way of example, in a hypoferremic or anemic individual, the level of one or more hematological parameters is below the range desired for healthy, non-anemic and non-hypoferremic individuals. For example, subjects who have decreased levels of red blood cells, total serum iron, hemoglobin, and/or serum ferritin (or other hematological parameters) may benefit from receiving the compositions comprising lactoferrin and/or preparations comprising Lf and a nutritional mixture, which may provide statistically, biologically, or clinically significant increases in one or more hematological parameters. In certain instances, other particular hematological parameters, such as the level of hepcidin, may be above the level or range of levels desired for or healthy, non-anemic and non-hypoferremic individuals. The immunological status of a subject before, during, and after treatment with the preparations described herein may be monitored. Inflammation and the inflammatory response, including cytokine induction and production can be determined by methods routinely practiced in the art. The increased or decreased level of inflammatory factors and cytokines in a biological sample obtained from the subject before, during, and after treatment may be readily determined by methods and assays described herein and practiced routinely in the art to monitor the effect of treatment. An immune response in a subject may be determined by any number of well-known immunological techniques and methods with which those having ordinary skill in the art will be readily familiar. Such assays include, but need not be limited to, in vivo or in vitro determination of soluble antibodies, C-reactive protein, NF-KB, TGF-β, soluble mediators such as cytokines (e.g., IL-6, IL-Ιβ, leukemia inhibitory factor, TNF-a, IFN- γ, IL-2, IL-4, IL-10, IL-12), lymphokines, chemokines, hormones, growth factors, and the like, as well as other small peptides (e.g., hepcidin and prohepcidin), carbohydrate, nucleotide and/or lipid mediators. Cellular activation state changes may also be determined, for example, by determining altered functional or structural properties of cells of the immune system, for example cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles or the onset of apoptosis (programmed cell death). Procedures for performing these and similar assays may be found, for example, in Lefkovits

(Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental

Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281 : 1309 (1998); and references cited therein.

A "biological sample" may include a sample from a subject, and may be a blood sample (from which serum or plasma may be prepared), a biopsy specimen, one or more body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid, amniotic fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. A biological sample may further refer to a tissue or cell preparation in which the morphological integrity or physical state has been disrupted, for example, by dissection, dissociation,

solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication, or any other means for processing a sample derived from a subject or biological source. In certain embodiments, the subject or biological source may be a human or non-human animal, a primary cell culture (e.g., immune cells), or culture adapted cell line, including but not limited to, genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiatable cell lines, transformed cell lines, and the like.

Formulations and Methods of Use

Lactoferrin and nutritional mixtures described herein may be formulated in physiologically acceptable (or suitable) compositions and preparations. As discussed in detail herein, the compositions and preparations do not include (i.e., exclude, lack) an inorganic source of a biologically effective amount of iron. A lactoferrin or a composition comprising lactoferrin, a nutritional mixture or a composition comprising the mixture, and a preparation of the lactoferrin and nutritional mixture, as described herein, may be administered to a subject in a manner appropriate, as determined by persons skilled in the medical, clinical, and nutritional arts, suitable for maintaining or improving hematological parameters, immunological status (e.g., reducing the likelihood of developing inflammation, or reducing or abrogating an inflammatory state), for treating and/or preventing a condition or disorder described herein, and supplementing daily nutritional requirements. An appropriate dose and a suitable duration and frequency of administration of the compositions and preparations described herein may be determined by such factors as the condition of the subject, the age of the subject, the type and severity of any disease or disorder or condition that affects the health of the subject, whether the subject is pregnant, desiring to become pregnant, post-partum, or lactating, the particular form of the active ingredient(s), and the method of administration.

Treatment of a subject or patient refers to the medical management of the disease, disorder, or condition (see, e.g., Stedman's Medical Dictionary). In general, an appropriate dose and treatment regimen provides the composition(s) and preparation(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., maintenance or improvement of health status; an improved clinical outcome (e.g., reduced incidence or occurrence or severity of a pregnancy-associated complication; reduced incidence or occurrence or severity of postpartum-associated condition;

reduced occurrence or severity of a thrombotic event); abatement or lessening in severity of the symptoms of the disease, disorder, or condition to be treated (e.g., hypoferremia or iron deficiency anemia or other anemia); decreased occurrence of symptoms; improved quality of life; longer disease-free status (i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); and/or overall survival. By way of example, administration of the preparations comprising Lf and a nutritional mixture described herein maintain, improve, or restore iron homeostasis (as indicated by one or more hematological parameters described herein and known in the art). Dosing of the compositions and preparations is determined with the intent to be sufficient to prevent (i.e., reduce the likelihood of occurrence), delay the onset of or diminish the severity of hypoferremia and/or iron deficiency anemia and/or other anemias including chronic anemia. Administration of the compositions described herein that comprise a lactoferrin may also inhibit, decrease, or reduce, production of at least one

proinflammatory cytokine (e.g., IL-Ιβ, IL-6, IL-8, and TNF-a) or other inflammatory mediator and thereby reduce, abrogate, or ameliorate inflammation (or an inflammatory state).

A "therapeutically effective amount" or "effective amount" means an amount of an active ingredient (e.g., an isolated lactoferrin and ingredients of the nutritional mixture), composition or formulation, that is sufficient, in the subject (e.g., a non-human mammal or a human) in need thereof and to which it is administered, to treat (i.e., effectively manage) or prevent (i.e., decrease or reduce the likelihood of occurrence of) the stated disease, disorder or condition (e.g., hypoferremia, IDA, or other anemia) and/or to supplement dietary requirements. In certain embodiments, when a preparation of a lactoferrin and a nutrient mixture excluding an inorganic source of a biologically (i.e., pharmacologically) effective amount of iron is administered to the subject, a therapeutically effective amount or effective amount of the preparation and compositions therein refers to the combined effect of the lactoferrin and nutrient mixture independent of whether the lactoferrin and nutrient mixture are administered concurrently or sequentially.

A dosing regimen, including optimal doses and frequency of dosing, may generally be determined using experimental models and/or clinical trials. With respect to the optimal doses and frequency of dosing of ingredients contained within the nutritional mixture, a considerable body of nutritional and dietary supplement information and knowledge is available in the art with which a skilled person will be familiar. The optimal dose of an active ingredient may depend upon the age, body mass, weight, or blood volume of the subject (i.e., patient). The use of the minimum dosage that is sufficient to provide effective therapy or prophylaxis is usually preferred. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays, techniques, and methods (including physical examination and observation) suitable for the condition being treated or prevented and with which persons skilled in the art are familiar. In addition, for example, clinical trials to determine the

effectiveness of administering a lactoferrin and/or a nutrient mixture are designed for the particular patient population to be treated. Design of clinical trials, including determination of exclusion criteria, inclusion criteria, primary endpoints, secondary endpoints, blinding, randomization of the patients, and analysis of clinical data is routinely performed by persons skilled in the clinical arts.

A subject (host or patient) in need of treatment as described herein may be a human or may be a non-human primate or other animal {i.e., veterinary use).

Examples of non-human primates and other animals include but are not limited to farm animals, pets, and zoo animals {e.g., horses, cows, buffalo, llamas, goats, rabbits, cats, dogs, chimpanzees, orangutans, gorillas, monkeys, elephants, bears, large cats, etc.). In certain embodiments, the subject is a human. In particular embodiments, the subject is a pregnant human or is a human desiring to become pregnant (and thus is at risk of becoming pregnant).

A lactoferrin and a nutritional mixture may be formulated together or separately in a composition(s) and preparation(s) according to well known

methodologies. Any physiological or pharmaceutically suitable excipient or carrier {i.e., a non-toxic material that does not interfere with the activity of the active ingredient) known to those of ordinary skill in the art for use in pharmaceutical compositions may be employed in the compositions and preparations described herein that comprise a lactoferrin and/or nutritional mixture. Excipients for therapeutic use are well known, and are described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21 ^st Ed. Mack Pub. Co., Easton, PA (2005)), and are described in greater detail herein. For example, saline and phosphate buffered saline at

physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the compositions and preparations. For example, sodium benzoate, sorbic acid and esters of /?-hydroxybenzoic acid may be added as

preservatives. In addition, antioxidants and suspending agents may be used. An injectable pharmaceutical composition is preferably sterile.

In one embodiment, the lactoferrin and nutrient mixture are combined in a single preparation or composition. The lactoferrin and the nutritional ingredients that comprise the nutrient mixture may be combined together in a single composition (or preparation) or the lactoferrin may be formulated separately into a first composition and the nutrient mixture formulated as a second composition and then the lactoferrin composition and the nutrient mixture composition are combined to form a single preparation. In certain embodiments, the preparations (and compositions thereof) may be formulated for oral, parenteral, lingual, buccal, intranasal, transdermal,

intramuscular, or subcutaneous administration. In a particular embodiment, the preparations and compositions are formulated for oral administration.

In another embodiment, when a preparation (which may also be called a kit) comprises a composition that comprises an isolated lactoferrin (which composition may be called a first composition solely for ease and clarity of description), that is formulated separately from a composition that comprises a nutritional mixture (which may be called a second composition), each of the first and second compositions may be administered concurrently or sequentially to provide the desired dose of the ingredients within the compositions. In certain embodiments, the lactoferrin (or composition comprising the lactoferrin) is formulated appropriately for administration by a route including, but not limited to, oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, intranasal, vaginal, rectal, and topical. In another certain embodiment, the lactoferrin or composition comprising the lactoferrin is formulated for oral administration. In other particular embodiments, the composition comprising the nutrient mixture is formulated for oral, lingual, buccal, intramuscular, subcutaneous, transdermal, intranasal, or parenteral administration. In another more specific embodiment, the nutrient mixture, or composition comprising the nutrient mixture, is formulated for oral administration.

When the preparation comprises a first composition that comprises the lactoferrin and a second composition that comprises the nutritional mixture, one composition may be administered prior to, concurrent with, or subsequent to administration of the second composition (also referred to as coordinate

administration). When the lactoferrin and nutritional mixture are formulated separately into two different compositions, each composition may be administered concurrently (i.e., at the same time of day and the same number of times during a given time period, such as a day or week) or at different times of day, and/or for different number of times during a given time period. By way of non-limiting example, the composition comprising the lactoferrin may be administered twice a day, and the composition comprising the nutritional mixture may be administered once a day, providing the desired daily dose of the preparation. In another non-limiting example, the dose of the preparation may be a weekly dose, wherein each composition is administered for a different number of days during the week (for illustration, one composition may be administered three times per week and the other composition may be administered seven times per week). The appropriate dosing of each separate composition and the preparation can be readily determined by a person skilled in the medical, nutritional, and clinical arts depending on an individual subject's health status, weight, age, gender, and any other relevant factor.

As described herein, methods that comprise administering lactoferrin (or compositions and preparations comprising lactoferrin) include administering at least one dose of the lactoferrin, two or more doses of the lactoferrin, or multiple doses of lactoferrin. The lactoferrin may also be administered by one or more different routes as discussed in greater detail herein. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and which are described herein.

When lactoferrin is administered by two or more (i.e., more than one) administrative routes, administration of the lactoferrin via each of the different routes may occur concurrently or sequentially (i.e., administration of lactoferrin by one route is accomplished prior to administration of lactoferrin by a different route). In certain specific embodiments, lactoferrin is administered orally and also administered concurrently or sequentially by any one of topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal routes. By way of non-limiting examples, lactoferrin may be administered orally and also administered concurrently or sequentially by a second route (any one of, for example, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal), such as vaginally (for example, for a subject who is pregnant and who is at risk of occurrence of at least one pregnancy-associated complication such as pre- term labor). Appropriate route or routes of administration and timing of administration (i.e., concurrent or sequential) of lactoferrin are readily determined by a person skilled in the medical and clinical arts. As described herein administration of lactoferrin refers to administration of one or more doses that comprise a course of lactoferrin therapy (i.e., lactoferrin therapeutic regimen).

In other embodiments, when the lactoferrin (or a composition that comprises a lactoferrin) and the nutritional mixture (or a composition that comprises the nutritional mixture) are formulated together, the preparation may be formulated for administration by a route including, but not limited to, oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In other embodiments, the preparation may be formulated for administration by a route including oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal. In a more specific embodiment, the preparation is formulated for oral administration. In certain specific embodiments, the preparation may be administered orally and also administered concurrently or sequentially by any one of topical, parenteral, lingual, buccal, transdermal, subcutaneous, intramuscular, and intranasal routes. By way of non-limiting example, the preparation may be

administered orally and also administered concurrently or sequentially by a second route (any one of, for example, parenteral, lingual, buccal, transdermal, subcutaneous, intramuscular, and intranasal). Appropriate route or routes of administration and timing of administration (i.e., concurrent or sequential) of the preparation are readily determined by a person skilled in the medical, nutritional, and clinical arts. As described herein, administration of the preparation refers to administration of one or more doses that comprise a course of therapy (i.e., therapeutic regimen).

In other certain specific embodiments, when the preparation comprises a composition comprising the nutritional mixture formulated separately from a composition comprising the lactoferrin, the composition comprising the nutritional mixture is delivered orally. In a particular embodiment, the nutritional mixture may be administered by two or more (i.e., more than one) administrative routes.

Administration of the nutritional mixture via each of the different routes may occur concurrently or sequentially (i.e., administration of the nutritional mixture by one route is accomplished prior to administration of the nutritional mixture by a different route). In certain specific embodiments, a composition comprising the nutritional mixture is administered orally and also administered concurrently or sequentially by any one of topical, parenteral, lingual, buccal, transdermal, subcutaneous, intramuscular, and intranasal routes. By way of non-limiting examples, the nutritional mixture may be administered orally and also administered concurrently or sequentially by a second route (any one of, for example, parenteral, lingual, buccal, transdermal, subcutaneous, intramuscular, and intranasal). Appropriate route or routes of administration and timing of administration (i.e., concurrent or sequential) of the nutritional mixture are readily determined by a person skilled in the medical, nutritional, and clinical arts. As described herein administration of the nutritional mixture refers to administration of one or more doses that comprise a course of therapy (i.e., therapeutic regimen).

In other embodiments, when the lactoferrin and the nutrient mixture are formulated separately into two different compositions, each may be administered by the same route or by different routes as described above and herein. In certain

embodiments, a lactoferrin (or composition comprising lactoferrin) may be

administered by two different routes as described above and the second composition comprising the nutrient mixture may be administered by one route (for example, by any one of orally, lingually, buccally, intramuscularly, subcutaneously, transdermally, or parenterally).

The term parenteral as understood in the art refers to delivery by a route other than via the alimentary canal and which is by injection, including subcutaneous injection, intravenous, intramuscular, intradermal, subdermal, intrasternal,

intracavernous, intrathecal, intrameatal, intraurethral injection, or infusion techniques.

The preparations and compositions described herein, including a composition comprising at least one isolated lactoferrin, and a composition comprising a nutrient mixture, are each formulated separately or together (as described herein) in a manner that allows the active ingredient(s) contained therein to be bioavailable upon administration of the preparation and compositions to a subject. Each preparation and composition is also formulated in a form appropriate for the route of administration. Each preparation and composition may be in the form of a solid, liquid, emulsion, ointment, suspension, gel, or gas (aerosol). For optimal bioavailability, the lactoferrin (and compositions and preparations comprising lactoferrin), is delivered in a manner and in a form that avoids proteolytic digestion by gastric enzymes. Accordingly, in certain embodiments, a composition or preparation comprising a lactoferrin is formulated with an enteric coating, which is a barrier capable of controlling the location in the digestive system where the lactoferrin is absorbed. Most enteric coatings present a surface that is stable at the strongly acidic pH found in the stomach, but breaks down rapidly at a less acidic (relatively more basic) pH. For example, an enteric coating will not dissolve in the acidic juices of the stomach (about pH 3) but will dissolve in the higher pH environment (above pH 5.5) present in the small intestine. Materials used for enteric coatings include fatty acids, waxes, polymers, and other materials known to a person skilled in the art.

In other particular embodiments, compositions and preparations that comprise lactoferrin are formulated in an acid-resistant formulation, such as but not limited to an acid resistant capsule. Delivery of lactoferrin in an acid-resistant formulation or vehicle maintains the integrity of the lactoferrin or increases the percent of proportion of undegraded lactoferrin molecules in the composition or preparation. An acid resistant formulation thereby decreases, reduces, minimizes, or abrogates degradation of the lactoferrin polypeptide. Such formulations may be used when the lactoferrin is to be administered orally to increase the bioavailability of lactoferrin.

In another more specific embodiment, the preparation comprising Lf and nutritional mixture, a composition comprising Lf, and/or a composition comprising the nutrient mixture may be used as a Medical Food. A Medical Food as defined by regulatory agencies, including the U.S. Food and Drug Administration (FDA), is a substance or composition prescribed by a physician when a patient has special nutrient needs in order to manage a disease or health condition, and the patient is under the physician's ongoing care. In another embodiment, a lactoferrin is formulated in a composition that is a pharmaceutical or a biological according to rules and regulations of a regulatory agency. Compositions and preparations described herein that comprise a lactoferrin and/or nutritional mixture, but lack an inorganic source of a biologically effective amount of iron, may provide dietary management of inflammation, hypoferremia, IDA, and other anemias, including chronic anemia, anemia due to B6 deficiency, and anemia due to B12 deficiency. These compositions and preparations thus provide nutritional requirements to subjects who are at risk of a thrombotic event occurring, to subjects who are pregnant, desirous of becoming pregnant, to subjects who are at risk of a pregnancy-associated complication occurring, to subjects who are at risk of a postpartum-associated complication occurring, and/or to subjects who have below normal levels of at least one hematological parameter (including but not limited to hemoglobin, hematocrit, serum ferritin, total serum iron).

The compositions and preparations described herein may include one or more nutrient minerals or vitamins in an amount that requires monitoring and oversight by a clinician. By way of example, under current regulatory practice in the United States, when a composition comprises folic acid in an amount that is intended to provide greater than 1.0 mg per day, a prescription is required.

The preparations and compositions described herein that comprise a lactoferrin and nutritional mixture may also be used as nutritional supplements. Such supplements may be available as over the counter (OTC) products.

The preparations comprising Lf and a nutritional mixture described herein may be formulated as sterile aqueous or non-aqueous solutions, suspensions or emulsions, which additionally comprise a physiologically acceptable or suitable carrier. A pharmaceutically acceptable or suitable carrier may include (or refer to) an excipient (i.e., a non-toxic material that does not interfere with the activity of the active ingredient) and/or a diluent. Such compositions may be in the form of a solid, liquid, or gas (aerosol). Alternatively, compositions described herein may be formulated as a lyophilate (i.e., a lyophilized composition or preparation) or as another dried or desiccated powder or granulated form (including for example, a dry infant formula that is solubilized with the addition of water or other appropriate solute), or may be encapsulated within liposomes using technology known in the art. Pharmaceutical compositions may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins (such as albumin), polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or preservatives. The preparation described herein, composition comprising a lactoferrin and/or a composition comprising a nutritional mixture (as described herein) may be encapsulated within one or more delivery materials appropriate for attaining and/or maintaining an effective therapeutic level of the lactoferrin and/or nutritional mixture in the subject. Similarly, a nutrient mixture (or a composition comprising the mixture) may be encapsulated within one or more delivery materials appropriate for attaining and/or maintaining an effective level of the nutrients in the subject.

In general, as discussed herein, the type of excipient is selected on the basis of the mode of administration. The preparations and compositions may be formulated for any appropriate manner of administration, including, for example, topical, buccal, lingual, oral, intranasal, intrathecal, rectal, vaginal, intraocular, subconjunctival, transdermal, sublingual or parenteral administration, including subcutaneous, intravenous, intramuscular, intrasternal, intracavernous, intrameatal or intraurethral injection or infusion.

The preparations and compositions may further comprise ingredients that act as delivery vehicles, including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. While any suitable excipient or carrier known and available to a person having skill in the art may be employed in the compositions described herein, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. Preparations and compositions described herein may be administered through use of insert(s), bead(s), timed-release

formulation(s), patch(es) or fast-release formulation(s).

For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer, and the preparation or composition is sterile. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.

Biodegradable microspheres (e.g., polylactic galactide) as well as nanoparticles may also be used as carriers for the preparations and compositions described herein. Suitable biodegradable microspheres described, for example, in U.S. Patent

Nos. 4,897,268 and 5,075,109. In particular embodiments in which the preparation or composition is combined with a microsphere, the microsphere is larger than

approximately 25 microns. A preparation or composition described herein may be lyophilized or otherwise formulated as a lyophilized product using one or more appropriate excipient solutions (e.g. , sucrose) as diluents upon administration.

Nanoparticles may be used to deliver the lyphophilized product and appropriate excipient(s).

The preparations and compositions (which are pharmaceutically or physiologically acceptable or suitable compositions, preparations, or formulations) disclosed herein may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin,

polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in pharmaceutical preparations or compositions for topical administration (e.g., oral or vaginal). The preparations and compositions described herein may be administered by using any one of several delivery vehicles described herein and used in the art, including but not limited to a sponge, gel cap, suppository, gauze (or other suitable fabric for application to the tissue to be treated), nanoparticles, and a lozenge. With respect to certain delivery vehicles, such as a sponge, fabric, or gauze, the preparation or composition is attached to, absorbed by, adsorbed to, or in some manner applied to the vehicle that permits release of the preparation or composition upon contact with the tissue to be treated.

A preparation or composition disclosed herein may be intended for rectal, oral, or vaginal administration, in the form, e.g., of a suppository or lozenge, which will melt in the rectum, oral, or vaginal space, respectively, and release the drug or components of the composition. A preparation or composition described herein that is administered orally may also be in the form of a liquid. The preparation or composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

The preparations and compositions described herein may be endotoxin free, particularly when delivered parenterally. An endotoxin free preparation or composition is substantially free of endotoxins and/or related pyrogenic substances (i.e., an endotoxin is not detectable by methods accepted by regulatory agencies to demonstrate with sufficient sensitivity whether an endotoxin is present). Endotoxins include toxins that are present in viable microorganisms and include toxins that are released only when the microorganisms lack cell integrity or die. Pyrogenic substances include fever-inducing, thermostable substances (lipopolysaccharides and

glycoproteins) located in the outer membrane of bacteria and other microorganisms. These substances can cause fever, hypotension, and shock when administered to humans. Manufacturing compositions and preparations that are endotoxin- free can require special equipment, expert artisans, and can be significantly more expensive than making formulations that are not endotoxin-free.

Also provided herein is a method for manufacturing a preparation comprising a lactoferrin (or a composition comprising the lactoferrin) and/or a composition comprising a nutritional mixture, wherein an inorganic source of a biologically (or pharmacologically) effective amount of iron is excluded from the composition and preparation. As described in detail herein, each composition and preparation does not include (i.e., excludes) an inorganic source of a biologically effective amount of iron). In one embodiment, the method of manufacture of the preparation comprises isolating a lactoferrin from a biological source as described herein or obtaining a lactoferrin by recombinant molecular biology methods and protein isolation methods that are described herein and/or are routinely practiced in the art. The isolated lactoferrin may then be combined with at least one physiologically suitable excipient to provide a composition comprising the lactoferrin. The method of manufacture may further comprise formulating the isolated lactoferrin (or composition comprising the isolated lactoferrin) with a nutritional mixture. The isolated lactoferrin may be combined (mixed) with each vitamin and any other ingredient, such as at least one mineral, and/or at least one other dietary ingredient of the nutritional mixture. Alternatively, each ingredient may be combined to provide the nutritional mixture, which mixture is then combined with the isolated lactoferrin (or composition comprising the isolated lactoferrin). The method may further comprise combining (mixing) at least one non-dietary ingredient with the nutritional mixture and isolated lactoferrin to formulate a preparation.

In another embodiment the method of manufacturing the preparation comprises obtaining an isolated lactoferrin and formulating the lactoferrin in a first composition, and in an additional step obtaining and formulating a nutritional mixture in a separate, second composition. In other embodiments, a method is provided for manufacture of a composition comprising the nutritional mixture. The method of manufacture comprises combining each of the ingredients as described herein, into a single nutrient mixture to provide the desired amount of each ingredient that is to be administered to a subject per dose.

In still other embodiments, a method is provided for manufacture of a composition that comprises a lactoferrin (or a composition comprising the lactoferrin) and the nutrient mixture (e.g., a nutrient mixture including vitamins, such as at least folic acid and Vitamin B6, and optionally, in addition, at least Vitamin B 12); the composition may further comprise at least one other vitamin, at least one mineral (excluding iron), and/or at least one other active nutritional ingredient. Optionally the composition may further comprise at least one non-dietary ingredient. Such a method of manufacture comprises combining or mixing together in a manner appropriate to provide a single composition that comprises the lactoferrin and nutritional mixture and optionally at least one non-dietary ingredient. In certain embodiments, the method of manufacture further comprises isolating a lactoferrin from a biological source as described herein or obtaining a lactoferrin by recombinant molecular biology methods and protein isolation methods described herein and routinely practiced in the art.

In certain embodiments, for example, when the nutrient mixture is a prenatal nutrient mixture intended for use by a pregnant, postpartum, or lactating subject, the method may further comprise addition of excipients and/or other non- dietary ingredients such as a stool softener and/or an anti-nausea agent.

The following Examples are offered for the purpose of illustrating the embodiments disclosed herein and are not to be construed to limit the scope of this invention.

EXAMPLES EXAMPLE 1

CLINICAL TRIAL WITH PREGNANT WOMEN AFFECTED BY HEREDITARY THROMBOPHILIA,

HYPOFERREMIA, AND IRON DEFICIENCY ANEMIA

A clinical trial was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. All patients gave written informed consent. The open, prospective, randomized, single center clinical trial was performed to obtain additional information on efficacy and safety of oral bovine lactoferrin (bLf ) versus ferrous sulfate in treating hypoferremia (also referred to herein and in the art as iron deficiency (ID)) and iron deficiency anemia (IDA) in pregnant women affected by hereditary thrombophilia (HT). Clinical Protocol

Pregnant women were recruited from obstetrical patients seen between December 2006 through December 2009 at Studio Diagnosi Medica, Viale Regina Margherita 270, Rome, Italy or between January 2010 and April 2010 at Clinica Fabia Mater, Via Olevano Romano, 25 Rome, Italy. Anemic pregnant women with a history of adverse outcomes, including recurrent miscarriages, were screened for thrombophilic abnormalities at Istituto Regina Elena, Dipartimento di Patologia Medica in Rome, Italy.

Women of 20-40 years of age were eligible for this study. Inclusion criteria included the following: pregnant women, regardless of trimester, with a diagnosis of hereditary thrombophilia and hypoferremia or IDA. A woman was defined as having hypoferremia and IDA when any of the following hematological values occurred: Red Blood Cells <4,000,000/mL, hemoglobin concentration < 11 g/dL, total serum iron < 30 mg/dL, and serum ferritin <12 ng/mL.

Exclusion criteria included women with uncomplicated pregnancies, non-pregnant women, lack of informed consent, unverified age, use of iron

supplements, concomitant diseases, recent blood transfusion(s), and allergies to milk proteins or iron products. Exclusion of pregnant women during the clinical trial was considered on the basis of voluntary declaration, lack of treatment effectiveness, side effects, protocol infringement, missed programmed visits, and miscarriage.

All enrolled HT pregnant women received low-molecular-weight heparin (0.3 U /day) to prevent and reduce the risk of venous thromboembolism (VTE). Treatment with bLf was initiated when at least one of the four hematological parameters indicated an iron deficiency.

Pregnant women affected by HT, hypoferremia and IDA were enrolled and randomized in two Arms. A capsule containing 100 mg of bLf (Lattoglobina, Grunenthal, Italy) was administered orally twice a day before meals (200 mg/day in total) to women in Arm A. bLf iron saturation was approximately 30%, as determined by optical spectroscopy. The amount of iron supplied by two capsules of bLf corresponded to 88 μg/day. To women in Arm B, a tablet, containing 520 mg of ferrous sulfate (Ferro-Grad, Abbot Laboratories, USA), was administered orally once a day with food, which is in accordance with the Italian Standard of Care. Iron supplied by one ferrous sulfate tablet was 156 mg/day.

A third group, Arm C, included pregnant women affected by HT, hypoferremia, and IDA, who were randomized to receive iron supplementation but who refused iron supplementation. Arm C therefore received no treatment for hypoferremia or IDA and was considered a control group for statistical purposes.

Laboratory tests performed during the trial included the following.

Genetic thrombophilia markers, which included factor V Leiden, prothrombin 2021 OA mutation, antiphospholipid antibodies, hyperhomocysteinemia, and deficiencies of antithrombin, protein C, or protein S, were determined according to the method described by Jackson et al. {BMC Clin. Pathol. 8:3; 1-7 (2008)) at Istituto Regina Elena, Dipartimento di Patologia Medica, Rome, Italy. The number of red blood cells and the concentration of hemoglobin, total serum iron, serum ferritin and hematocrit (%) were assessed using venous blood according to techniques as previously described (Meier et al, Clin. Med. Res. 1 : 29-36 (2003)). Serum IL-6 levels were determined by standard ELISA Quantitative kits (R&D Systems, Wiesbaden, Germany). Determinations of prohepcidin concentrations in serum were carried out by using commercially available ELISA kits obtained from DRG (Heidelberg, Germany). Laboratory tests were performed upon enrolment (Time 0) and after 30 days of treatment and every 30 days thereafter until delivery.

Safety and compliance analysis was determined and registered by monitoring vital sign assessments, adverse events, and clinical laboratory evaluations. Clinical supply use was monitored during scheduled visits and registered in each patient's file.

Calculations for statistical differences between the various hematological parameters and statistical analysis among the various parameters in the different Arms before and after therapy were carried out using ANOVA test. P values <0.0001 were considered significant. Calculations for evaluating miscarriage statistical differences between Arms were conducted on intent to treat basis. Analyses were carried out using Yates corrected, chi-square, one degree of freedom, Fishers exact two-tailed test. P values < 0.050 were considered significant. Statistical analysis of miscarriages between groups was also determined using the numbers of women in each group who completed the study (see Results). Results

Over a study period of 40 months, pregnant women with previous adverse pregnancy outcomes, including recurrent miscarriages, were screened for HT genetic markers. Pregnant women with hypoferremia or IDA who were positive for at least one genetic HT marker were enrolled in the clinical trial. Two hundred and ninety six (296) enrolled patients were randomized in two Arms and self-selected to a third control Arm by their refusal of iron supplementation.

(1) Arm A: 128 pregnant women entered the clinical trial to receive oral administration of 100 mg of bLf (Lattoglobina, Grunenthal, Italy) twice each day before meals.

(2) Arm B: 124 pregnant women entered the clinical trial to receive oral administration of 520 mg of ferrous sulfate (FerroGrad, Abbott Laboratories, USA), once each day with food.

(3) Arm C: (control group) 44 pregnant women who refused iron supplementation and remained untreated with iron supplementation or bLf throughout the trial were considered as a control group. The use of a placebo is considered unethical in treating hypoferremia or IDA pregnant women diagnosed with hereditary thrombophilia; therefore, a control group for this trial included women who refused iron supplementation, which is the current standard of care for treating hypoferremia or IDA pregnant women diagnosed with hereditary thrombophilia.

Among the enrolled study participants 98.4% (126/128) of Arm A, 71.7 % (86/124) of Arm B, and 88.6 % (39/44) of Arm C completed the study (see Figure 1). The highest compliance was recorded in pregnant women receiving bLf (no patient (0%) was lost to study due to adverse events). The lowest compliance was recorded in pregnant women receiving oral ferrous sulfate (19% of patients were lost to study due to adverse events). Of the eight (8) total pregnant women excluded for miscarriage, three (3) (6.8%>) were untreated controls (Arm C), and five (5) (4.03%>) were treated with ferrous sulfate (Arm B). None of the women (0%) treated with bLf experienced a miscarriage (Arm A) (see Figure 1). In an intent-to-treat analysis (Yates corrected, Fishers exact two-tailed test), the incidence of miscarriages in the group treated with bLf (Arm A) to Arm B plus Arm C results in a P value of 0.0374; comparison of the bLf treatment group versus Arm B (ferrous sulfate) results in a P value of 0.0722; and comparison of the bLf treatment group vs. Arm C provides a P value of 0.026.

Statistical analyses using the numbers of women who completed the study also indicates the significance of using bLf to reduce the likelihood of a miscarriage occurring. Comparison of the incidence of miscarriages in the group treated with bLf (Arm A; 0/126) to the group treated with ferrous sulfate (Arm B; 5 miscarriages/86 women who completed the trial) results in a P value of 0.0121 (2x2 contingency Fishers exact 2-tailed analysis). If the more conservative statistical analysis, Yates (corrected chi-square (4.856), 2-tailed) test is used, the difference between Arm A and Arm B is still highly significant (P = 0.0276). A comparison between Arm A and Arm C (untreated subjects; 3 miscarriages/39 women who completed the trial) also demonstrates that administration of bLf provided a statistically significant decrease in the incidence of miscarriage (P = 0.0148, 2x2 contingency Fishers exact 2-tailed analysis; P = 0.0186, Yates corrected chi-square, 2-tailed).

Of the pregnant women in this study, 5 were excluded due to loss of analysis (two in the bLf treatment group (Arm A) and three in the ferrous sulfate treatment group (Arm B)), and 10 were excluded for protocol violation(s) (eight in the ferrous sulfate treatment group (Arm B) and two in the untreated group (Arm C)) (see Figure 1). Baseline clinical characteristics and hematological values, irrespective or pregnancy trimester, are reported in Table 1.

The 296 study participants with a history of adverse pregnancy outcomes were found to carry specific HT genetic markers, including protein C, protein S, antithrombin deficiencies, elevated coagulation factors, F5 R506Q (factor V Leiden), and F2 G20210A (prothrombin G20210A) mutations. Although study participants were found to carry different HT genetic markers, as a group they were evaluated as HT challenged pregnant women at risk of life-threatening thrombotic events. Baseline characteristics for the three treatment arms were similar except for a lower number of women who refused therapy or were treated with ferrous sulfate compared to treatment with bLf. Baseline laboratory measurements prior to therapy were similar in all Arms.

Study participants were treated when any of the hematological parameters became positive for hypoferremia or IDA during pregnancy. This resulted in patient enrollment during all pregnancy trimesters. Laboratory studies for the number of red blood cells, hemoglobin, total serum iron, serum ferritin were assayed at the time of enrollment (time 0), after 30 days of treatment, and every 30 days thereafter until delivery. Hematocrit percentage was only measured at the time of delivery. The hematological parameters included in Arm A, Arm B, and Arm C are reported as mean values ± standard deviation in Tables 2, 3, and 4 respectively, in relation to the trimester of pregnancy in which the therapy (Arm A and Arm B) was started.

Oral administration of bLf led to significant improvements of all hematological values (see Table 2) according to the trimester of pregnancy and to the severity of hypoferremia and IDA. During the first and second trimester of pregnancy, bLf treatment for 30 days resulted in a significant increase of total serum iron

( =0.0001). In the third trimester, 30 days of bLf treatment resulted in a significantly increase in all hematological values ( =0.0001). In stark contrast, ferrous sulfate treatment of study participants during all trimesters of pregnancy failed to significantly improve hematological values compared to bLf, and in many cases treatment with ferrous sulfate resulted in hematological decreases (see Table 3). No side effects or adverse events were observed in bLf treated pregnant women compared to women treated with ferrous sulfate, who reported typical side effects and toxicities associated with ferrous sulfate therapy. In the control arm of women refusing therapy, all hematological values decreased during pregnancy (see Table 4).

The highest efficacy in treating hypoferremia and IDA was obtained with oral administration of bLf. As discussed herein, upon study entry, participants were treated until delivery, and hematological values were evaluated every 30 days and again at delivery (see Figure 2). All hematological parameters were significantly higher in bLf treated women compared to ferrous sulfate treated women or untreated controls ( =<0.0001). Moreover, hematocrit values following bLf treatment significantly increased over baseline (Table 1) and, at delivery, corresponded to a mean value of 38±2 (P =0.0001) while the mean hematocrit values in women treated with ferrous sulfate were similar to baseline values (28+3). In untreated women, hematocrit mean values decreased significantly (20±2) (P=0.0001).

In a previous study, women who were diagnosed with hypoferremia and IDA and with uncomplicated pregnancies had elevated serum IL-6 concentration, which decreased after bLf oral administration. In contrast, IL-6 concentration increased following ferrous sulfate treatment (see Paesano et al, Biochimie 91 :44-51 (2009). Epub 2008 Jun 14). In the present study, baseline values of serum IL-6 in pregnant women affected by HT, hypoferremia, and IDA were greater than uncomplicated hypoferremia and IDA pregnancies (mean values 94±10 and 22±6 pg/ml, respectively).

Forty-nine (49) women were enrolled during their third trimester of pregnancy. For this subgroup, circulating IL-6 levels and hematological parameters were assayed in 20 women belonging to Arm A and 20 women in Arm B, as well as in 9 women belonging to Arm C. In the 20 pregnant women belonging to Arm A, oral bLf significantly reduced serum IL-6 levels ( =0.0001) and significantly increased the number of red blood cells, hemoglobin, total serum iron, and serum ferritin values ( =0.0001) (Table 5). By contrast, oral ferrous sulfate increased serum IL-6 (P=0.015). Moreover, ferrous sulfate treatment failed to significantly increase hemoglobin concentration (P = 0.03) and was ineffective in improving the number of red blood cells, total serum iron, and serum ferritin concentrations (see Table 5). In Arm C, serum IL-6 concentrations remained at high levels without significant variations (mean value 98±13). The hematological values of these pregnant women are reported in Table 4. Similar to the findings with respect to serum IL-6 levels, baseline mean value of serum prohepcidin in HT pregnant women was found to be higher than that reported in uncomplicated pregnancies (775±44 and 102.0±30.7 ng/ml, respectively) (see, e.g., Chelchowska et al, Ginekol Pol. 79(11):754-57 (2008)). After 30 days of therapy, bLf increased levels of serum prohepcidin (mean value from 770±40 to 840±36 ng/ml), while patients treated with ferrous sulfate showed decreased levels (mean value decreased from 780±48 to 720±60 ng/ml) (see Table 5). However, the changes in prohepcidin levels trended but did not reach statistical significance in Arms A and B ( =0.0018 and =0.0038, respectively). In the 9 women belonging to Arm C, the values of serum prohepcidin did not change (mean value 770±49 ng/ml). The hematological values of these pregnant women are reported in Table 4.

In summary, after 30 days of oral bLf therapy, all hematological parameters (RBCs, hemoglobin, total serum iron, and serum ferritin) significantly increased (P<0.0001) and serum IL-6 concentrations significantly decreased (P<0.0001) (see Table 5). After 30 days of oral ferrous sulfate therapy, hematological parameters increased slightly and were not statistically significant, and serum IL-6 levels did not decrease, but slightly increased (P = 0.015). In the absence of treatment (Arm C), almost all hematological parameters decreased, and serum IL-6 slightly increased; the changes were not statistically significant.

Table 1. Baseline hematological values of enrolled HT affecting pregnant women who completed the study

Table 2. Hematological values before and after 30 days of oral bLf in HT women at different trimesters of pregnancy (Arm A).

RBC: red blood cells; Hb: hemoglobin (g/dl); TSI: total serum iron ^g/dl); SF: serum ferritin (ng/ml). The values are expressed meant standard deviation. Statistical analysis was performed using ANOVA test.

Table 3. Hematological values before and after 30 days of oral ferrous sulfate in HT women at different trimesters of pregnancy (Arm B).

RBC: red blood cells; Hb: hemoglobin (g/dl); TSI: total serum iron ^g/dl); SF: serum ferritin (ng/ml). The values are expressed as meant standard deviation. Statistical analysis was performed using ANOVA test.

Table 4. Hematological values before and after 30 days in HT women refusing therapy at different trimesters of pregnancy (Arm C).

RBC: red blood cells; Hb: hemoglobin (g/dl); TSI: total serum iron ^g/dl); SF: serum ferritin (ng/ml). The values are expressed as meant standard deviation. Statistical analysis was performed using ANOVA test.

Table 5. Serum IL-6 levels and hematological parameters of 49 third trimester pregnant women affected by HT, hypoferremia or IDA and treated with oral bLf or ferrous sulfate

RBC: red blood cells; Hb: hemoglobin (g/dl); TSI: total serum iron ^g/dl); SF: serum ferritin (ng/ml).

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, non-U.S. patents, non-U.S. patent applications, and non-patent

publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, each in its entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. From the foregoing a person skilled in the art will appreciate that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.