WO2011150393A2 - Méthodes d'amélioration de la santé maternelle et fœtale - Google Patents

Méthodes d'amélioration de la santé maternelle et fœtale Download PDF

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WO2011150393A2
WO2011150393A2 PCT/US2011/038438 US2011038438W WO2011150393A2 WO 2011150393 A2 WO2011150393 A2 WO 2011150393A2 US 2011038438 W US2011038438 W US 2011038438W WO 2011150393 A2 WO2011150393 A2 WO 2011150393A2
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Prior art keywords
vitamin
lactoferrin
subject
iron
nutritional
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PCT/US2011/038438
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WO2011150393A9 (fr
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Rosalba Paesano
Jeffrey L. Lillard
William A. Goolsbee
Piera Valenti
Miriam Pietropaoli
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Bmg Hematology Llc
Microbo S.R.L.
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Publication of WO2011150393A2 publication Critical patent/WO2011150393A2/fr
Publication of WO2011150393A9 publication Critical patent/WO2011150393A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the text file is 80 KB, was created on May 27, 2011 and is being submitted electronically via EFS-Web.
  • 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.
  • VTE thromboembolism
  • DVT deep venous thrombosis
  • PE pulmonary embolism
  • VTE thromboembolism
  • VT deep venous thrombosis
  • PE pulmonary embolism
  • VTE thromboembolism
  • VT deep venous thrombosis
  • PE pulmonary embolism
  • VTE thromboembolism
  • a pulmonary embolism occurs when a thrombus or a portion of the thrombus detaches from a vein wall and lodges within a pulmonary artery.
  • 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.
  • 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
  • thrombophilia which may be hereditary thrombophilia, acquired thrombophilia, or resulting from both hereditary and acquired thrombophilia.
  • 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)).
  • hypoferremia and IDA represent a risk factor for maternal and infant health.
  • 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)).
  • pregnancy-associated anemia results in preterm delivery, retardation of fetal growth, low birth weight, and inferior neonatal health.
  • Exemplary drugs include those that suppress platelet aggregation (anti-platelet therapeutics), for example, aspirin, ticlopidine, eicosapentaenoic acid (EPA), dipyridamole, and dilazep hydrochloride.
  • anti-platelet therapeutics for example, aspirin, ticlopidine, eicosapentaenoic acid (EPA), dipyridamole, and dilazep hydrochloride.
  • an antiplatelet therapeutic such aspirin suppresses formation of thrombus at the impaired site of the blood vessel by
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • additional active nutritional ingredients selected from (a) at least one mineral selected from calcium, chromium, chloride, copper,
  • 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 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.
  • risk factor selected from infertility, thrombophilia, recurrent miscarriage, Crohn's disease, ulcerative colitis, type 1 or type 2 diabetes, hypertension, renal disease, microcyth
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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, micro
  • 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.
  • 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-
  • 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-
  • 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.
  • thrombophilia an endothelial injury, surgery, malignancy,
  • 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.
  • 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-
  • Embodiment 45 The method according to any one of Embodiments 38-
  • 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.
  • 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-
  • 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-
  • 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-
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • provided herein is a use of isolated lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a postpartum subject.
  • provided herein is a use of isolated lactoferrin for preventing or treating a postpartum-related psychological condition in a postpartum subject.
  • an isolated lactoferrin for use in preventing or treating a postpartum-related psychological condition in a postpartum 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 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.
  • a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient
  • 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.
  • 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.
  • 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.
  • a first composition that comprises an isolated lactoferrin and a physiologically suitable excipient
  • 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.
  • a preparation in still another embodiment, 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.
  • 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, wherein the method comprises administering a lactoferrin to the subject, thereby reducing the likelihood that the pregnancy-associated complication will occur.
  • the subject also has hypoferremia or iron deficiency anemia.
  • the thrombophilia is hereditary thrombophilia.
  • 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.
  • the lactoferrin is administered orally.
  • the lactoferrin is administered by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • 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.
  • 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.
  • the prenatal nutritional mixture further comprises Vitamin B 12.
  • 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.
  • the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation.
  • 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.
  • 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.
  • the composition comprising the prenatal nutritional mixture is formulated with the lactoferrin to form a preparation (or medicament).
  • 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 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.
  • 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
  • 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.
  • 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 Will
  • 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.
  • 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.
  • 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
  • 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.
  • the first composition and the second composition are each formulated separately for oral administration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the risk factor is at least one prior occurrence of miscarriage or intrauterine fetal death.
  • 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.
  • 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.
  • 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.
  • the nutritional mixture further comprises Vitamin B 12.
  • 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.
  • the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • the lactoferrin is administered orally.
  • the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • the composition comprising a prenatal nutritional mixture is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal.
  • a lactoferrin 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.
  • 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).
  • 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 a lactoferrin to the subject, thereby reducing the likelihood that the thrombotic event will occur.
  • 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
  • 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.
  • the risk factor is thrombophilia.
  • thrombophilia is hereditary thrombophilia.
  • the subject is pregnant or postpartum.
  • the subject also has hypoferremia or iron deficiency anemia.
  • 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.
  • 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.
  • the lactoferrin is administered by at least one route selected from oral, parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • parenteral lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • composition is also administered, the composition is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal.
  • a lactoferrin for reducing the likelihood of occurrence of a thrombotic event in a subject who is at risk of a thrombotic event occurring.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprising a nutritional mixture is formulated with the lactoferrin to form a preparation (or a medicament).
  • a method for preventing or treating colic in a neonate or infant 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.
  • the mother receives lactoferrin prenatally and postpartum.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a use 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.
  • a method for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum, said method comprising administering a lactoferrin to the subject.
  • 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.
  • 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.
  • 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
  • the lactoferrin is administered orally and by at least one route selected from parenteral, lingual, transdermal, vaginal, rectal, intramuscular, subcutaneous, buccal, intranasal, and topical.
  • the composition when the composition is administered, the composition is administered by a route selected from oral, parenteral, lingual, transdermal, intramuscular, subcutaneous, buccal, and intranasal.
  • a lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum.
  • a use for a lactoferrin for preventing or treating hypoferremia or iron deficiency anemia in a subject postpartum is provided.
  • 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.
  • 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.
  • 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.
  • 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 is formulated with the lactoferrin to form a preparation (or a medicament).
  • a method for preventing or treating a postpartum related psychological condition in a postpartum subject, said method comprising administering a lactoferrin to the subject.
  • the postpartum related psychological condition comprises postpartum depression
  • the postpartum psychological condition comprises postpartum psychosis.
  • 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.
  • 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.
  • 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.
  • 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,
  • a lactoferrin for preventing or treating postpartum depression in a postpartum subject.
  • a use for a lactoferrin for preventing or treating postpartum depression in a postpartum subject is provided.
  • a use for a lactoferrin in the manufacture of a medicament for preventing or treating postpartum depression in a postpartum subject 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.
  • 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.
  • 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 is formulated with the lactoferrin to form a preparation (or a medicament).
  • 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 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.
  • 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.
  • 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.
  • a preparation 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.
  • the lactoferrin is human lactoferrin or bovine lactoferrin.
  • the lactoferrin is recombinant human lactoferrin or recombinant bovine lactoferrin.
  • 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.
  • 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.
  • a composition includes at least one composition, one or more compositions, or a plurality of compositions, respectively.
  • 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.
  • 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.
  • 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.
  • 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.
  • HT hereditary thrombophilia
  • ID iron deficiency
  • IDA iron deficiency anemia
  • 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.
  • 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 thromboembolic disease
  • VTE thromboembolism
  • 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 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
  • 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)).
  • 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.
  • hypoferremia also referred to herein and in the art as iron deficiency in the absence of anemia (ID)
  • IDA iron deficiency anemia
  • 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).
  • IL interleukin
  • TNF tumor necrosis factor
  • 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).
  • lactoferrin 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.
  • a thrombotic event e.g., VTE, including deep venous thrombosis and pulmonary embolism, or an arterial thrombosis
  • iron deficiency without anemia also called hypoferremia herein and in the art
  • iron deficiency anemia IDA
  • hypoferremia total serum iron levels decrease but hemoglobin levels remain normal.
  • IDA 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, 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.
  • 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.
  • treatment of hypoferremia, IDA, and chronic anemia typically comprises administering large quantities of iron from an inorganic source, for example, ferrous sulfate.
  • 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)).
  • 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).
  • proinflammatory mediators such as inflammatory cytokines (e.g., IL-6)
  • Iron toxicity has been considered a risk factor for a variety of different conditions in non-pregnant individuals and in pregnant women (see, e.g., Weinberg, Oxidative Medicine and Cellular Longevity 2(2) : 107- 109 (2009)) .
  • Toxic effects of iron on mother and fetus relate, at least in part, to catalysis of free radical formation and oxidative stress (see, e.g., Weinberg, Metallomics 2(11):732- 40 (2010); Epub 2010 Sep 24; Favier et al, supra).
  • 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.
  • 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)).
  • bovine lactoferrin bLf
  • 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.
  • 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
  • postpartum-related psychological conditions e.g., postpartum mild depression, postpartum depression, and postpartum psychosis.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • a stool softener or anti-nausea agent or other ingredient/agent generally regarded as safe (GRAS) e.g., a stool softener or anti-nausea agent or other ingredient/agent generally regarded as safe (GRAS)
  • infant formulas may 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.
  • 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 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.
  • lactoferrin may reduce inflammation by reducing and/or maintaining the production of proinflammatory factors such as IL- ⁇ , 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.
  • lactoferrin 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.,
  • 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.
  • 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.
  • bovine lactoferrin 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.
  • 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.
  • 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 (CO3 2 ), requisite to stabilize ferric ion binding.
  • Fe 3 ions also referred to as ferric ions, iron III, or Felll
  • CO3 2 carbonate ions
  • 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;
  • 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
  • a lactoferrin such as bLf
  • 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).
  • 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- ⁇ , IL-8, and TNF- ⁇ ) in uncomplicated pregnancies (see, e.g., Paesano et al, Biometals, supra;
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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).
  • amino acid sequences of mature bovine lactoferrin i.e., without the 19-amino acid signal peptide
  • SEQ ID NOS:4-7 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.
  • 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).
  • 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)).
  • lactoferrin 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 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.
  • 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.
  • 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.
  • “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.
  • a truncated lactoferrin retains at least one iron binding site.
  • methods for using compositions that comprise a polypeptide that comprises a fragment of lactoferrin as described herein are also provided 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).
  • 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
  • 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.
  • 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.
  • regions of lactoferrin that exhibit particular activities e.g., the N-terminal region comprising anti-microbial activity
  • 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
  • lactoferrin variants and fragments having the desired biological activities can be made readily and without undue
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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 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.
  • a thrombotic event such as thrombosis
  • 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.
  • 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.
  • 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.
  • VTE venous thromboembolism
  • VTD venous thromboembolic disease
  • 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.
  • 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.
  • 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.
  • VTE deep venous thrombosis
  • PE pulmonary embolism
  • 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
  • DVT diaphoresis
  • 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.
  • 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.
  • 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)).
  • ELISA enzyme-linked immunosorbant assays
  • 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.
  • duplex ultrasonography see, e.g., Lensing et al, N. Engl. J. Med. 320:342-45 (1989)
  • contrast venography impedance plethysmography
  • MMRV magnetic resonance venograph
  • CTV computed tomography venography
  • CTA combined CTV-CT angiography
  • 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.
  • a person skilled in the medical and clinical art may also use one or any combination of diagnostic methods, including physical examination, clinical symptoms, and analytical tests and methods described above, for monitoring the health status of the subject and for monitoring whether the likelihood of a thrombotic event occurring is increasing, decreasing, or remains unchanged.
  • a method 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).
  • the subject has thrombophilia as a risk factor.
  • thrombophilia also called hypercoagulability
  • 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.
  • 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.
  • anti-phospholipid antibodies e.g., a lupus anticoagulant and anticardiolipin antibodies
  • factor VIII levels elevated factor VIII levels.
  • anti-phospholipid antibodies, heparin- induced thrombocytopenia, and paroxysmal nocturnal hemoglobinuria are types of acquired thrombophilia that are suggested to pose a high risk for thrombosis. Elevated levels of homocysteine due to MTHFR mutation or vitamin deficiency (e.g., Vitamin B6, B12, and folic acid) also considered risk factors for thrombosis.
  • Analytical techniques and tests are available in the art for determining thrombophilia, including genetic testing to determine if a subject has inherited thrombophilia and which gene (or genes) is affected.
  • pregnancy and/or the postpartum period is a risk factor that increases the risk of a thrombotic event occurring in a subject.
  • 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).
  • TAT thrombin-antithrombin
  • VTE 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.
  • 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.
  • COPD chronic obstructive pulmonary disease
  • 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.
  • COPD chronic obstructive pulmonary disease
  • 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)).
  • 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).
  • Epstein-Barr Virus infection e.g., Epstein-Barr Virus infection, a chronic bacterial infection, a chronic viral infection (e.g., hepatitis B, hepatitis C, human immunodeficiency virus (HIV) and AIDS), ging
  • 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)).
  • therapies that treat renal disease, or treat conditions associated with renal disease 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • lactoferrin 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).
  • 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.
  • a method 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).
  • a lactoferrin i.e., an isolated lactoferrin
  • a composition comprising the lactoferrin to the subject (i.e., female subject or patient).
  • a method for reducing the likelihood of occurrence of a pregnancy-associated complication in a subject 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.
  • 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.
  • 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).
  • infertility 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 receive
  • 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 may also be risk factors that predispose a pregnant subject to the occurrence of a pregnancy-associated complication.
  • a subject who is receiving an inorganic source of iron e.g., via ferrous sulfate, ferrous fumarate or other iron salts
  • 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.
  • lactoferrin or preparations and compositions comprising a lactoferrin 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- ⁇ , 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.
  • 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.
  • the methods described herein for reducing the likelihood of occurrence of a pregnancy-associated complication comprise
  • 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.
  • 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)).
  • hereditary thrombophilia 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)).
  • 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.
  • increased levels of inflammatory cytokines, including IL-6 present additional maternal and fetal/neonate or infant risks during pregnancy.
  • pregnancy-associated complication which may also be called a pregnancy-associated pathology
  • 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.
  • 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.
  • a method for reducing the likelihood of occurrence of a miscarriage or intrauterine fetal death by administering a lactoferrin to a subject 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.
  • 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).
  • the subject may receive lactoferrin during at least the first trimester, which is the time during which most miscarriages occur.
  • lactoferrin may be administered to the subject during the first two trimesters or during the entire pregnancy.
  • fetal death refers in the medical art and vary among different countries and can vary within a country.
  • state governments within the United States vary in the definitions related to fetal death.
  • the loss of a fetus at any stage is a fetal demise.
  • fetal losses may be classified according to gestational stage.
  • a spontaneous abortion when not an induced termination of pregnancy
  • 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).
  • 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.
  • Placental abruption 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.
  • Intrauterine growth restriction 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.
  • symmetrically impaired or asymmetrically impaired 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.
  • 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.
  • SGA gestational age
  • 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.
  • 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.
  • a lactoferrin which may be combined with a prenatal nutritional mixture as described herein
  • 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.
  • 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
  • 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.
  • 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 e.g., multiple births, poor nutrition, smoking, and alcohol or drug use.
  • a method 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
  • 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).
  • hypoferremia and IDA represent risk factors that can adversely affect maternal and/or infant health.
  • 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.
  • lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron may be administered prior to conception, during pregnancy, and during the postpartum period.
  • 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.
  • 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.
  • methods 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.
  • administering 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.
  • 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 may reduce miscarriage, spontaneous abortion (including recurrent spontaneous abortion), or preterm delivery, in a statistically, biologically, or clinically significant manner.
  • 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.
  • lactoferrin alone or lactoferrin in combination with a nutritional mixture and excluding an inorganic source of iron may be administered to the subject during the first two trimesters or during the entire pregnancy.
  • hypoferremia and IDA represent risk factors that can adversely affect maternal and/or infant health (see, e.g., Siega-Riz et al., supra).
  • 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)).
  • 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.
  • 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).
  • TfR-1 transferrin receptors
  • 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.
  • IDA iron deficiency anemia
  • IDA may also be characterized by low serum transferrin saturation, elevated serum transferrin, low hematocrit, and hypochromic, microcytic red blood cells.
  • hepcidin and ferroportin 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)).
  • Hepcidin 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)).
  • hepcidin 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.
  • 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).
  • 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)).
  • 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)).
  • anemia and hypoxia see, e.g., Nicolas et al, J. Clin. Invest. 110: 1037- 1044 ((2002)).
  • IL-6 upregulates the hepcidin gene in hepatocytes (see, e.g., Nemethet al, J. Clin. Invest. 113: 1271-1276 (2004)).
  • IL- 6-induced hepcidin 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)).
  • 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).
  • 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
  • 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.
  • the neonate/infant also receives an infant formula that further comprises the lactoferrin.
  • Components (i.e., ingredients) of infant formulas are well known and described in the art and are sold commercially.
  • 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.
  • the neonate/infant receives a combination of the breast milk from the birth mother and an 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 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).
  • a postpartum subject e.g., a thrombotic event, postpartum-related psychological conditions, e.g., postpartum mild depression (i.e., postpartum "blues"), postpartum depression and/
  • 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.
  • maternity blues or "postpartum blues”
  • postpartum blues 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 develop postpartum psychosis, such as severe manic-depression, which often requires hospitalization. See, for example, (see, e.g., Weiss, Best Pract. Res. Clin. Haematol.
  • 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 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.
  • 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).
  • 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.
  • lactoferrin or the preparation comprising lactoferrin and the nutritional mixture may be performed as described in greater detail herein.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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).
  • compositions and preparations comprising a lactoferrin and nutritional mixture lack (i.e., do not include; exclude) an inorganic source of a biologically (or
  • the subject is female and is postpartum.
  • 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
  • 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).
  • ID iron deficiency
  • IDA iron deficiency anemia
  • 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.
  • 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.
  • infant formula and nutrient mixtures liquid or solid
  • formulations may depend, at least in part, on the age and health status of the infant or child.
  • lactoferrin 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.
  • 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.
  • 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.
  • 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.
  • the preparations described above and herein may further comprise at least one (i.e., one or more) other dietary (i.e., nutritional) ingredient and/or at least one (i.e., one or more) non-dietary ingredient (e.g., a stool softener or anti-nausea agent or other ingredient/agent generally regarded as safe (GRAS)).
  • other dietary (i.e., nutritional) ingredient e.g., one or more) non-dietary ingredient
  • non-dietary ingredient e.g., a stool softener or anti-nausea agent or other ingredient/agent generally regarded as safe (GRAS)
  • Anemia generally, is characterized by an insufficient number of healthy red blood cells to carry adequate oxygen to the body's tissues.
  • iron deficiency anemia typically, the red blood cells are smaller than normal; IDA is, therefore, an example of microcytic anemia.
  • chronic anemia including anemia related to kidney disease, the red blood cell size is normal (normocytic anemia).
  • pernicious anemia related to vitamin B 12 deficiency
  • administering 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.
  • 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.
  • hematological parameters e.g., red blood cell count and/or morphology, hemoglobin, total serum iron, serum ferritin, hematocrit
  • lactoferrin to improve or restore hematological status includes the capability to improve, increase, or restore the level of a reduced
  • hematological parameter in a statistically, clinically, or biologically significant manner to a level that is within range of the level of the hematological parameter in the absence of hypoferremia or anemia (including iron deficiency anemia).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (%).
  • 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.
  • 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.
  • the level of one or more hematological parameters is below the range desired for healthy, non-anemic and non-hypoferremic individuals.
  • 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.
  • 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- ⁇ , soluble mediators such as cytokines (e.g., IL-6, IL- ⁇ , leukemia inhibitory factor, TNF-a, IFN- ⁇ , 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.
  • cytokines e.g., IL-6, IL- ⁇ , leukemia inhibitory factor, TNF-a, IFN- ⁇ , IL-2, IL-4, IL-10, IL-12
  • lymphokines e.g., hepcidin and prohepcidin
  • carbohydrate e.g.,
  • 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
  • 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.
  • body fluids e.g., lung lavage, ascites, mucosal washings, synovial fluid
  • body fluids e.g., lung lavage, ascites, mucosal washings, synovial fluid
  • bone marrow e.g., 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.
  • 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.
  • a primary cell culture ⁇ e.g., immune cells
  • 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.
  • 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
  • a composition 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • GRAS hydroxy-3-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)
  • compositions and preparations 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.
  • 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).
  • 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.
  • 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 thrombo
  • 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.
  • 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.
  • 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.
  • 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.,
  • 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.
  • 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.
  • the dose or dosing regimen of lactoferrin administered to an infant 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.
  • colic i.e., delay onset, reduce or delay severity and/or duration of a colic episode
  • hypoferremia and/or IDA i.e., delay onset, reduce or delay severity and/or duration of a hypoferremia and/or IDA
  • 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.
  • 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).
  • compositions described herein that comprise a lactoferrin may also inhibit, decrease, or reduce, production of at least one proinflammatory cytokine (e.g., IL- ⁇ , IL-6, IL-8, and TNF-a) or other inflammatory mediator and thereby reduce, abrogate, or ameliorate inflammation (or an inflammatory state).
  • proinflammatory cytokine e.g., IL- ⁇ , IL-6, IL-8, and TNF-a
  • other inflammatory mediator e.g., IL- ⁇ , IL-6, IL-8, and TNF-a
  • 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
  • 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.
  • lactoferrin described herein also may be produced recombinantly according to methods routinely practiced in the molecular biology, protein expression, and protein isolation arts.
  • 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.
  • 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 prevent, reduce the likelihood of occurrence or likelihood of worsening of, ameliorate, or mitigate inflammation and/or the
  • a preparation 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • the preparations are formulated appropriately for a subject who is pregnant (or desiring to become pregnant), or postpartum, or lactating.
  • DRI Dietary Reference Intake
  • RDA Recommended Dietary Allowance
  • AI Adequate Intake
  • UL Tolerable Upper Intake Level
  • 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.
  • 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.
  • certain health organizations have increased the UL of Vitamin D.
  • 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.
  • 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.
  • 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.
  • a subject who is at risk of a thrombotic event occurring and who is receiving warfarin must carefully regulate vitamin K intake, a vitamin that is involved in clot formation.
  • warfarin e.g., COUMADIN®
  • vitamin E has blood-thinning effects and may increase risk of excess bleeding.
  • 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.
  • the preparation and nutritional mixtures may comprise at least one of calcium, chromium, copper, iodine (iodide), magnesium, manganese, molybdenum, selenium, and zinc.
  • 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.
  • 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.
  • the isolated lactoferrin and the nutritional mixture are combined or mixed together (i.e., formulated) to provide the preparation.
  • 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.
  • 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.
  • the composition comprising the isolated lactoferrin (which can be referred to as a first composition) and the
  • compositions comprising the nutritional mixture are formulated separately and administered to the subject separately.
  • One composition may be administered prior to, concurrent with, or subsequent to
  • 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).
  • 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.
  • 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.
  • active nutritional ingredients also include enzymes, for example, amylases, proteases, various pancreatic and digestive enzymes, lipase, among others.
  • 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.
  • 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.
  • DHA docosahexaenoic acid
  • omega-3 omega-3 fatty acid
  • EPA eicosapentaenoic acid
  • ALA a- linolenic 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
  • a prenatal preparation comprising Lf and a nutritional mixture 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.
  • 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 ⁇ g folic acid
  • the RDA for a non-pregnant adult is 400 ⁇ g (see, e.g., Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes:
  • folic acid 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 ⁇ g. 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 ⁇ g folic acid per day. Compositions comprising greater than 1.0 mg folic acid are typically obtained by prescription.
  • 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 ⁇ g).
  • Vitamin supplements for non-pregnant adults males and females greater than 19 years of age
  • teenagers from about 14-18 years of age typically include approximately 400 ⁇ g folate.
  • the folic acid ingredient is included in the composition in an amount sufficient to provide approximately 400 ⁇ g folate or between about 300-500 ⁇ g per day to the subject.
  • 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 ⁇ g (i.e., 1.5 times the RDA).
  • folic acid is present in an amount sufficient to provide a daily value of about between 600-980 ⁇ g, 600-650 ⁇ g, 650-700 ⁇ g, 700-750 ⁇ g, 750-800 ⁇ g, 800-850 ⁇ g, 850-900 ⁇ g, 900-950 ⁇ g, 900-975 ⁇ g, 950-975 ⁇ g, 975-980 ⁇ g, 980-990 ⁇ g, or 975-995 ⁇ g.
  • folic acid is present in an amount sufficient to provide a daily value of about 600 ⁇ g, 650 ⁇ g, 700 ⁇ g, 750 ⁇ g, 800 ⁇ g, 850 ⁇ g, 900 ⁇ g, 950 ⁇ g, 960 ⁇ g, 980 ⁇ g, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, or 4.0 mg.
  • the nutritional mixture may be either a nutritional mixture formulated for a non-pregnant adult (i.e., to provide about 400 ⁇ g folic acid per day or about between 300-500 ⁇ g 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 ⁇ g folic acid, which is 1.5 times the recommended RDA for a non-pregnant adult).
  • the nutritional mixture is formulated for use by an infant or child.
  • 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 ⁇ g per day and for infants between about 7 to 12 months is about 80 ⁇ g per day.
  • the RDA for children between 1 and 3 years of age is about 150 ⁇ g per day, for children 4-8 years old is about 200 ⁇ g per day, and for children 9-13 years of age is about 300 ⁇ g per day.
  • each vitamin (or source thereof) that may be formulated in the nutritional mixture can be readily determined by persons skilled in the art.
  • 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.
  • 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 ⁇ g, 2.0-2.5 ⁇ g, 2.5-3 ⁇ g, 3 -5 ⁇ g, 5-10 ⁇ g, 10- 20 ⁇ g, or 20-30, 30-40, 40-50, 50-75, 75-100, 100-200, 200-400, 400-600, 600-800, 300-1200, 1200-1500 ⁇ , 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.
  • 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 ⁇ g per day.
  • 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.
  • the nutrient mixture when the subject is in need thereof, may be formulated to provide significantly greater amounts of vitamins B6 and B12 than RDAs and than typically present in a dietary supplement.
  • a subject may receive such a nutrient mixture and lactoferrin when the subject has thrombophilia (including hereditary thrombophilia).
  • 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.
  • the nutrient mixture may comprise folic acid formulated to provide approximately 400 ⁇ g 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.
  • the nutrient mixture comprises at least biotin, and in other embodiments, comprises at least biotin, Vitamin B6, folic acid, and Vitamin B 12.
  • 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)).
  • nutritional mixtures comprising biotin are formulated to provide a daily amount of about 30 ⁇ per day, such as for a non-pregnant adult.
  • biotin may be included in an amount to provide between about 30 ⁇ g to about 300 ⁇ g per day.
  • biotin may be included in the nutritional mixture to provide a level of biotin between about 50 ⁇ g to about 300 ⁇ g per day, about 100 ⁇ g to about 300 ⁇ g per day, about 125 ⁇ g to about 300 ⁇ g per day, about 150 ⁇ g to about 300 ⁇ g per day, about 175 ⁇ g to about 300 ⁇ g per day, about 200 ⁇ g to about 300 ⁇ g per day, about 225 ⁇ g to about 300 ⁇ g per day, about 250 ⁇ g to about 300 ⁇ g per day, about 275 ⁇ g to about 300 ⁇ g per day, or about 290-310 ⁇ g 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 ⁇ g per day.
  • biotin may be included at a level to provide between about 1.5 to about 8 ⁇ g per day.
  • a nutrient mixture formulated for infants from 0 to about 6 months comprises biotin in an amount to provide about 5 ⁇ g per day and for infants from about 7 to about 12 months, the nutritional formula may provide about 6 ⁇ g per day.
  • Nutritional mixtures formulated for use by children may include biotin at a level to provide about between 8 to 30 ⁇ g per day.
  • a nutritional mixture for administration to children from about 1-3 years may be formulated to provide biotin at about 7-9 ⁇ g per day (e.g., about 8 ⁇ g per day).
  • a nutritional mixture for children from about 4-8 years may be formulated to provide biotin at about 10-14 ⁇ g per day (e.g., about 12 ⁇ g per day); for children about 9-13 years, the nutritional mixture may be formulated to provide about 15-25 ⁇ g biotin per day (e.g., about 18, 20, or 22 ⁇ g per day); and for children of about 14-18 years, the nutritional mixture maybe formulated to provide about 20-30 ⁇ g per day of biotin (e.g., about 20, 22, 25, 26, 28, or 30 ⁇ g biotin per day).
  • Vitamin D also called calciferol
  • Vitamin D is an essential vitamin required for bone health, including health and development of fetal bone.
  • 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)).
  • IU International Units
  • 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.
  • 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.
  • 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.
  • an inorganic source of a biologically (or pharmacologically) effective amount of iron is excluded from the preparation.
  • the nutritional mixture comprises one or more of calcium, chromium, copper, iodine (iodide), magnesium, manganese, molybdenum, selenium, and zinc.
  • the prenatal preparation includes the mineral, calcium.
  • 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.
  • 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
  • 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).
  • GRAS a regulatory agency
  • non-dietary ⁇ i.e., non-nutritional) ingredients are excluded from the preparations and compositions described herein.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a nutrient mixture comprises about between 2-25 or 9-11 mg vitamin B6 and 400-980 or 600-980 ⁇ g folic acid.
  • the mixture 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 ⁇ g folic acid.
  • the nutrient mixture further comprises 2-15 ⁇ g vitamin B 12.
  • 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 ⁇ g folic acid and which may further comprise between 2-15 ⁇ g vitamin B12, may also further comprise between about 125-300 mg calcium.
  • vitamin B6 and folic acid are between about 2-25 mg vitamin B6 and 600-980 ⁇ g folic acid, respectively.
  • 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 ⁇ g per day.
  • these nutrient compositions may further comprise a source of Vitamin D sufficient to provide about 2000 IU per day.
  • 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.
  • the preparation and/or nutritional mixture is in the form of an infant formula and further comprises the additional vitamins, A, C, D, E, K, thiamin (Bi), biotin, and riboflavin (B 2 ).
  • the infant formulation preparation may also comprise one or more minerals, for example, but not limited to calcium, chromium, chloride, copper, iodide, fluoride, magnesium, manganese, molybdenum, potassium, phosphorous, selenium, sodium, and zinc.
  • 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.
  • 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
  • nucleotides e.g., lactose in a milk based
  • 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).
  • 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.
  • 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.
  • 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).
  • an amount of inorganic iron that exhibits or has a detectable biological activity or effect 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).
  • inflammatory immune modulators including for example, inflammatory cytokines, C-reactive protein
  • hematological parameters
  • 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 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)).
  • ICP-MS inductively coupled plasma - mass spectrometry
  • the methods described herein for reducing the likelihood of occurrence of a pregnancy-associated complication comprise administering to the subject a composition comprising an isolated lactoferrin and a physiologically suitable (i.e., pharmaceutically acceptable) excipient.
  • 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.
  • the preparation is formulated to supplement the diet of a pregnant subject.
  • the nutritional mixture may comprise an amount of folic acid sufficient to provide at least 1.5 times greater (i.e., at least 600 ⁇ g) than the daily recommended amount for a non-pregnant adult.
  • 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.
  • compositions 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • lactoferrin delivered per dose and per day to an infant or child can be determined readily by a person skilled in the art.
  • 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.
  • methods that comprise administering 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
  • lactoferrin 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).
  • 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.
  • 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).
  • a second route any one of, for example, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal
  • 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.
  • 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).
  • 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.).
  • the subject is a human.
  • the subject is a pregnant human or is a human desiring to become pregnant (and thus is at risk of becoming pregnant).
  • 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.
  • saline and phosphate buffered saline at physiological pH may be used.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the composition.
  • sodium benzoate, sorbic acid and esters of /?-hydroxybenzoic acid may be added as
  • An injectable pharmaceutical composition is preferably sterile.
  • 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.
  • lactoferrin is delivered orally.
  • lactoferrin 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).
  • 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.
  • 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).
  • a second route any one of, for example, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal
  • 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.
  • administration of lactoferrin refers to administration of one or more doses that comprise a course of lactoferrin therapy (
  • the preparations described herein may be formulated for oral, parenteral, lingual, buccal, intranasal, transdermal, intramuscular, or subcutaneous administration.
  • the preparations and compositions are formulated for oral administration.
  • 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.
  • the lactoferrin or composition comprising the lactoferrin
  • the lactoferrin or composition comprising the lactoferrin is formulated for oral administration.
  • the composition comprising the nutrient mixture is formulated for oral, lingual, buccal, intramuscular, subcutaneous, transdermal, intranasal, or parenteral administration.
  • the nutrient mixture, or composition comprising the nutrient mixture is formulated for oral administration.
  • 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.
  • lyophilate i.e., a lyophilized composition or preparation, e.g., lyophilized infant formula
  • one composition may be administered prior to, concurrent with, or subsequent to administration of the second composition (also referred to as coordinate
  • 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.
  • the composition comprising the lactoferrin may be administered twice a day
  • the composition comprising the nutritional mixture may be administered once a day, providing the same daily dose of the preparation.
  • 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.
  • lactoferrin 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).
  • 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.
  • 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).
  • a second route any one of, for example, topical, parenteral, lingual, buccal, rectal, vaginal, transdermal, subcutaneous, intramuscular, and intranasal
  • 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.
  • administration of lactoferrin refer
  • the preparation 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
  • 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).
  • 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.
  • administration of the preparation refers to administration of one or more doses that comprise a course of therapy (i.e., therapeutic regimen).
  • 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.
  • 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).
  • 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.
  • 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).
  • administration of the nutritional mixture refers to administration of one or more doses that comprise a course of therapy ⁇ i.e., therapeutic regimen).
  • each may be administered by the same route or by different routes as described above and herein.
  • a lactoferrin (or composition comprising lactoferrin) may be
  • 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).
  • parenteral 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 are intracavernous, intrathecal, intrameatal, intraurethral injection, or infusion techniques.
  • 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).
  • a lactoferrin (and compositions and preparations comprising lactoferrin), is delivered in a manner and in a form that avoids proteolytic digestion by gastric enzymes.
  • 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.
  • 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.
  • 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.
  • 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.
  • FDA U.S. Food and Drug Administration
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • regulatory agencies health agencies, or medical organizations
  • recommendations provided by regulatory agencies, health agencies, or medical organizations with respect to content of nutrients to be included in a nutrient mixture.
  • the American Academy of Pediatrics Committee on Nutrition has published recommendations for nutrient content and recommended amounts of each nutrient in infant formulas.
  • 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.
  • infants e.g., pre-term neonates
  • infants who are at increased risk of developing hypoferremia or IDA or who have been diagnosed with hypoferremia or IDA.
  • 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.
  • 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.
  • OTC over the counter
  • 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.
  • 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.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • polypeptides or amino acids such as glycine
  • antioxidants such as chelating agents such as EDTA or glutathione
  • stabilizers dyes
  • flavoring agents e.g., pepperminophene, glycine
  • composition comprising the lactoferrin
  • 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.
  • 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.
  • 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).
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer, and the
  • composition and/or preparation is sterile.
  • 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
  • nanoparticles may also be used as carriers for the compositions described herein.
  • the microsphere is larger than
  • 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.
  • excipient solutions e.g. , sucrose
  • Nanoparticles may be used to deliver the lyphophilized product and appropriate excipient(s).
  • compositions which are pharmaceutically or physiologically acceptable or suitable compositions, preparations, or formulations
  • the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin,
  • 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.
  • 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.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • VTE venous thromboembolism
  • 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 ⁇ g/day.
  • a tablet, containing 520 mg of ferrous sulfate Fero-Grad, Abbot Laboratories, USA
  • Iron supplied by one ferrous sulfate tablet was 156 mg/day.
  • 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.
  • 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 Maria, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RBC red blood cells
  • Hb hemoglobin (g/dl)
  • TSI total serum iron ⁇ g/dl
  • SF serum ferritin (ng/ml).

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Abstract

L'invention porte sur des méthodes pour le maintien, l'amélioration et le développement de la santé de femmes enceintes, de nourrissons et d'enfants par l'administration de compositions qui comportent une lactoferrine isolée. Les compositions peuvent comporter en outre un mélange nutritionnel formulé pour compléter le régime du sujet à traiter, et les compositions ne comprennent pas de source de fer inorganique. Les compositions de mélange de nutriments peuvent comporter au moins de la vitamine B6 et de l'acide folique et/ou de la vitamine B12. Les méthodes selon l'invention comprennent des méthodes pour la prévention (la réduction du risque de survenue) d'une complication associée à la grossesse, telle que la fausse couche, l'hématome rétro-placentaire, la naissance prématurée ou l'accouchement prématuré. L'invention porte également sur des méthodes de prévention (réduction du risque de survenue) ou de traitement d'un état associé au post-partum. L'invention porte également sur des méthodes de prévention ou de réduction du risque de survenue d'un événement thrombotique chez un sujet par l'administration d'une composition comportant une lactoferrine ou par l'administration d'une préparation qui comporte de la lactoferrine et un mélange de nutriments formulé pour compléter le régime du sujet et qui ne comprend pas de source de fer inorganique.
PCT/US2011/038438 2010-05-28 2011-05-27 Méthodes d'amélioration de la santé maternelle et fœtale WO2011150393A2 (fr)

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CN103550760A (zh) * 2013-11-07 2014-02-05 无锡科捷诺生物科技有限责任公司 乳铁蛋白在制备炎症因子偏高肥胖人群贫血药物中的应用
ITMI20122152A1 (it) * 2012-12-17 2014-06-18 Progine Farmaceutici S R L Composizione per uso topico.
ITMI20122151A1 (it) * 2012-12-17 2014-06-18 Progine Farmaceutici S R L Preparazione antiabortiva.
RU2632435C1 (ru) * 2016-09-12 2017-10-04 Федеральное бюджетное учреждение науки "Московский научно-исследовательский институт эпидемиологии и микробиологии им. Г.Н. Габричевского" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Способ персонализированного ведения беременных с инфекционной патологией урогенитального тракта на ранних сроках гестации
EP3205347A4 (fr) * 2014-10-08 2018-06-06 Keio University Inhibiteur de formation de piege extracelluaire de leucocytes
CN111257445A (zh) * 2020-01-22 2020-06-09 上海交通大学医学院附属仁济医院 用于sle孕妇病情监测及其胎儿结局预测的产品及方法
US10980786B2 (en) * 2014-01-10 2021-04-20 Nestec S.A. Maternal vitamin B6 administration for the prevention of increased adiposity, overweight or obesity in the offspring
IT202100013898A1 (it) 2021-05-27 2022-11-27 Hulka S R L Composizione comprendente un estere di alfa-tocoferolo per la riduzione del rischio di parto pretermine
CN116253805A (zh) * 2023-03-23 2023-06-13 深圳霁因生物医药转化研究院 一种多肽、水凝胶和用途

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20122152A1 (it) * 2012-12-17 2014-06-18 Progine Farmaceutici S R L Composizione per uso topico.
ITMI20122151A1 (it) * 2012-12-17 2014-06-18 Progine Farmaceutici S R L Preparazione antiabortiva.
WO2014097123A1 (fr) * 2012-12-17 2014-06-26 Progine Farmaceutici S.R.L. Composition pour application topique comprenant de la lactoferrine
CN103550760A (zh) * 2013-11-07 2014-02-05 无锡科捷诺生物科技有限责任公司 乳铁蛋白在制备炎症因子偏高肥胖人群贫血药物中的应用
US10980786B2 (en) * 2014-01-10 2021-04-20 Nestec S.A. Maternal vitamin B6 administration for the prevention of increased adiposity, overweight or obesity in the offspring
EP3205347A4 (fr) * 2014-10-08 2018-06-06 Keio University Inhibiteur de formation de piege extracelluaire de leucocytes
RU2632435C1 (ru) * 2016-09-12 2017-10-04 Федеральное бюджетное учреждение науки "Московский научно-исследовательский институт эпидемиологии и микробиологии им. Г.Н. Габричевского" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Способ персонализированного ведения беременных с инфекционной патологией урогенитального тракта на ранних сроках гестации
CN111257445A (zh) * 2020-01-22 2020-06-09 上海交通大学医学院附属仁济医院 用于sle孕妇病情监测及其胎儿结局预测的产品及方法
CN111257445B (zh) * 2020-01-22 2022-09-09 上海交通大学医学院附属仁济医院 用于sle孕妇病情监测及其胎儿结局预测的产品及方法
IT202100013898A1 (it) 2021-05-27 2022-11-27 Hulka S R L Composizione comprendente un estere di alfa-tocoferolo per la riduzione del rischio di parto pretermine
WO2022248269A1 (fr) 2021-05-27 2022-12-01 Hulka S.R.L. Composition comprenant un ester d'alpha-tocophérol pour réduire le risque de naissance prématurée
CN116253805A (zh) * 2023-03-23 2023-06-13 深圳霁因生物医药转化研究院 一种多肽、水凝胶和用途
CN116253805B (zh) * 2023-03-23 2023-10-24 深圳霁因生物医药转化研究院 一种多肽、水凝胶和用途

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