WO2011150393A2

WO2011150393A2 - Methods for improving maternal and fetal health

Info

Publication number: WO2011150393A2
Application number: PCT/US2011/038438
Authority: WO
Inventors: Rosalba Paesano; Jeffrey L. Lillard; William A. Goolsbee; Piera Valenti; Miriam Pietropaoli
Original assignee: MICROBO Srl; BMG HEMATOLOGY LLC
Current assignee: MICROBO Srl; BMG HEMATOLOGY LLC
Priority date: 2010-05-28
Filing date: 2011-05-27
Publication date: 2011-12-01
Anticipated expiration: 2012-11-28
Also published as: WO2011150393A9

Abstract

Methods are provided herein for maintaining, enhancing, and improving the health of pregnant women, infants, and children by administering compositions that comprise an isolated lactoferrin. The compositions may further comprise a nutritional mixture formulated to supplement the diet of the subject to be treated, and the compositions lack an inorganic source of iron. The nutrient mixture compositions may comprise at least vitamins B6 and folic acid and/or vitamin B12. The methods provided herein include methods for preventing (reducing the likelihood of occurrence of) a pregnancy-associated complication, such as miscarriage, placental abruption, premature birth, or preterm labor. Also provided are methods for preventing (reducing the likelihood of occurrence) or treating a postpartum-associated condition. Methods are also provided for preventing or reducing the likelihood of occurrence of a thrombotic event in a subject by administering a composition comprising a lactoferrin or administering a preparation that comprises lactoferrin and a nutrient mixture formulated to supplement the diet of the subject and which lacks an inorganic source of iron.

Description

METHODS FOR IMPROVING MATERNAL AND FETAL HEALTH

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/349,666 filed on May 28, 2010, and U.S. Provisional Application No. 61/423,982 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_405PC_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

The methods described herein relate generally to preventing the occurrence of pregnancy and/or postpartum associated or related complications and to preventing occurrence of a thrombotic event in a subject.

Description of the Related Art

In the United States, approximately 250,000 cases of venous

thromboembolism (VTE), 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. Because signs and symptoms of VTE are nonspecific and difficult to diagnose, the exact incidence of VTE is unknown but may have an annual incidence of 0.1-0.2% (see, e.g., Anderson et al., Arch. Intern. Med. 151 :933-38 (1991); Silverstein et al, Arch. Intern. Med. 158:585-93 (1998)).

Risk factors that predispose an individual to VTE include stasis or endothelial injury (e.g., resulting from indwelling venous device; major trauma or injury), medical conditions, (e.g., malignancy, pregnancy, cardiovascular conditions or events), administration of drugs (e.g., chemotherapy or hormones), and thrombophilia, which may be hereditary thrombophilia, acquired thrombophilia, or resulting from both hereditary and acquired thrombophilia. In pregnant women, the risk of both arterial and venous thrombosis is increased compared with women who are not pregnant (see, e.g., James, Hematology Am. Soc. Hematol. Educ. Program 277-85 (2009); Rosenberg et al, Obstet Gynecol. Clin. North Am. 34:481-500, xi (2007)). Risk of VTE occurs during all three trimesters of pregnancy, although typically greatest during the third trimester, and also during the postpartum period (see, e.g., Heit et al, Ann. Intern. Med. 143:697-706 (2005); Pomp et al, J. Thromb. Haemost. 7:632-37 (2008)). Reports have indicated that VTE accounts for approximately 10% of all maternal deaths (see, e.g., James et al., Am. J. Obstet. Gynecol. 194: 1311-15 (2006); see also, e.g., James, Hematology Am. Soc. Hematol. Educ. Program, supra).

Women who have thrombophilia may also have hypoferremia and iron deficiency anemia (IDA). The standard of care for treating hypoferremia and IDA comprises administering large quantities of inorganic iron, 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 alternative therapies for pregnant women to prevent or treat hypoferremia and IDA.

Various drugs are currently used for the treatment of thrombosis.

Exemplary drugs include those that suppress platelet aggregation (anti-platelet therapeutics), for example, aspirin, ticlopidine, eicosapentaenoic acid (EPA), dipyridamole, and dilazep hydrochloride. An antiplatelet therapeutic such as aspirin suppresses formation of thrombus at the impaired site of the blood vessel by

suppressing development of blood coagulation triggered by platelet aggregation.

However, because platelets also prevent hemorrhage from the blood vessel, excessive suppression of the platelet can result in decreased effectiveness of platelets in preventing hemorrhage. Anticoagulants used for treatment or prevention of thrombosis act by suppressing a blood coagulation factor and include warfarin, heparin, low molecular weight heparin, and argatroban. Anticoagulants are useful in preventing formation of intravascular fibrin clots, whereas fibrinolytics (e.g., plasminogen activators) are useful for dissolution of fibrin clots. Uncontrolled bleeding may occur after long-term administration of large doses of an anticoagulant or fibrinolytic. When heparin is used, complications include heparin resistance, bleeding, heparin-induced thrombocytopenia, and osteoporosis.

A need therefore exists for safe and efficacious therapeutic agents for managing the care of subjects who have VTE or who have an increased risk of thromboembolism.

BRIEF SUMMARY

Described herein are the following embodiments related to methods for improving maternal health and fetal health.

Embodiment 1. A method for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia, said method comprising administering an isolated lactoferrin to the subject, thereby reducing the likelihood that the pregnancy-associated complication will occur.

Embodiment 2. The method according to Embodiment 1 , wherein the subject also has hypoferremia or iron deficiency anemia.

Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the thrombophilia is hereditary thrombophilia.

Embodiment 4. The method according to any one of Embodiments 1-3, wherein the pregnancy-associated complication is 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, and intrauterine growth restriction.

Embodiment 5. The method according to any one of Embodiments 1-4, wherein the lactoferrin is administered orally.

Embodiment 6. The method according to any one of Embodiments 1-4, wherein the lactoferrin is administered by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. Embodiment 7. The method according to any one of Embodiments 1-4, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 8. The method according to any one of Embodiments 1-7, further comprising administering a prenatal nutritional mixture that is formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded.

Embodiment 9. The method according to Embodiment 8, wherein the prenatal 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 10. The method according to Embodiment 9, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6, and Vitamin B 12.

Embodiment 11. The method according to Embodiment 9 or

Embodiment 10, wherein the nutritional mixture further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc; and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. Embodiment 12. A method for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant, said method comprising administering to the subject a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation, thereby reducing the likelihood of occurrence of the pregnancy-associated complication.

Embodiment 13. The method according to Embodiment 12 wherein the subject has at least one risk factor selected from infertility, thrombophilia, recurrent miscarriage, Crohn's disease, ulcerative colitis, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, lupus erythematosus, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, rheumatoid arthritis, psoriasis, asthma, chronic bronchitis, and chronic obstructive pulmonary disease.

Embodiment 14. The method according to either Embodiment 12 or Embodiment 13, wherein the pregnancy-associated complication is 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, and intrauterine growth restriction.

Embodiment 15. The method according to any one of Embodiments 12- 14, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6 and Vitamin B12.

Embodiment 16. The method according to Embodiment 15, wherein the prenatal nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 17. The method according to any one of Embodiments 12- 14, wherein the prenatal 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 18. The method according to any one of Embodiments 12- 17, wherein the first composition and the second composition are formulated together for administration by a route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, and subcutaneous.

Embodiment 19. The method according to any one of Embodiments 12- 17, wherein the first composition and the second composition are formulated together for oral administration.

Embodiment 20. The method according to any one of Embodiments 12- 17, wherein the first composition and the second composition are each formulated separately and each is administered by a route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, vaginal, rectal, intramuscular, topical, and

subcutaneous.

Embodiment 21. The method according to any one of Embodiments 12- 17, wherein the first composition and the second composition are each formulated separately and are each administered orally. Embodiment 22. The method according to any one of Embodiments 12- 17, wherein the first composition and the second composition are each formulated separately, and wherein the first composition is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 23. A method for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death; and (b) administering an isolated lactoferrin to the subject, thereby decreasing the likelihood of occurrence of miscarriage or intrauterine fetal death.

Embodiment 24. The method according to Embodiment 23, wherein the risk factor is at least one prior occurrence of miscarriage or intrauterine fetal death.

Embodiment 25. The method according to Embodiment 23 or

Embodiment 24, wherein the risk factor is selected from infertility, thrombophilia, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, lupus erythematosus, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, asthma, chronic bronchitis, and chronic obstructive pulmonary disease.

Embodiment 26. The method according to Embodiment 25, wherein the risk factor is thrombophilia.

Embodiment 27. The method according to Embodiment 26, wherein thrombophilia is hereditary thrombophilia.

Embodiment 28. The method according to Embodiment 23 or

Embodiment 24, wherein the risk factor is that at least one paternal histocompatability antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus.

Embodiment 29. The method according to Embodiment 23 or

Embodiment 24, wherein the risk factor is that at least one fetal antigen or at least one embryonic antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus.

Embodiment 30. The method according to any one of Embodiments 23- 29, further comprising administering a prenatal nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

Embodiment 31. The method according to Embodiment 30, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6, and Vitamin B 12.

Embodiment 32. The method according to Embodiment 31, wherein the prenatal nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 33. The method according to any one of Embodiments 30-

32, wherein the prenatal 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 34. The method according to any one of Embodiments 23- 29, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 35. The method according to any one of Embodiments 30-

33, wherein the lactoferrin is administered separately or together with the nutritional mixture by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 36. The method according to any one of Embodiments 23- 35, wherein the lactoferrin is administered orally.

Embodiment 37. The method according to any one of Embodiments 23- 35, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 38. A method for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring, said method comprising (a) identifying the subject who is at risk of a thrombotic event occurring; and (b) administering an isolated lactoferrin to the subject, thereby reducing the likelihood that the thrombotic event will occur.

Embodiment 39. The method according to Embodiment 38, wherein the subject at risk of a thrombotic event occurring has at least one risk factor selected from thrombophilia, an endothelial injury, surgery, malignancy, cardiovascular disease, hypertension, obesity, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, nephritic syndrome, renal disease, prior venous thromboembolism (VTE), type 1 or type 2 diabetes, heparin-induced thrombocytopenia, paroxysmal nocturnal hemoglobinuria, microcythemia, von Willebrand disease, schizophrenia, hyperthyroidism, cystic fibrosis, a microbial infection, asthma, chronic bronchitis, chronic obstructive pulmonary disease, oral contraceptive use, hormone replacement therapy, chemotherapy, and radiation therapy.

Embodiment 40. The method according to Embodiment 38, wherein the risk factor is a microbial infection selected from Epstein-Barr Virus infection, a chronic bacterial infection, a chronic viral infection, gingivitis or periodontitis, a vaginal microbial infection, and a urogenital microbial infection.

Embodiment 41. The method according to Embodiment 38, wherein the risk factor is thrombophilia.

Embodiment 42. The method according to Embodiment 41, wherein the thrombophilia is (a) hereditary thrombophilia; (b) acquired thrombophilia; or (c) hereditary and acquired thrombophilia.

Embodiment 43. The method according to Embodiment 38, wherein the subject is pregnant or desirous of becoming pregnant or postpartum.

Embodiment 44. The method according to any one of Embodiments 38-

43, wherein the subject also has hypoferremia or iron deficiency anemia.

Embodiment 45. The method according to any one of Embodiments 38-

44, further comprising administering to the subject a nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

Embodiment 46. The method according to Embodiment 45, wherein the nutritional mixture comprises (a) at least folic acid and Vitamin B6 or (b) at least folic acid, Vitamin B6, and Vitamin B12.

Embodiment 47. The method according to Embodiment 46, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 48. The method according to any one of Embodiments 45- 47, 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 49. The method according to any one of Embodiments 45-

48, wherein the nutritional mixture is formulated for a subject who is pregnant or who is desirous of becoming pregnant or who is postpartum.

Embodiment 50. The method according to any one of Embodiments 38- 44, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 51. The method according to any one of Embodiments 45-

49, wherein the lactoferrin is administered separately or together with the nutritional mixture and by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 52. The method according to any one of Embodiments 38-

50, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 53. A method for preventing or treating colic in an infant, said method comprising administering to the infant (a) breast milk from the birth mother of the infant, wherein an isolated lactoferrin is administered to the mother (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin.

Embodiment 54. The method according to Embodiment 53, wherein the lactoferrin is administered to the mother prenatally and postpartum.

Embodiment 55. The method of either Embodiment 53 or Embodiment 54, further comprising administration of a prenatal nutritional mixture to the mother wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

Embodiment 56. The method of Embodiment 55, wherein the prenatal nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12.

Embodiment 57. The method according to Embodiment 56, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc. Embodiment 58. The method according to any one of Embodiments 55- 57, 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 59. A method for preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject, said method comprising administering an isolated lactoferrin to the postpartum subject.

Embodiment 60. A method for preventing or treating a postpartum- related psychological condition in a postpartum subject, said method comprising administering an isolated lactoferrin to the subject.

Embodiment 61. The method according to Embodiment 60, wherein the psychological condition comprises postpartum depression.

Embodiment 62. The method according to clam 60, wherein the psychological condition comprises postpartum psychosis.

Embodiment 63. The method of according to any one of Embodiments 59-62, further comprising administering a prenatal nutritional mixture to the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

Embodiment 64. The method of Embodiment 63, wherein the prenatal nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12.

Embodiment 65. The method according to Embodiment 64, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 66. The method according to any one of Embodiments 63- 65, 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 67. The method according to any one of Embodiments 59- 62, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 68. The method according to any one of Embodiments 63- 66, wherein the lactoferrin is administered separately or together with the nutritional mixture by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 69. The method according to any one of Embodiments 59- 68, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

Embodiment 70. The method according to any one of Embodiments 8- 11, 30-33, 45-49, 55-58, and 63-66, wherein the nutritional mixture is formulated with the lactoferrin to form a preparation.

Embodiment 71. A method 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, comprising administering to the infant or child a preparation that comprises (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture formulated to supplement the diet of the infant or child, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation.

Embodiment 72. The method according to Embodiment 71, wherein the nutritional composition comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12.

Embodiment 73. The method according to Embodiment 72, wherein the preparation is formulated as an infant formula further comprising at least one additional active nutritional ingredient.

Embodiment 74. A method for reducing the likelihood of occurrence of gestational diabetes in a pregnant subject, comprising administering a preparation, comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the pregnant subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation. Embodiment 75. The method according to Embodiment 74, wherein the nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12.

Embodiment 76. The method according to any one of Embodiments 71- 75, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

Embodiment 77. The method according to any one of Embodiments 71- 75, 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 78. The method according to any one of Embodiments 1- 77, wherein the lactoferrin is human lactoferrin or bovine lactoferrin.

Embodiment 79. The method according to Embodiment 78, wherein the lactoferrin is recombinant human lactoferrin or recombinant bovine lactoferrin.

Embodiment 80. The method according to Embodiment 78 or

Embodiment 79, 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 81. The method according to any one of Embodiments 78- 80, wherein the lactoferrin has any percent of saturation with one or more of iron(III), zinc, copper, and manganese.

Embodiment 82. The method according to Embodiment 81, wherein the lactoferrin has 10-30% saturation with iron(III).

Embodiment 83. The method according to Embodiment 81, wherein the lactoferrin is apolactoferrin.

In other specific embodiments, provided herein is a use of isolated lactoferrin for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In another embodiment provided herein is a use for isolated lactoferrin for the manufacture of a medicament for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In still another embodiment, is an isolated lactoferrin for use in reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia.

Also provided herein is a use of a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant. In another embodiment, also provided herein is a use of (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the subject in the manufacture of a preparation or medicament, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation, for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant. In still another embodiment is provided is a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for a use in reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant.

In other specific embodiments, provided herein is a use of isolated lactoferrin for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death. In another embodiment provided herein is a use for isolated lactoferrin for the manufacture of a medicament for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death. In still another embodiment, is provided an isolated lactoferrin for use in reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death.

In still other embodiments, provided herein is a use of isolated lactoferrin for reducing the likelihood of occurrence of a thrombotic event in a subject who is identified as at risk of a thrombotic event occurring. In another embodiment provided herein is a use for isolated lactoferrin for the manufacture of a medicament for reducing the likelihood of occurrence of a thrombotic event in a subject who is identified as at risk of a thrombotic event occurring. In still another embodiment, is provided an isolated lactoferrin for use in reducing the likelihood of occurrence of a thrombotic event in a subject who is identified as at risk of a thrombotic event occurring.

In still other embodiments, provided herein is an isolated lactoferrin alone or in combination with breast milk from the birth mother of the infant, for use in preventing or treating colic in an infant, wherein wherein the mother received the isolated lactoferrin (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum.

In still other embodiments, provided herein is a use of isolated lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject. In another embodiment provided herein is a use for isolated lactoferrin for the manufacture of a medicament for preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject. In still another embodiment, is provided an isolated lactoferrin for use in preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject.

In still other embodiments, provided herein is a use of isolated lactoferrin for preventing or treating a postpartum-related psychological condition in a postpartum subject. In another embodiment provided herein is a use for isolated lactoferrin for the manufacture of a medicament for preventing or treating a

postpartum-related psychological condition in a postpartum subject. In still another embodiment, is provided an isolated lactoferrin for use in preventing or treating a postpartum-related psychological condition in a postpartum subject.

Also provided herein is a use for a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the pregnant subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for reducing the likelihood of occurrence of gestational diabetes in a pregnant subject. Also provided herein is (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the pregnant subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for the manufacture of a preparation or a medicament for reducing the likelihood of occurrence of gestational diabetes in a pregnant subject. In still another embodiment, is provided a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the pregnant subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation, for use in reducing the likelihood of occurrence of gestational diabetes in a pregnant subject.

Also provided herein is a use for a preparation comprising that comprises (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture formulated to supplement the diet of the infant or child, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation 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. In another embodiment, is provided (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture formulated to supplement the diet of the infant or child, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for the manufacture of a preparation or a medicament for 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. In still another embodiment, a preparation is provided that comprises (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture formulated to supplement the diet of the infant or child, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation for use in 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.

In another embodiment, a method is provided for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia, wherein the method comprises administering a lactoferrin to the subject, thereby reducing the likelihood that the pregnancy-associated complication will occur. In a particular embodiment, the subject also has hypoferremia or iron deficiency anemia. In other particular embodiments, the thrombophilia is hereditary thrombophilia. In certain embodiments, the pregnancy-associated complication is 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, and intrauterine growth restriction. In other particular embodiments, the lactoferrin is administered orally. In yet other embodiments, the lactoferrin is administered by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In still other embodiments, the lactoferrin is administered both orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In other embodiments, the method further comprises administering a composition comprising a prenatal nutritional mixture that comprises at least folic acid and Vitamin B6, wherein a biologically effective amount of inorganic iron is excluded from the composition. In other particular embodiments, the prenatal nutritional mixture further comprises Vitamin B 12. In still other specific embodiments, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodine, fluorine, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In other certain embodiments, the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation. In still other embodiments, provided herein is lactoferrin for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In other embodiments, a use of lactoferrin is provided for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In yet another certain embodiment, provided herein is lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In still another embodiment, a use for lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia is provided. In other certain embodiments, the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation. In still other embodiments, provided herein is a use of lactoferrin for the manufacture of a medicament for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In other embodiments, a use of lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein is provided for the manufacture of a medicament or separate medicaments for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia. In other certain embodiments, the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation (or medicament).

In another embodiment, a method is provided for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant, said method comprising administering to the subject a preparation comprising (a) a first composition that comprises a lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12, wherein a biologically effective amount of inorganic iron is excluded from the preparation, thereby reducing the likelihood of occurrence of the pregnancy-associated complication. In another particular

embodiment, the subject has at least one risk factor selected from infertility, thrombophilia, recurrent miscarriage, Crohn's disease, ulcerative colitis, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, lupus erythematosus, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, rheumatoid arthritis, psoriasis, asthma, chronic bronchitis, and chronic obstructive pulmonary disease. In still another specific embodiment, the pregnancy-associated complication is 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, and intrauterine growth restriction. In a more particular embodiment, provided herein is a method for reducing the likelihood of occurrence of gestational diabetes in a pregnant subject, comprising administering a preparation, comprising (a) a first composition that comprises a lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the preparation. In certain embodiments, the first composition and the second composition are formulated together for administration by a route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, and subcutaneous. In other certain embodiments, the first composition and the second composition are formulated together for oral

administration. In still other certain embodiments, the first composition and the second composition are each formulated separately for administration by at least one route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, vaginal, rectal, intramuscular, topical, and subcutaneous. In yet another specific embodiment, the first composition and the second composition are each formulated separately for oral administration. In still another embodiment, the first composition and the second composition are each formulated separately, and wherein the first composition is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In a more specific embodiment, the second composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodine, fluorine, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In another embodiment, the first and second compositions described above are provided for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant. In yet another specific embodiment, a use is provided for the first and second compositions described above for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant. In yet another embodiment, a use for the first and second compositions described above is provided for the manufacture of a medicament for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject who is pregnant or who is at risk of becoming pregnant. In another embodiment, provided herein is a method for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death; and (b) administering a lactoferrin to the subject, thereby decreasing the likelihood of occurrence of miscarriage or intrauterine fetal death. In certain embodiments, the risk factor is at least one prior occurrence of miscarriage or intrauterine fetal death. In other certain embodiments, the risk factor is selected from infertility, thrombophilia, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, lupus erythematosus, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, asthma, chronic bronchitis, and chronic obstructive pulmonary disease. In still other specific embodiments, the risk factor is thrombophilia. In more specific embodiments, thrombophilia is hereditary thrombophilia. In yet another embodiment, the risk factor is that at least one paternal histocompatability antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus. In yet another specific embodiment, the risk factor is that at least one fetal antigen or at least one embryonic antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus. In still another embodiment, the method further comprises administering a composition that comprises a prenatal nutritional mixture comprising at least folic acid and Vitamin B6, wherein a biologically effective amount of inorganic iron is excluded from the composition. In a more specific embodiment, the nutritional mixture further comprises Vitamin B 12. In another specific embodiment, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodine (iodide), fluorine, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In still another embodiment, the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In yet another embodiment, the lactoferrin is administered orally. In still another embodiment, the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In yet another embodiment, the composition comprising a prenatal nutritional mixture is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal. In still another embodiment a lactoferrin is provided for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death; in another particular embodiment, a use of lactoferrin for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death is provided. In still another embodiment, a use for lactoferrin in the manufacture of a medicament for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death is provided herein. In yet another embodiment, a lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein are provided for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death; in another particular embodiment, a use of lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death are provided. In still another embodiment, a use for lactoferrin and a composition comprising a prenatal nutritional mixture as described above and herein in the manufacture of the same or separate medicaments for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death are provided herein. In certain particular embodiments, the composition is formulated with the lactoferrin to form a preparation (or medicament).

In another embodiment, a method is provided for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring, said method comprising (a) identifying the subject who is at risk of a thrombotic event occurring; and (b) administering a lactoferrin to the subject, thereby reducing the likelihood that the thrombotic event will occur. In a more specific embodiment, the subject at risk of a thrombotic event occurring has at least one risk factor selected from thrombophilia, an endothelial injury, surgery, malignancy, cardiovascular disease, hypertension, obesity, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, nephritic syndrome, renal disease, prior venous thromboembolism (VTE), type 1 or type 2 diabetes, heparin-induced thrombocytopenia, paroxysmal nocturnal hemoglobinuria, microcythemia, von

Willebrand disease, schizophrenia, hyperthyroidism, cystic fibrosis, a microbial infection, asthma, chronic bronchitis, chronic obstructive pulmonary disease, oral contraceptive use, hormone replacement therapy, chemotherapy, and radiation therapy. In another specific embodiment, the risk factor is a microbial infection selected from Epstein-Barr Virus infection, a chronic bacterial infection, a chronic viral infection, gingivitis or periodontitis, a vaginal microbial infection, and a urogenital microbial infection. In still another more specific embodiment, the risk factor is thrombophilia. In particular embodiments, thrombophilia is hereditary thrombophilia. In another specific embodiment, the subject is pregnant or postpartum. In still another specific embodiment, the subject also has hypoferremia or iron deficiency anemia. In another embodiment, the method further comprises administering a composition that comprises a nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the composition. In another specific embodiment, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide (iodine), fluoride (fluorine), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In yet another specific embodiment, the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In still another specific embodiment, the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In another particular embodiment, when the

composition is also administered, the composition is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal. Also provided herein is a lactoferrin for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring. In another embodiment, a use for a lactoferrin for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring is provided. In certain embodiments, a use for a lactoferrin in the

manufacture of a medicament for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring is provided. In other embodiments, is provided a lactoferrin and a composition comprising a nutritional mixture for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring. In still another embodiment, a use for a lactoferrin and a composition comprising a nutritional mixture for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring is provided. In still another embodiment, a use for a lactoferrin and a composition comprising a nutritional mixture in the same or separate medicaments for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring is provided. In other particular embodiments, the composition comprising a nutritional mixture is formulated with the lactoferrin to form a preparation (or a medicament).

In another embodiment, a method is provided for preventing or treating colic in a neonate or infant, said method comprising administering to the neonate or infant (a) breast milk from the birth mother of the neonate or infant, wherein the mother receives a lactoferrin (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin. In certain embodiments, the mother receives lactoferrin prenatally and postpartum. In yet other embodiments, the mother also receives a composition that comprises a prenatal nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the composition. In still other embodiments, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide (iodine), fluoride (fluorine), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In other certain embodiments, is provided breast milk from the birth mother of the neonate or infant, wherein the mother receives a lactoferrin (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin for preventing or treating colic in a neonate or infant. In other certain embodiments, a use is provided for breast milk from the birth mother of the neonate or infant, wherein the mother receives a lactoferrin (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin for preventing or treating colic in a neonate or infant. In yet other embodiments, a use is provided for breast milk from the birth mother of the neonate or infant, wherein the mother receives a lactoferrin (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin for preventing or treating colic in a neonate or infant for the manufacture of a medicament.

In still another embodiment, a method is provided for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum, said method comprising administering a lactoferrin to the subject. In a more specific embodiment, the method further comprises administering a composition comprising a prenatal nutritional mixture that comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the composition. In yet another specific embodiment, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide (iodine), fluoride (fluorine), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In still another embodiment, the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In a more specific embodiment, the lactoferrin is administered orally. In still another specific

embodiment, the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In a more specific embodiment, when the composition is administered, the composition is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal. Also provided herein is a lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum. In another embodiment, a use for a lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum is provided. In certain embodiments, a use for a lactoferrin in the manufacture of a medicament for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum is provided. In other embodiments, is provided a lactoferrin and a composition comprising a prenatal nutritional mixture for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum. In still another embodiment, a use for a lactoferrin and a composition comprising a prenatal nutritional mixture for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum is provided. In still another embodiment, a use for a lactoferrin and a composition comprising a prenatal nutritional mixture in the same or separate medicaments for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum. In other particular embodiments, the composition comprising a nutritional mixture is formulated with the lactoferrin to form a preparation (or a medicament).

In another embodiment, a method is provided for preventing or treating a postpartum related psychological condition in a postpartum subject, said method comprising administering a lactoferrin to the subject. In certain embodiments, the postpartum related psychological condition comprises postpartum depression, and in other certain embodiments, the postpartum psychological condition comprises postpartum psychosis. In a more specific embodiment, the method further comprises administering a composition comprising a prenatal nutritional mixture that comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the composition. In yet another specific embodiment, the composition further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide (iodine), fluoride (fluorine), magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E. In still another

embodiment, the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In a more specific embodiment, the lactoferrin is administered orally. In still another specific embodiment, the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical. In a more specific embodiment, when the composition is administered, the composition is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular,

subcutaneous, buccal, and intranasal. Also provided herein is a lactoferrin for preventing or treating postpartum depression in a postpartum subject. In another embodiment, a use for a lactoferrin for preventing or treating postpartum depression in a postpartum subject is provided. In certain embodiments, a use for a lactoferrin in the manufacture of a medicament for preventing or treating postpartum depression in a postpartum subject is provided. In other embodiments, is provided a lactoferrin and a composition comprising a prenatal nutritional mixture for preventing or treating postpartum depression in a postpartum subject in a subject postpartum. In still another embodiment, a use for a lactoferrin and a composition comprising a prenatal nutritional mixture for preventing or treating postpartum depression in a postpartum subject is provided. In still another embodiment, a use for a lactoferrin and a composition comprising a prenatal nutritional mixture in the same or separate medicaments for preventing or treating postpartum depression in a postpartum subject. In other particular embodiments, the composition comprising a nutritional mixture is formulated with the lactoferrin to form a preparation (or a medicament). In another embodiment, a method is provided 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, comprising administering to the infant or child a preparation that comprises (a) a first composition that comprises a lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12, wherein a biologically effective amount of inorganic iron is excluded from the preparation. In particular embodiments, the preparation is formulated as an infant formula further comprising at least one additional active nutritional ingredient that provides the daily nutritional requirements of the infant. Also provided herein, is a preparation that comprises (a) a first composition that comprises a lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the preparation 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. In another particular embodiment, a preparation is provided for use in the manufacture of a medicament 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, wherein the preparation comprises (a) a first composition that comprises a lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture comprising (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B12, wherein a biologically effective amount of inorganic iron is excluded from the preparation.

With respect to all methods and uses described above and herein, in certain embodiments, the lactoferrin is human lactoferrin or bovine lactoferrin. In another specific embodiment, the lactoferrin is recombinant human lactoferrin or recombinant bovine lactoferrin. In other specific embodiments, the lactoferrin is a polypeptide fragment of lactoferrin, and the polypeptide fragment is either lobe N or lobe C of the lactoferrin, which lactoferrin in certain embodiments is human lactoferrin or is bovine lactoferrin, which in other specific embodiments is recombinant human lactoferrin or recombinant bovine lactoferrin, respectively. In still other embodiments, the lactoferrin has any degree of saturation with one or more of iron(III), zinc, copper, and manganese. In another particular embodiment, the degree of saturation of iron(III) is 10-30%. In still another specific embodiment, the lactoferrin is apolactoferrin. 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 Arm A (treated with bLf). The circle in the "B" column designates 86 HT affected pregnant women in Arm B (treated with ferrous sulfate). The circle in the "C" column designates 39 HT affected pregnant women in Arm C (control group; absence of treatment). Each point represents the mean, and the bars represent 95% confidence intervals.

DETAILED DESCRIPTION

Methods are provided herein 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, wherein the method comprises administering a lactoferrin to the subject. 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. Exemplary risk factors that a subject may have include by way of nonlimiting example thrombophilia, pregnancy, postpartum, an autoimmune disease, hypertension, chronic microbial infection, decreased pulmonary function, 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.

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., Biometals 23:411-17 (2010); Epub 2010 April 21); Paesano et al, Biochimie, 91 :44-51 (2009); Epub 2008 June 14; Paesano et al, Biochem. Cell Biol. 84:377-380 (2006);

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)).

Lactoferrin exhibits multiple biological activities that may be dependent or independent of its iron binding capacity (see, e.g., Valenti et al, Cell Mol. Life Sci. 62:2576-87 (2005)). For example, lactoferrin has been used to treat pregnant women suffering from hypoferremia (also referred to herein and in the art as iron deficiency in the absence of anemia (ID)) and iron deficiency anemia (IDA) by, at least in part, restoring the physiological transport of iron from tissues to circulation (see, e.g., Paesano et al., Biometals, supra; Paesano et al., Biochimie, supra; Paesano et al, Biochem. Cell Biol., supra; Valenti et al, Riv. It Ost. Gin. 17:783-90 (2008); Paesano et al., // Ginecologo Rivista di Ostetricia e Ginecologia 3 Suppl. 1 Marzo, 1-6 (2008)). Administration of lactoferrin also appears to down-regulate production of inflammatory mediators, such as pro-inflammatory cytokines (e.g., interleukin (IL)-ip, IL-6, IL-8, tumor necrosis factor (TNF)-a). In reported studies, lactoferrin effected a decrease in serum IL-6 concentration, and thereby may exert an anti-inflammatory effect (see, e.g., Paesano et al., Biometals, supra; Paesano et al., Biochimie, supra; International Patent Application Publication No. WO 2007/065482). Unappreciated in the art, until the disclosure provided herein, is the capability of lactoferrin to reduce the likelihood of a thrombotic event (e.g., VTE, including deep venous thrombosis and pulmonary embolism, or an arterial thrombosis) in a subject.

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 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.

Subjects who are at risk of a thrombotic event occurring include subjects who have chronic anemia, hypoferremia, or IDA. As discussed in greater detail herein, subjects who are pregnant or desiring to become pregnant and who are at risk that a pregnancy-associated complication will occur include subjects who have hypoferremia or IDA. Presently, treatment of hypoferremia, IDA, and chronic anemia typically comprises administering large quantities of iron from an inorganic source, for example, ferrous sulfate. However, oral ferrous sulfate often fails to improve hypoferremia and IDA due, at least in part, to poor bioavailability.

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: 1653-57 (1995); Oldenburg et al, Eur. J. Clin. Invest. 30:505-10 (2000); Paesano et al, Biochem. Cell Biol. 84:377- 380 (2006); Reifen et al, Dig. Dis. Sci. 45:394-97 (2000)). 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. lO'.i^' i^' l- l (2001)). Approximately one-third of the women who received the oral iron containing supplement were unable to receive the iron salt 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)). 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).

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.

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)) . Iron

supplementation has been suggested to result in an increased risk that a woman will develop gestational diabetes during pregnancy (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)). 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(11):732- 40 (2010); Epub 2010 Sep 24; Favier et al, supra).

The combination of inefficient absorption and poor tolerability of iron from inorganic sources 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. Therefore, the methods described herein comprise administration of compositions and preparations that include lactoferrin and exclude an inorganic source of iron and thus provide benefits of lactoferrin to the subject receiving the preparation in the absence of any undesired effect of iron.

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 supra; Paesano et al., Biochem. Cell Biol. 84:377-380 (2006)). Unexpectedly, 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 significantly decreased incidence of miscarriage than women who were not treated with lactoferrin (i.e., women who were either treated with ferrous sulfate or women who did not receive either lactoferrin or ferrous sulfate).

Accordingly, also provided herein in another embodiment, is a method for reducing the likelihood of occurrence of a pregnancy-associated complication by administering a lactoferrin to a subject who is pregnant or desires to become pregnant. Pregnancy-associated complications include, but are not limited to, preeclampsia, preterm labor, 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. In another particular embodiment, methods are provided herein for decreasing or reducing the likelihood of occurrence of gestational diabetes comprising administering the compositions and preparations described herein that include lactoferrin. Methods are also provided herein for preventing {i.e., reducing the likelihood of occurrence, severity, or frequency) and/or treating postpartum

complications (such as but not limited to postpartum-related psychological conditions, e.g., postpartum mild depression, postpartum depression, and postpartum psychosis). These methods are described in greater detail herein.

In other certain embodiments, the methods described above and herein comprise administering a lactoferrin (Lf) in combination with a nutrient mixture that is formulated to supplement the diet of the subject to be treated. Accordingly, the nutrient mixture may be formulated to supplement the diet of a subject who is desiring to become pregnant, is pregnant, or who is postpartum; a subject who is an infant

(including a neonate/newborn) or a child; or a subject who is at risk of developing a thrombotic event. Unless stated otherwise, the nutrient mixture and preparations and compositions comprising the nutrient mixture described herein lack {i.e., do not include; exclude) an inorganic source of a biologically (or pharmacologically) effective amount of iron.

The preparations and compositions described herein that comprise a nutritional mixture comprise one or more vitamins, for example, folic acid, Vitamin B6, Vitamin B 12, biotin, thiamine (also called Vitamin Bl), riboflavin (also called Vitamin B2), niacin, pantothenic acid (also called 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, 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 nutritional mixture 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)). The compositions and preparations comprising a nutritional mixture are described in greater detail herein.

Infants, particularly infants born from women who have hypoferremia or IDA, 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 a 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. Perinatol. 2009 March:36(l):27-42 doi: 10.1016/j.clp.2008.09.013).

In particular embodiments, methods are described herein for reducing the likelihood of occurrence or treating hypoferremia and/or IDA in an infant or child, wherein the methods comprise administering to an infant or a child in need thereof a lactoferrin (Lf) in combination with the nutrient mixture. The nutrient mixture is appropriately formulated with nutritional ingredients that supplement the diet of the infant or child with the exception of including an inorganic source of iron. The compositions and preparations comprising a nutritional mixture, such as infant formula and children's nutritional mixture and compositions, are described in greater detail herein.

Lactoferrin

The methods described herein comprise administering a lactoferrin, which is a globular, cationic, non-heme iron-binding protein. Lactoferrin 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, KCR (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 bind tightly, but reversibly, two Fe ³ ions (also referred to as ferric ions, iron III, or Felll) together with two carbonate ions (CO3² ), 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 ion (approximately 10²² 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-α) 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%>), 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 Fe (II), Zn, Cu, and Mn. In another certain embodiment, lactoferrin has any degree of saturation with one or more of Zn, Cu, and Mn.

Lactoferrin, which as used in the methods described herein is an isolated lactoferrin and may be 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. Isolated 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 described herein, including bovine lactoferrin, also may be produced recombinantly according to methods routinely practiced in the molecular biology, protein expression, and protein isolation arts.

Full-length lactoferrin 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 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 methods described herein comprise administering lactoferrin, wherein the lactoferrin 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: 11-14), 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 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 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 indicator 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 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 for Reducing Occurrence of Thrombotic Events

In one embodiment, methods are provided herein for preventing (i.e., reducing or decreasing the likelihood of occurrence in a statistically, clinically, and/or biologically significant manner) a thrombotic event (such as thrombosis) in a subject (i.e., patient) who is at risk of a thrombotic event occurring, wherein the method comprises administering a lactoferrin to the subject. In another embodiment, the lactoferrin is administered in combination with a nutrient mixture that is formulated to supplement the diet of the subject who is at risk of a thrombotic event occurring and which nutrient mixture lacks (i.e., does not include; excludes) an inorganic source of a biologically (or pharmacologically) effective amount of iron. The compositions and preparations comprising lactoferrin alone and in combination with a nutritional mixture are described in greater detail herein.

Subjects at risk for occurrence of a thrombotic event include, for example, subjects who have at least one risk factor for venous thromboembolism (VTE) (also called venous thromboembolic disease (VTD)) or an arterial embolism. 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); 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 for reducing the likelihood of a thrombotic event occurring further comprises identifying a subject who has one or more of the aforementioned risk factors or other risk factor described herein or in the art, and who is therefore at risk for a thrombotic event occurring. Risk factors 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.

In a specific embodiment, a method is provided for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring by administering a lactoferrin (or a composition or preparation comprising an isolated lactoferrin). In certain embodiments, the subject has 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. Hereditary thrombophilia may result from any one of several genetic mutations as described herein and in the art. 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.

In other specific embodiments, 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 hereditary and acquired). 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 (HIV) 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).

According to the methods described herein, the lactoferrin may be administered to a subject (i.e., patient), who has any one or more of risk factors, at a time that is 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 a lactoferrin 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, lactoferrin may be administered prior to conception, during pregnancy (including any one, any two, or all three trimesters), and/or during the postpartum period.

The lactoferrin (or a composition comprising the lactoferrin and a pharmaceutically suitable excipient or preparation as described herein) may be administered to the subject who is at risk of a thrombotic event occurring via any one or more of several administrative routes appropriate for treating the subject. The lactoferrin may be administered by one or more of oral, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, intramuscular, subcutaneous, and intranasal routes. 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, intramuscular, subcutaneous, and intranasal routes.

Methods for Reducing Occurrence of Pregnancy-Associated Complications

In one embodiment, a method is provided herein 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, which method comprises administering a lactoferrin (i.e., an isolated lactoferrin) or a composition comprising the lactoferrin to the subject (i.e., female subject or patient). In another particular embodiment, a method is provided for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject, which method comprises administering to the subject in need thereof a composition that comprises a lactoferrin wherein an inorganic source of a biologically effective amount of iron is excluded from the composition. In still other embodiments, the lactoferrin is administered with a nutritional mixture that lacks (i.e., does not include; excludes) an inorganic source of a biologically (pharmacologically) effective amount of iron. Nutrient mixtures that are formulated for a subject who is pregnant, desiring to become pregnant, or who is postpartum are also called herein prenatal nutrient mixtures. Compositions and preparations comprising lactoferrin alone and in combination with a nutrient mixture that may be used in methods for reducing the likelihood of a pregnancy-associated complication occurring are described in greater detail herein.

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 prevent miscarriages or intrauterine fetal death that may occur due to a fetal chromosomal abnormality.

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. In a particular embodiment, the condition or risk factor is thrombophilia (tendency to develop thrombosis), and/or hypoferremia or iron deficiency anemia, and/or an iron-related toxic effect.

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, chronic bronchitis, cystic fibrosis, and chronic obstructive pulmonary disease) may also be risk factors that predispose a pregnant subject to the occurrence of a pregnancy-associated complication. As discussed herein, a subject who is receiving an inorganic source of iron (e.g., via ferrous sulfate, ferrous fumarate or other iron salts) may be at greater risk of developing a pregnancy-associated complication such as gestational diabetes due to toxic effects of the elemental iron.

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. Methods are provided wherein lactoferrin or preparations and compositions comprising a lactoferrin, and which may include a nutritional mixture and excluding an inorganic source of a biologically (or pharmacologically) effective amount of iron (described in greater detail 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 and compositions 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. Accordingly, in certain embodiments, the methods described herein further comprise identification of a subject who has one or more conditions, disorders, diseases, or risk factors, for example, those discussed herein, that increase the likelihood that the subject will experience one or more pregnancy-associated complications.

In certain embodiments, the methods described herein for reducing the likelihood of occurrence of a pregnancy-associated complication comprise

administering a lactoferrin to a subject who is pregnant, at risk or desirous of becoming pregnant, and who also has thrombophilia. A subject who has thrombophilia may have hereditary thrombophilia, acquired thrombophilia, or have aspects of both acquired and hereditary thrombophilia. In certain particular embodiments, thrombophilia is hereditary thrombophilia, which 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. The hypercoagulable state represents one of the physiological changes in problem pregnancies. 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)).

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 be treated by and benefit from the methods 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, methods are provided herein 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, by administering a lactoferrin or by administering a lactoferrin and a nutritional mixture, excluding an inorganic source of a biologically effective amount of iron, to the subject.

Accordingly, in a particular embodiment, a method is provided for reducing the likelihood of occurrence of a miscarriage or intrauterine fetal death by administering a lactoferrin to a subject. In other certain embodiments, the method for reducing the likelihood of occurrence of a miscarriage or intrauterine fetal death comprises administering a lactoferrin in combination with a nutrient mixture that is formulated to supplement the diet of a pregnant subject {i.e., prenatal nutritional mixture). The nutrient mixture and preparations and compositions comprising the nutrient mixture lack {i.e., do not include; exclude) a biologically (or pharmacologically) effective amount of a source of inorganic iron. The compositions and preparations comprising a nutritional mixture are described in greater detail herein.

As described herein, lactoferrin alone or lactoferrin in combination with a nutritional mixture 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). Lactoferrin alone or lactoferrin in combination with a nutritional mixture 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 miscarriage, spontaneous abortion, or preterm delivery, 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 lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron (as described above and herein) prior to conception and/or may receive one or more doses of lactoferrin subsequent to conception (including for any length of time during the pregnancy). For example, the subject may receive lactoferrin during at least the first trimester, which is the time during which most miscarriages occur. In other embodiments, lactoferrin may be administered to the subject during the first two trimesters or during the entire pregnancy.

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. Methods described herein that comprise administering a lactoferrin to a subject who is pregnant, or who desires to become pregnant and thus is at risk of becoming pregnant, may be used for preventing (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). As described herein, a lactoferrin, which may be combined with a prenatal nutritional mixture as 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 methods described herein may also be useful 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 the methods described herein includes preterm labor.

Methods described herein may also be used for reducing or decreasing the likelihood that the pregnancy-associated complication, intrauterine growth restriction of the fetus, will occur. 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 methods described herein may be used 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 (which may be combined with a prenatal nutritional mixture as described herein) 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.

In another particular embodiment, a method is provided for reducing the likelihood of occurrence or development of (or preventing) gestational diabetes by administering to a pregnant subject in need thereof (i.e., at risk of developing gestational diabetes) a lactoferrin in combination with a prenatal nutrient mixture. The nutrient mixture and preparations and compositions comprising the nutrient mixture lack (i.e., do not include; exclude) an inorganic source of a biologically (or

pharmacologically) effective amount of iron. The compositions and preparations comprising a nutritional mixture are described in greater detail herein.

Gestational diabetes is typically described in the art as a condition in which women who have not previously been diagnosed with diabetes exhibit high blood glucose levels during pregnancy. Infants born to mothers who have gestational diabetes are at increased risk of complications at birth, including for example, large size for gestational age (which may lead to complications during birth), low blood sugar, and jaundice. All pregnant women may develop gestational diabetes; however, women at greater risk include women who have a previous diagnosis of gestational diabetes (i.e., during a previous pregnancy); have a family member with type 2 diabetes; increased maternal age (particularly greater than 35 years of age); have non-Caucasian ethnic background; are overweight, obese, or morbidly obese; had a previous pregnancy resulting in birth of a high birth weight baby (typically greater than 90^th percentile or greater than 4000 grams); and poor obstetric history. Women are typically monitored during pregnancy by clinical testing methods and techniques routinely practiced in the art (e.g., fasting glucose test, 2-hour postgrandial glucose test, random glucose test, screening glucose challenge test, and oral glucose tolerance test). Persons skilled in the medical and clinical arts can determine on the basis of the clinical tests, medical history, symptomatology, and examination whether a subject is at greater risk of developing gestational diabetes, has gestational diabetes, and/or whether the subject is responding to prevention and/or treatment with the compositions described herein, which may be administered in combination with other therapeutic practices, such as dietary

adjustments and exercise, for example.

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 methods 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.

For a subject who is pregnant, or is desirous, and thus at risk, of becoming pregnant, lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron (as described above and herein) may be administered prior to conception, during pregnancy, and during the postpartum period. As described herein, the dosing of lactoferrin may be determined by a person skilled in the clinical and medical arts. The use of the minimum dosage that is sufficient to provide effective therapy is usually preferred. As described herein, methods that comprise administering at least one lactoferrin includes administering at least one dose of the lactoferrin, two or more doses of the lactoferrin, or multiple doses of lactoferrin per day. The lactoferrin may also be administered by one or more different routes as discussed in greater detail herein.

In still another embodiment, as described herein, methods are provided for reducing the likelihood of occurrence of at least one pregnancy-associated complication comprising administering a lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron 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 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, such as, and in particular, miscarriage.

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 methods described herein that comprise administering a lactoferrin to the subject, therefore, may reduce miscarriage, spontaneous abortion (including recurrent spontaneous abortion), or preterm delivery, in a statistically, biologically, or clinically significant manner. As described herein with respect to methods for reducing the risk of other pregnancy-associated complications occurring, the subject who has or who is at risk of developing maternal immune intolerance, may receive lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron (as described above and herein) during at least the first trimester, which is the time during which most miscarriages occur. In other embodiments, lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron (as described above and herein) may be administered to the subject during the first two trimesters or during the entire pregnancy.

Pregnancy, Hypoferremia, and IDA

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., supra). 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.

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: 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)).

Postpartum Subjects and Infants

In another embodiment, the preparations comprising lactoferrin (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 an infant (including a

neonate/newborn). The method comprises administering to the infant breast milk from the birth mother who had received the compositions/preparations prenatally, postpartum, (i.e., postnatally), or both prenatally and postpartum. In another embodiment, the neonate/infant also receives an infant formula that further comprises the lactoferrin. Components (i.e., ingredients) of infant formulas (both milk based and plant based) are well known and described in the art and are sold commercially. In certain particular embodiments, an infant formula is prepared that comprises lactoferrin and nutrients required by the infant with the exception of an inorganic source of a biologically effective amount of iron. In still another embodiment, the neonate/infant receives a combination of the breast milk from the birth mother and an infant formula supplemented with lactoferrin. 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 lactoferrin 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.

Administration to a postpartum subject (mother) of lactoferrin alone or lactoferrin in combination with a nutritional mixture, which is described in detail herein, may be continued if lactoferrin with or without the nutrient mixture had been administered during pregnancy, or may be initiated after birth of the neonate and continued during the postpartum period, to reduce (i.e., decrease in a statistically, biologically, or clinically significant manner) the likelihood of occurrence, severity, or duration of a postpartum complication of the mother (e.g., a thrombotic event, postpartum-related psychological conditions, e.g., postpartum mild depression (i.e., postpartum "blues"), postpartum depression and/or psychosis, postpartum fatigue, hypoferremia, IDA).

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).

Accordingly, methods are provided herein for reducing the likelihood of occurrence or severity of a postpartum-related psychological condition occurring in the mother during the postpartum period by administering a lactoferrin or by administering a lactoferrin and a nutritional mixture and excluding an inorganic source of iron (which nutritional mixture is described in greater detail herein). In particular embodiments, when for example, but not limited to, the subject has thrombophilia, the nutritional mixture may comprise at least folic acid, Vitamin B6, and Vitamin B 12.

The likelihood of occurrence, severity, or duration of a postpartum- related psychological condition, such as but not limited to postpartum mild depression, postpartum-related depression, or postpartum psychosis, may also be reduced (i.e., decreased in a statistically, biologically, or clinically significant manner) by administering a lactoferrin, optionally in combination with a nutritional mixture, during pregnancy, and/or during the postpartum period. Also, provided herein is a method for preventing or treating postpartum fatigue and/or postpartum-related psychological conditions, such as but not limited to postpartum mild depression, postpartum depression, and postpartum psychosis in a postpartum subject by administering to the postpartum subject (mother) lactoferrin alone or lactoferrin in combination with a nutritional mixture (as described above and herein). In another embodiment, the preparations comprising lactoferrin (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 of lactoferrin or the preparation comprising lactoferrin and the nutritional mixture may be performed as described in greater detail herein. For example, lactoferrin 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, MCNAm. 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 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)). In addition to performing hematological 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 a postpartum-related psychological condition (e.g., postpartum depression or postpartum psychosis) 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)).

Lactoferrin-containing compositions and/or 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. In another embodiment, methods are provided for preventing (i.e., decreasing the likelihood of occurrence) or treating hypoferremia or iron deficiency anemia in a subject by administering a lactoferrin (or composition comprising the lactoferrin) alone or lactoferrin in combination with a nutritional mixture (as described above and herein). As described in detail herein, compositions and preparations comprising a lactoferrin and nutritional mixture lack (i.e., do not include; exclude) an inorganic source of a biologically (or

pharmacologically) effective amount of iron. In a certain embodiment, the subject is female and is postpartum.

In certain embodiments, methods are provided for administering compositions and preparations described herein (which comprise lactoferrin and a nutritional mixture of vitamins and minerals and exclude an inorganic source of a biologically effective amount of iron) to an infant or child to reduce the likelihood of occurrence (i.e., reduce in a statistically, biologically, or clinically significant manner) of hypoferremia (also called iron deficiency (ID)) or iron deficiency anemia (IDA). Infants who are at risk of developing hypoferremia or IDA include infants whose birth mothers had hypoferremia 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 lactoferrin and lack a source of inorganic 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 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

The preparations and nutritional compositions for administration to an infant or child 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 to be administered to an infant or child 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)).

Methods for Treating/Preventing Anemias and Improving Hematological Status

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 a lactoferrin may increase serum iron levels to a level that is improved in a statistically, biologically, or clinically significant manner and/or that is normal (i.e., the level of serum iron is restored to a level that falls within range of levels considered normal for the particular subject or for similar subjects). Levels of total serum iron, as well as the level of other hematological parameters discussed below and herein, that are considered normal can be readily ascertained by persons skilled in the art. In a subject who has iron deficiency anemia, 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 methods 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/or morphology, hemoglobin, total serum iron, serum ferritin, hematocrit) to the level of the respective indicator 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 (indicator) 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 parameter 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 parameters that are indicators of hematological status may be increased or elevated, such as 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 health and well-being of the subject, including a subject at risk of developing a thrombotic event, such as 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 may also be assessed by monitoring fetal movement, fetal heart tones, growth, and which include physical examination and may also include ultrasonography. Monitoring the subject 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 lactoferrin alone or lactoferrin in combination with a nutrient mixture and excluding an inorganic source of a biologically (pharmacologically) effective amount of iron (which compositions and preparations are described in greater detail herein) 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 (e.g., red blood cells (including red blood cell count, morphology), hemoglobin level, hematocrit, total serum iron, serum ferritin) 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 at least one lactoferrin or lactoferrin in combination with a nutrient mixture, the hematological and/or immunological 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.

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.

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, serum 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 parameters (i.e., 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 of hepcidin and/or prohepcidin levels are described in the literature. For certain assays and techniques, assay kits are commercially available. The level of each hematological parameter in a sample from a subject may then be compared with predetermined levels and ranges 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. The levels and ranges that are considered normal may also vary with gender, age, and weight (or mass) of the subject.

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, 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 compositions comprising lactoferrin and/or 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 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 lactoferrin and the preparations and compositions described herein comprising lactoferrin and a nutritional mixture 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), 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.

Compositions, Preparations, and Formulations

A lactoferrin is formulated in a physiologically {i.e., pharmaceutically) acceptable (or suitable) composition and/or preparation as described in greater detail herein and is administered to a subject in a manner appropriate, as determined by persons skilled in the medical arts, for reducing the likelihood of occurrence of a pregnancy-associated complication (including the numerous complications described herein, for example, miscarriage and intrauterine fetal death); for reducing the likelihood of occurrence of a thrombotic event; for preventing or treating colic in a neonate or infant; and for preventing or treating a postpartum condition or

complication, such as hypoferremia or IDA and/or postpartum-related psychological conditions such as postpartum psychosis and postpartum depression and/or fatigue in a subject postpartum; and for preventing or treating hypoferremia and/or IDA in an infant or child. In another particular embodiment, a composition is provided that comprises at least one physiologically or pharmaceutically acceptable or suitable excipient and the lactoferrin (which is an isolated lactoferrin) but which composition does not include {i.e., lacks or excludes) an inorganic source of a biologically (or pharmacologically) effective amount of iron. An appropriate dose and a suitable duration and frequency of administration of the lactoferrin may be determined by such factors as the condition of the patient (i.e., subject); age of the subject; the type and severity of the patient's condition, disease or disorder that affects the health of the subject; whether the subject is pregnant, desiring to become pregnant, postpartum, or lactating; the particular form of the active ingredient; and the method of administration.

In another embodiment, lactoferrin is administered in combination with a nutritional mixture. The nutritional mixture comprises 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 B12 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)). Each of the lactoferrin and nutritional mixture is formulated in physiologically acceptable (or suitable)

compositions and preparations. As discussed in detail herein, compositions and preparations that comprise the nutritional mixture do not include (i.e., exclude, lack) an inorganic source of a biologically (or pharmacologically) 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), and supplementing daily nutritional requirements, as provided by vitamins, and optionally minerals, and optionally at least one other active nutritional (i.e., dietary) ingredient. An appropriate dose and a suitable duration and frequency of administration of the compositions and preparations described herein will be determined by such factors as the condition of the patient (i.e., subject); the age of the subject; the type and severity of any disease or disorder or condition that affects the health of the subject (for example, whether the subject is at risk of a thrombotic event occurring); 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 preparations in an amount sufficient to provide therapeutic and/or prophylactic benefit. Therapeutic and/or prophylactic benefit includes, for example, an improved clinical outcome (e.g., reduced or decreased occurrence of a pregnancy-associated or postpartum complication; reduced or decreased occurrence of a thrombotic event; or reduced or decreased risk that a pregnancy-associated complication and/or thrombotic event will occur; reduced or decreased occurrence or severity of hypoferremia and/or IDA in subject, include an infant or child); abatement or lessening in severity of the symptoms of the disease, disorder, or condition to be treated; 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.

In certain embodiments with respect to methods for reducing the likelihood of occurrence (or preventing or prophylaxis) of a thrombotic event, a dose or dosing regimen of lactoferrin should be sufficient to prevent, delay the onset of, or diminish the severity of a thrombotic event, or of a symptom, condition, or sequelae thereof. In other certain embodiments, with respect to methods for reducing the likelihood of occurrence (i.e., preventing) of a pregnancy-associated complication or pathology, the dose or dosing regimen of lactoferrin should be sufficient to prevent the occurrence of at least one pregnancy-associated complication in a statistically, clinically, or biologically significant manner. Accordingly, in certain specific embodiments, the dose or dosing regimen of lactoferrin administered to a subject in need thereof should be sufficient to reduce the occurrence of (i.e., prevent) a miscarriage or intrauterine fetal death in a statistically, clinically, or biologically significant manner. In another embodiment, with respect to methods for inducing or maintaining immune tolerance in a subject (including inducing maternal immune tolerance to a fetal or paternal histocompatability antigen), dosing is designed and intended to be sufficient to prevent, delay the onset of, or diminish the severity of immune intolerance and one or more associated conditions or sequelae (e.g.,

miscarriage).

In yet another embodiment, the dose or dosing regimen of lactoferrin administered to a subject in need thereof should be sufficient to prevent, delay, reduce or decrease the occurrence of, delay onset of, and/or reduce severity of a postpartum complication such as hypoferremia, IDA, a postpartum-related psychological condition (e.g., postpartum mild depression, postpartum depression, or postpartum psychosis), and/or postpartum fatigue in a statistically, clinically, or biologically significant manner. In still another embodiment, the dose or dosing regimen of lactoferrin administered to an infant (including a neonate/newborn) should be sufficient to prevent or treat colic (i.e., delay onset, reduce or delay severity and/or duration of a colic episode) and/or to prevent or treat hypoferremia and/or IDA (i.e., delay onset, reduce or delay severity and/or duration of a hypoferremia and/or IDA)in an infant or child in a statistically, clinically, or biologically significant manner. Similarly, the dose or dosing regimen of lactoferrin and the dose or dosing of a nutrient mixture (as described herein) when lactoferrin and the nutrient mixture are administered for use in these methods should be sufficient to provide a desired clinical and biological effect in a statistically, clinically, or biologically significant manner.

Administration of lactoferrin and the compositions and preparations described herein that comprise a lactoferrin may also 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 may also be 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 anemia (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).

Compositions comprising lactoferrin:

Lactoferrin, which as used in the methods described herein is typically an isolated lactoferrin and may be human lactoferrin, bovine lactoferrin, murine lactofemn, or buffalo lactoferrin. In certain embodiments, the compositions described herein comprise bovine lactoferrin. In other embodiments, the compositions comprise human lactoferrin. The compositions may further comprise one or more

physiologically or pharmaceutically acceptable excipients, which are described in greater detail herein. In certain specific embodiments, a composition comprising the lactoferrin lacks an inorganic source of a biologically effective amount of iron such that a biological or pharmacological activity of iron from the inorganic source is not detected or observed.

Any lactoferrin described herein also may be produced recombinantly according to methods routinely practiced in the molecular biology, protein expression, and protein isolation arts. In other embodiments, the lactoferrin is a fragment or a variant {i.e., comprises one or more insertions, deletions, or substitutions of an amino acid) of a human lactoferrin, bovine lactoferrin, murine lactoferrin, or buffalo lactoferrin. As described in detail herein, a lactoferrin fragment or variant retains {i.e., in a statistically or biologically significant manner) the biological activities of the full- length and mature lactoferrin polypeptides. Such biological activities include antimicrobial activity; the capability to prevent the increased production or to reduce or decrease the production of one or more inflammatory mediators including

proinflammatory cytokines and thus prevent, reduce the likelihood of occurrence or likelihood of worsening of, ameliorate, or mitigate inflammation and/or the

inflammatory response; the capability to restore or maintain iron homeostasis and/or to modulate the level of a hematological parameter and thus reduce, ameliorate, or mitigate hypoferremia or IDA.

Preparations:

A preparation is provided that comprises an isolated lactoferrin (or a composition comprising the lactoferrin and at least one physiologically suitable excipient) and a nutritional mixture (also called herein a nutrient mixture) (or a composition comprising the nutrient mixture and at least one physiologically suitable excipient). The nutrient mixture is formulated with ingredients sufficient to supplement the diet of the subject who is to receive the lactoferrin and nutritional mixture. The preparations and nutritional mixture 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 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, such as biotin and/or Vitamin D.

ingredient/agent generally regarded as safe (GRAS)). Other nutrients may include bioflavonoids referred to as Vitamin P. For ease of description, the compositions may be referred to as first and second compositions, such as a composition comprising lactoferrin and at least one physiologically suitable excipient may be referred to as a first composition, and a composition comprising the nutrient mixture and optionally at least one physiologically suitable excipient may be called a second composition.

The nutritional ingredients of the nutrient mixture are deemed 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 B12.

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 B12/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 in detail herein, lactoferrin is a biologically and physiologically active ingredient of the preparations that also comprise folic acid, Vitamin B6, and may further comprise Vitamin B 12.

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.

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 contribute to a toxic or undesired effect and care is taken not to exceed a particular daily value that is based on the recommended dietary allowance. 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. 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 carefully regulate 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 in the nutrient mixture. 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.

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 example, preparations may be formulated for (1) a pregnant or lactating subject; (2) an infant (which includes newborn (neonate)) or child, particularly a child or infant 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; (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.

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 an 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 B 12, 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 a biologically effective amount of an inorganic source 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 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 960 μg, 980 μg, 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 μ§, 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 μ 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 μg 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 μg per day (e.g., about 12 μg 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 mixture 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 comprises one or more of calcium, chromium, copper, iodine (iodide), magnesium, manganese, molybdenum, selenium, and zinc. In a more specific embodiment, the prenatal preparation 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. 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 the compositions and preparations described herein that are intended to be administered to infants and children (e.g., 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 or methods for reducing the likelihood of occurrence, duration or severity of colic), 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 B 12, 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 (B₂). 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 and medical arts.

The nutritional mixtures 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 inorganic iron 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) 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 toxic effect or undesired effect or condition in a subject (including but not limited to inducing an inflammatory status of the subject, gastrointestinal discomfort, nausea, vomiting, diarrhea, gestational diabetes, 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)).

The methods described herein for reducing the likelihood of occurrence of a pregnancy-associated complication (including, for example, methods for reducing the likelihood of occurrence of miscarriage and intrauterine fetal death); and the methods for reducing the likelihood of occurrence of a thrombotic event, comprise administering to the subject a composition comprising an isolated lactoferrin and a physiologically suitable (i.e., pharmaceutically acceptable) excipient. In a specific embodiment, the compositions lack an inorganic source of a biologically effective amount of iron. The methods may further comprise administering a preparation comprising the isolated lactoferrin and a nutritional mixture as described above and herein, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation. When the subject who is receiving the preparation is pregnant or desirous of becoming pregnant, the preparation is formulated to supplement the diet of a pregnant subject. For example, the nutritional mixture may comprise an amount of folic acid sufficient to provide at least 1.5 times greater (i.e., at least 600 μg) than the daily recommended amount for a non-pregnant adult. With respect to a method for preventing or treating colic in an infant (including a neonate/newborn), the composition comprising an isolated lactoferrin and/or the preparations as described herein and above may be administered to the pregnant subject during pregnancy. The subject may receive the composition and/or preparation during only one trimester, which may be the last (third) trimester, or during two or all three trimesters. A method for treating colic in a neonate or infant may further include administering to the infant/neonate lactoferrin in the breast milk from the birth mother or may further include administering to the infant lactoferrin in the breast milk from the birth mother and lactoferrin present in an infant formula.

In other certain embodiments, methods are provided for reducing the likelihood of occurrence of iron deficiency or iron deficiency anemia in an infant or child (including a neonate/newborn), by administering the composition comprising a lactoferrin and in the same or different composition comprise a nutritional mixture that lack (i.e., exclude) an inorganic source of a biologically effective amount of iron. These compositions and preparations may be administered in a liquid infant formula that contains all the nutritional requirements for an infant or that contains the amount of nutrients for the infant or child who is transitioning from a diet of infant formula to solid food. In other particular embodiments, the preparations and compositions described herein may be administered to an infant or child in a concentrated supplement that may be administered as liquid drops or in a chewable solid form or a powdered form added to other foodstuffs, or as any other form that is appropriate and safe for consumption by an infant or child.

A "therapeutically effective amount" or "effective amount" means an amount of an active ingredient (e.g., lactoferrin; active nutritional ingredients that are included in a nutrient 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., reduce or decrease the likelihood of occurrence of) the stated disease, disorder or condition. 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. An optimal dose of lactoferrin that is included in the preparations and compositions for use in the methods described herein may also 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 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.

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 at least 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. In particular embodiments, about 10 mg, 25 mg, 50 mg, 100 mg, about 200, 300, 400, 500, 600, 700, 750 mg, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 or more mg 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, at least about 200, 300, 400, 500, 600, 700, 750 mg, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 mg or more per day. In one particular embodiment, the total daily dose of lactoferrin that may be administered may be 50, 100, or 200 mg per day, which may be delivered in a single dose or in two separate doses in the compositions and preparations described herein. 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 is prepared and administered in two individual doses of 100 mg each. In another certain embodiment lactoferrin may be formulated for administration at a dose 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.

The amount of lactoferrin delivered per dose and per day to an infant or child can be determined readily by a person skilled in the art. By way of an additional example, when a preparation or composition described herein that comprises lactoferrin is delivered 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, 50 mg, 100 mg, or 200 mg.

The use of the minimum dosage that is sufficient to provide effective therapy (i.e., clinical or biological benefit) or prophylaxis is usually preferred. As described herein, methods that comprise administering lactoferrin (or compositions or 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. Subject (i.e., 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.

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 preterm 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). 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). In other particular embodiments, the subject is a human infant (including a neonate) or child.

A lactoferrin and a nutritional mixture may be formulated in a composition or preparation 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 described herein that comprise a lactoferrin and/or a 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 composition. 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.

With respect to each embodiment of the methods described herein, the lactoferrin or a composition comprising the lactoferrin may be administered to the subject via any one or more of the following routes: oral, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal, and any other route appropriate for treating the subject. In certain specific embodiments, lactoferrin is delivered orally. When lactoferrin is administered by two or more {i.e., more than one) administrative route, 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 preterm 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, wherein the methods described herein comprise administering the preparations described herein that comprise an isolated lactoferrin and a nutrient mixture, the preparations 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.

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. By way of example, the compositions described herein may be formulated as a liquid, solid, semi-solid, gel, capsule, or lyophilate (i.e., a lyophilized composition or preparation, e.g., lyophilized infant formula) or other dehydrated form that is solubilized with the addition of water or other appropriate solute for oral administration.

When the preparation comprises one 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 same 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, and any other relevant factor.

When lactoferrin is administered by two or more (i.e., more than one) administrative route, 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 preterm 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 preparation, described in greater detail herein, comprises the lactoferrin (or a composition that comprises a lactoferrin) and a 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 compositions and preparations described herein, including a composition comprising the 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 composition to a subject. Each composition and preparation is also formulated in a form appropriate for the route of administration. Each composition and preparation may be in the form of a solid, liquid, emulsion, ointment, suspension, gel, or gas (aerosol). For optimal bioavailability, a 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 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.

A composition comprising Lf, a preparation comprising Lf and nutritional mixture, 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 comprising a lactoferrin and preparations comprising a lactoferrin and a nutritional mixture, described herein, may provide dietary management of inflammation, hypoferremia, IDA, and other anemias, including chronic anemia. Preparations comprising a lactoferrin and the nutritional mixture described herein may also provide dietary management of 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, or to subjects who are pregnant, desirous of becoming pregnant and who are at risk of a pregnancy-associated complication occurring (such as, by way of example, a pregnant subject who also has thrombophilia), or who are postpartum. These compositions and preparations thus provide nutritional requirements 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.

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 from an inorganic source 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 a biologically effect amount of iron.

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 compositions and preparations 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 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. A lactoferrin (and a

pharmaceutical composition comprising the lactoferrin) may be encapsulated within one or more delivery materials appropriate for attaining and/or maintaining an effective therapeutic level of the lactoferrin in the subject. Similarly, a prenatal mixture of nutrients excluding iron (and a pharmaceutical composition comprising the mixture) may be encapsulated within one or more delivery materials appropriate for attaining and/or maintaining an effective therapeutic 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 compositions and preparations described herein 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 compositions and preparations 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. In the methods described herein,

compositions and preparations 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

composition and/or preparation 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 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 composition or preparation is combined with a microsphere, the microsphere is larger than

approximately 25 microns. A composition or preparation 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 a pharmaceutical composition or preparation for topical administration (e.g., oral or vaginal). The compositions and preparations 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 composition or preparation is attached to, absorbed by, adsorbed to, or in some manner applied to the vehicle that permits release of the composition or preparation upon contact with the tissue to be treated.

A composition or preparation 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 composition or preparation described herein that is administered orally may also be in the form of a liquid. The composition or preparation 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 compositions and preparations described herein may be endotoxin free, particularly when delivered parenterally. An endotoxin free composition or preparation 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 composition that comprises a lactoferrin. In one embodiment, the method of manufacture of the composition comprises isolating a lactoferrin from a biological source as described herein (e.g., isolating bovine lactoferrin from cow's milk) 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.

Also provided herein are methods 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. 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 a nutrient mixture. 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 the embodiments described herein.

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 (P=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 preg (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).

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.

Claims

CLAIMS We claim the following:

1. A method for reducing the likelihood of occurrence of a pregnancy- associated complication in a subject who is pregnant or who is desirous of becoming pregnant and who has thrombophilia or who is at risk of developing thrombophilia, said method comprising administering an isolated lactoferrin to the subject, thereby reducing the likelihood that the pregnancy-associated complication will occur.

2. The method according to claim 1, wherein the subject also has hypoferremia or iron deficiency anemia.

3. The method according to claim 1, wherein the thrombophilia is hereditary thrombophilia.

4. The method according to claim 1, wherein the pregnancy-associated complication is 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, and intrauterine growth restriction.

5. The method according to claim 1, wherein the lactoferrin is

administered orally.

6. The method according to claim 1, wherein the lactoferrin is

administered by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

7. The method according to claim 1, wherein the lactoferrin is

administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

8. The method according to any one of claims 1-7, further comprising administering a prenatal nutritional mixture that is formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded.

9. The method according to claim 8, wherein the prenatal 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.

10. The method according to claim 9, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6, and Vitamin B 12.

11. The method according to claim 9, wherein the nutritional mixture further comprises one or more additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc; and (b) at least one vitamin selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin D, and Vitamin E.

12. A method for reducing the likelihood of occurrence of a pregnancy- associated complication in a subject who is pregnant or who is at risk of becoming pregnant, said method comprising administering to the subject a preparation comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation,

thereby reducing the likelihood of occurrence of the pregnancy-associated complication.

13. The method according to claim 12 wherein the subject has at least one risk factor selected from infertility, thrombophilia, recurrent miscarriage, Crohn's disease, ulcerative colitis, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, lupus erythematosus, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, rheumatoid arthritis, psoriasis, asthma, chronic bronchitis, and chronic obstructive pulmonary disease.

14. The method according to claim 12, wherein the pregnancy-associated complication is 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, and intrauterine growth restriction.

15. The method according to any one of claims 12-14, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6 and Vitamin B 12.

16. The method according to claim 15, wherein the prenatal nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

17. The method according to claim 12, wherein the prenatal 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.

18. The method according to claim 12, wherein the first composition and the second composition are formulated together for administration by a route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, and subcutaneous.

19. The method according to claim 12, wherein the first composition and the second composition are formulated together for oral administration.

20. The method according to claim 12, wherein the first composition and the second composition are each formulated separately and each is administered by a route selected from oral, parenteral, lingual, buccal, intranasal, transdermal, vaginal, rectal, intramuscular, topical, and subcutaneous.

21. The method according to claim 12, wherein the first composition and the second composition are each formulated separately and are each administered orally.

22. The method according to claim 12, wherein the first composition and the second composition are each formulated separately, and wherein the first composition is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

23. A method for reducing the likelihood of occurrence of miscarriage or intrauterine fetal death, comprising: (a) identifying a subject who is pregnant or who is desirous of becoming pregnant, and who has at least one risk factor that increases the risk of occurrence of miscarriage or intrauterine fetal death; and (b) administering an isolated lactoferrin to the subject, thereby decreasing the likelihood of occurrence of miscarriage or intrauterine fetal death.

24. The method according to claim 23, wherein the risk factor is at least one prior occurrence of miscarriage or intrauterine fetal death.

25. The method according to claim 23, wherein the risk factor is selected from infertility, thrombophilia, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, lupus erythematosus, type 1 or type 2 diabetes, hypertension, renal disease, microcythemia, von Willebrand disease, schizophrenia, hyperthyroidism, a malignancy, cystic fibrosis, Epstein-Barr Virus infection, chronic bacterial infection, gingivitis or periodontitis, a vaginal microbial infection, urogenital microbial infection, chronic viral infection, asthma, chronic bronchitis, and chronic obstructive pulmonary disease.

26. The method according to claim 25, wherein the risk factor is thrombophilia.

27. The method according to claim 26, wherein thrombophilia is hereditary thrombophilia.

28. The method according to claim 23, wherein the risk factor is that at least one paternal histocompatability antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus.

29. The method according to claim 23, wherein the risk factor is that at least one fetal antigen or at least one embryonic antigen induces an immune response in the subject, thereby reducing maternal immune tolerance to a conceptus or fetus.

30. The method according to any one of claims 23-29, further comprising administering a prenatal nutritional mixture, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

31. The method according to claim 30, wherein the prenatal nutritional mixture comprises (a) at least folic acid and Vitamin B6; or (b) at least folic acid, Vitamin B6, and Vitamin B 12.

32. The method according to claim 31 , wherein the prenatal nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

33. The method according to claim 30, wherein the prenatal 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.

34. The method according to claim 23, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

35. The method according to claim 30, wherein the lactoferrin is administered separately or together with the nutritional mixture by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

36. The method according to claim 23, wherein the lactoferrin is administered orally.

37. The method according to claim 23, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

38. A method for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring, said method comprising (a) identifying the subject who is at risk of a thrombotic event occurring; and (b) administering an isolated lactoferrin to the subject, thereby reducing the likelihood that the thrombotic event will occur.

39. The method according to claim 38, wherein the subject at risk of a thrombotic event occurring has at least one risk factor selected from thrombophilia, an endothelial injury, surgery, malignancy, cardiovascular disease, hypertension, obesity, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, nephritic syndrome, renal disease, prior venous thromboembolism (VTE), type 1 or type 2 diabetes, heparin-induced thrombocytopenia, paroxysmal nocturnal hemoglobinuria, microcythemia, von Willebrand disease, schizophrenia, hyperthyroidism, cystic fibrosis, a microbial infection, asthma, chronic bronchitis, chronic obstructive pulmonary disease, oral

contraceptive use, hormone replacement therapy, chemotherapy, and radiation therapy.

40. The method according to claim 38, wherein the risk factor is a microbial infection selected from Epstein-Barr Virus infection, a chronic bacterial infection, a chronic viral infection, gingivitis or periodontitis, a vaginal microbial infection, and a urogenital microbial infection.

41. The method according to claim 38, wherein the risk factor is thrombophilia.

42. The method according to claim 41, wherein the thrombophilia is (a) hereditary thrombophilia; (b) acquired thrombophilia; or (c) hereditary and acquired thrombophilia.

43. The method according to claim 38, wherein the subject is pregnant or desirous of becoming pregnant or postpartum.

44. The method according to claim 38, wherein the subject also has hypoferremia or iron deficiency anemia.

45. The method according to any one of claims 38-44, further comprising administering to the subject a nutritional mixture formulated to supplement the diet of the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

46. The method according to claim 45, wherein the nutritional mixture comprises (a) at least folic acid and Vitamin B6 or (b) at least folic acid, Vitamin B6, and Vitamin B 12.

47. The method according to claim 46, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

48. The method according to claim 45, 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.

49. The method according to claim 45, wherein the nutritional mixture is formulated for a subject who is pregnant or who is desirous of becoming pregnant or who is postpartum.

50. The method according to claim 38, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

51. The method according to claim 45, wherein the lactoferrin is administered separately or together with the nutritional mixture and by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

52. The method according to claim 38, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

53. A method for preventing or treating colic in an infant, said method comprising administering to the infant (a) breast milk from the birth mother of the infant, wherein an isolated lactoferrin is administered to the mother (i) prenatally, (ii) postpartum, or (iii) prenatally and postpartum; or (b) a combination of the breast milk of (a) and an infant formula comprising the lactoferrin.

54. The method according to claim 53, wherein the lactoferrin is administered to the mother prenatally and postpartum.

55. The method of either claim 53 or claim 54, further comprising administration of a prenatal nutritional mixture to the mother wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

56. The method of claim 55, wherein the prenatal nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12.

57. The method according to claim 56, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

58. The method according to claim 55, 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.

59. A method for preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject, said method comprising administering an isolated lactoferrin to the postpartum subject.

60. A method for preventing or treating a postpartum-related psychological condition in a postpartum subject, said method comprising administering an isolated lactoferrin to the subject.

61. The method according to clam 60, wherein the psychological condition comprises postpartum depression.

62. The method according to clam 60, wherein the psychological condition comprises postpartum psychosis.

63. The method of according to any one of claims 59-62, further comprising administering a prenatal nutritional mixture to the subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the mixture.

64. The method of claim 63, wherein the prenatal nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12.

65. The method according to claim 64, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.

66. The method according to claim 63, 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.

67. The method according to claim 59 or claim 60, wherein the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

68. The method according to claim 63, wherein the lactoferrin is administered separately or together with the nutritional mixture by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

69. The method according to claim 59 or claim 60, wherein the lactoferrin is administered (a) orally and (b) by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.

70. The method according to any one of claims 8, 30, 45, 55, and 63, wherein the nutritional mixture is formulated with the lactoferrin to form a preparation.

71. A method 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, comprising administering to the infant or child a preparation that comprises (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a nutritional mixture formulated to supplement the diet of the infant or child, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation.

72. The method according to claim 71, wherein the nutritional composition comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12.

73. The method according to claim 72, wherein the preparation is formulated as an infant formula further comprising at least one additional active nutritional ingredient.

74. A method for reducing the likelihood of occurrence of gestational diabetes in a pregnant subject, comprising administering a preparation, comprising (a) a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient; and (b) a second composition that comprises a prenatal nutritional mixture formulated to supplement the diet of the pregnant subject, wherein an inorganic source of a biologically effective amount of iron is excluded from the preparation.

75. The method according to claim 74, wherein the nutritional mixture comprises (i) at least folic acid and Vitamin B6 or (ii) at least folic acid, Vitamin B6, and Vitamin B 12.

76. The method according to any one of claims 71-75, wherein the nutritional mixture further comprises at least one or more minerals selected from calcium, omium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, assium, phosphorous, selenium, sodium, and zinc.

77. The method according to any one of claims 71-75, wherein the ritional mixture comprises (a) folic acid, Vitamin B6, and Vitamin B12; (b) one or more imins selected from biotin, thiamine, riboflavin, niacin, Vitamin B5, Vitamin A, Vitamin Vitamin D, and Vitamin E; and (c) one or more minerals selected from calcium, omium, copper, magnesium, manganese, molybdenum, selenium, and zinc.

78. The method according to any one of claims 1, 12, 23, 38, 53, 59, 60, and 74, wherein the lactoferrin is human lactoferrin or bovine lactoferrin.

79. The method according to claim 78, wherein the lactoferrin is ombinant human lactoferrin or recombinant bovine lactoferrin.

80. The method according to claim 78 or claim 79, wherein the lactoferrin L polypeptide fragment of lactoferrin, and wherein the polypeptide fragment is either lobe )r lobe C of the lactoferrin.

81. The method according to claims 78-80, wherein the lactoferrin has any cent of saturation with one or more of iron(III), zinc, copper, and manganese.

82. The method according to claim 81, wherein the lactoferrin has 10-30% uration with iron(III).

83. The method according to claim 81 , wherein the lactoferrin is

)lactoferrin.