EP3436032A1 - Mineralische zusammensetzungen und deren verwendung - Google Patents

Mineralische zusammensetzungen und deren verwendung

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Publication number
EP3436032A1
EP3436032A1 EP17715100.8A EP17715100A EP3436032A1 EP 3436032 A1 EP3436032 A1 EP 3436032A1 EP 17715100 A EP17715100 A EP 17715100A EP 3436032 A1 EP3436032 A1 EP 3436032A1
Authority
EP
European Patent Office
Prior art keywords
composition
brain
potential
pregnancy
female subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17715100.8A
Other languages
English (en)
French (fr)
Inventor
Nora Schneider
Jonas HAUSER
Irma SILVA ZOLEZZI
Tinu Mary SAMUEL
Sean DEONI
Tamas Bartfai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Societe des Produits Nestle SA
Original Assignee
Nestec SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nestec SA filed Critical Nestec SA
Publication of EP3436032A1 publication Critical patent/EP3436032A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • 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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • 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
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • 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
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to a composition for promoting, supporting or optimizing de novo myelination, and/or brain structure, and/or brain connectivity, and/or one or more of cognitive potential, learning potential, and intellectual potential and or cognitive functioning in an offspring of a female subject.
  • Neurodevelopment in particular brain development, in-utero and over the first 2 or 3 years of life is rapid and places exceptionally high demands on the supply of key nutrients to the infant. Failure to meet these nutrient demands during this crucial period may result in sub-optimal neurodevelopment, in particular brain development.
  • brain structure in particular the amount and/or spatial distribution of myelinated matter throughout the brain, for cognitive functioning and intelligence is well documented.
  • myelin in the brain provides an insulating sheet along neurons permitting much faster conduction of nerve impulses.
  • brain structure in particular the amount and/or spatial distribution of myelin throughout the brain, that affects brain connectivity e.g. via what pathway and how quickly and efficiently, messages in the form of neural impulses are communicated within the brain and in particular between different brain regions.
  • This interbrain communication can play a role in cognitive functioning and learning, and may affect and even serve to physiologically limit intellectual, cognitive and/or learning potential.
  • certain mineral nutrients may promote, support or optimise de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter throughout the brain, in an infant.
  • mary of Invention The present invention is based on a maternal supplement comprising one or more mineral nutrients, which is for administration to a female subject prior to or during pregnancy, and/or while an offspring is receiving breast milk from the female subject.
  • the maternal supplements of the invention are understood to have a beneficial effect on the de novo myelination in the offspring. Promoting, supporting and/or optimizing de novo myelination by way of maternal supplements enables the mother to provide her offspring with health benefits, including long- term benefits to the infant later in life when the infant is no longer breast-fed.
  • a first aspect of the invention therefore relates to a composition
  • a composition comprising one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, for use in promoting, supporting or optimizing one or more of the following:
  • composition in an offspring of a female subject, wherein said composition is for administration to the female subject.
  • compositions comprising one or more of the above mentioned mineral nutrients can promote, support or optimize de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and/or temporal-spatial distribution of myelinated matter throughout the brain, in a subject.
  • the current finding stems from the results of the nutritional analysis of the results of a longitudinal cognitive and brain imaging study wherein de novo myelination, in particular the de novo myelination trajectory, and/or brain structures, including the amount and spatial distribution of myelinated matter throughout the brain were examined and compared. Further details of this study and the results are given in the accompanying examples.
  • a second aspect of the invention relates to one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, for use in promoting, supporting or optimizing one or more of the following:
  • a third aspect of the invention relates to a method of promoting, supporting or optimizing one or more of the following:
  • a fourth aspect of the invention relates to the use of one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, in the manufacture of a composition to promote, support or optimize one or more of the following:
  • composition in an offspring of a female subject, wherein said composition is for administration to the female subject.
  • a fifth aspect of the invention relates to a composition
  • a composition comprising one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, wherein said composition is for use as a pre-pregnancy supplement for promoting, supporting or optimizing one or more of the following:
  • Fig. 1 - Shows the mean whole brain (all white matter) myelination trajectories in infants and young children breastfed vs fed with two commercial formulas comprising different levels of iron.
  • Fig. la - Shows the mean regional myelination trajectories in infants and young children breastfed vs fed with two commercial formulas comprising different levels of iron.
  • Fig. lb - Is a brain image showing the myelinated brain regions associated with iron.
  • Fig. lc - Shows the mean whole brain (all white matter) myelination trajectories in infants and young children breastfed vs fed with two commercial formulas comprising different levels of zinc.
  • Fig. Id - Shows the mean regional myelination trajectories in infants and young children breastfed vs fed with two commercial formulas comprising different levels of zinc.
  • Fig. le - Is a brain image showing the myelinated brain regions associated with zinc.
  • Fig. lg - Is a brain image showing the myelinated brain regions associated with phosphorus.
  • Fig. lh - Is a brain image showing the myelinated brain regions associated with magnesium.
  • Fig. li - Is a brain image showing the myelinated brain regions associated with folic acid.
  • Fig. lj - Is a brain image showing the myelinated brain regions associated with vitamin B12.
  • Fig. Ik - Is a brain image showing the myelinated brain regions associated with Sphingomyelin.
  • Fig. 1L - Is a brain image showing the myelinated brain regions associated with
  • Fig. 1M - Is a brain image showing the myelinated brain regions associated with phosphatidylcholine.
  • Fig. In - Is a brain image showing the myelinated brain regions associated with choline.
  • Fig. 10 - Is a brain image showing the myelinated brain regions associated with docosahexaenoic acid.
  • Fig. lp - Is a brain image showing the myelinated brain regions associated with arachidonic acid.
  • Figure 2 Shows the effect of nervonic acid on neuronal cell density and astrocyte cell density
  • Figure 3 Shows the effect of stearic acid on neuronal cell density and astrocyte cell density
  • Figure 4 Shows the effect of octanoic acid on neuronal cell density and astrocyte cell density
  • Figure 5 Shows the effect of sphingomyelin on number of neurospheres and neuronal proliferation
  • Figure 8 Shows the impact of DHA on MBP, NF, and/or MBP/NF at day 18 and/or day 30.
  • Figure 9 Shows the impact of stearic acid on A2B5, MBP, MAG, NF, MBP/NF, and/or MAG/NF at day 12, day 18 and/or day 30.
  • Figure 10 Shows the impact of vitamin B12 on A2B5, NF, MBP/NF, and/or MAG at day 12, day 18 and/or day 30.
  • Figure 11 Shows the impact of folic acid on A2B5, NF, MAG, MAG/NF, and/or MBP/NF at day 12, day 18 and/or day 30.
  • Figure 12 Shows the impact of choline on A2B5, MAG and/or MBP at day 12, day 18 or day 30.
  • Figure 13 Shows the impact of Iron on A2B5, MBP, MAG, NF, and/or MAG/NF at day 12, day 18 and/or day 30.
  • Figure 14 Shows the impact of Zinc on MBP, NF and/or MBP/NF at day 12, day 18 and/or day 30.
  • Figure 15 Shows the impact of phosphorus on MAG, NF, and/or MAG/NF at day 12, day 18 and/or day 30.
  • Figure 16 Shows the impact of magnesium on A2B5, MBP, NF, MAG, MBP/NF and/or MAG/NF at day 12, day 18 and/or day 30.
  • Figure 17 Shows the impact of copper on A2BF, MAG, and/or MAG/NF at day 12 and/or day 18.
  • Figure 18 shows the impact of phosphatidylcholine on A2B5 at day 12 and on MAG at day 18.
  • Figure 19 Shows the impact of phosphatidylinositol on A2B5, MBP, MAG, NF, MAG/NF at day 12, day 18 and/or day 30.
  • Figure 20 Shows the impact of phosphatidylserine on A2B5, NF, and/or MAG/NF at day 12 and/or D18.
  • Figure 21 Shows the impact of sphingomyelin on A2B5, MAG, and/or MBP at day 12, day 18 and/or day 30.
  • Figure 22 Shows the impact of ceramide on A2B5 at day 12, and on MAG at day 18.
  • Figure 23 Shows the impact of galactoceramide on A2B5, MBP, NF, and/or MBP/NF at day 12 and/or day 30.
  • Figure 24 Shows the impact of glucoceramide on A2B5 at day 12 and NF at day 12 and day 18.
  • Figure 25 Shows the impact of D-erythroceramide on A2B5 at day 12 and on MAG at day 18.
  • Figure 26 Shows the impact of Ceramide-l-phosphate on A2B5 at day 12, and on NF and MAG at day 18.
  • Figure 27 Shows the impact of monosialoganglioside-3 (GM3) on A2B5, MBP, MAG, and/or MBP/NF at day 12, day 18 and/or day 30.
  • GM3 monosialoganglioside-3
  • Figure 28 Shows the impact of disialogangliosides 3 (GD3) on A2B5, MBP, NF and/or MAG at day 12, day 18 and/or day 30.
  • GD3 disialogangliosides 3
  • Figure 29 Shows the fatty acid profile of Phosphatidylinositol (PI), Phosphatidylcholine, Phosphatidyl (PC), Phosphatidylserine (PS), and Sphingomyelin used in example 6.
  • PI Phosphatidylinositol
  • PC Phosphatidyl
  • PS Phosphatidylserine
  • Sphingomyelin used in example 6.
  • Figure 30 Shows the impact of vitamin B12 on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 31 Shows the impact of ARA on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 32 Shows the impact of stearic acid on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 33 Shows the impact of zinc on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 34 Shows the impact of phosphatidylinositol on MAG and MBP mRNA expression.
  • Figure 35 Shows the impact of GD3 on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 36 Shows the impact of DHA on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 37 Shows the impact of nervonic acid on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 38 Shows the impact of Iron on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 39 Shows the impact of phosphatidylcholine on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 40 Shows the impact of phosphatidylserine on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 41 Shows the impact of folic acid on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 42 Shows the impact of choline on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 43 Shows the impact of ceramide on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 44 Shows the impact of galactoceramide on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 45 Shows the impact of glucoceramide on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 46 Shows the impact of Ceramide-l-phosphate on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 47 Shows the impact of D-erythroceramide on MAG and MBP mRNA expression and on MBP and Betalll Co-expression.
  • Figure 48 Shows the impact of sphingomyelin on MBP and Betalll Co-expression.
  • Figure 49 Shows the impact of GM3 on MBP and Betalll Co-expression.
  • Fig. 50a - Is a brain image showing the myelinated brain regions associated with maternal DHA intake.
  • Fig. 50b - Is a brain image showing the myelinated brain regions associated with maternal choline intake.
  • Fig. 50c - Is a brain image showing the myelinated brain regions associated with maternal Iron intake.
  • Fig. 50d - Is a brain image showing the myelinated brain regions associated with maternal folic acid intake.
  • Fig. 50e - Is a brain image showing the myelinated brain regions associated with maternal vitamin B12 intake.
  • a synthetic composition comprising one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, for use in promoting supporting or optimizing de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and spatial distribution of myelinated matter throughout the brain, in particular as determined by de novo myelination and the de novo myelination trajectory, and/or brain connectivity, and/or intellectual potential and/or cognitive potential and/or learning potential and/or cognitive functioning in an offspring of a female subject, wherein said choline is for administration to the female subject.
  • the composition of the present invention may prevent, reduce the risk and/or mitigate a sub-optimal de novo myelination, in particular de novo myelination trajectory, and/or brain structure, in particular the amount and spatial distribution of myelinated matter throughout the brain, in particular as determined by de novo myelination and the de novo myelination trajectory, and/or the intellectual potential and/or cognitive potential and/or learning potential and/or cognitive functioning in said subject.
  • This may be non-therapeutic or therapeutic.
  • composition of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise depending on the needs.
  • promote refers to a factor or a number of factors causing a certain process to occur.
  • support or “supporting” used herein refers to a factor or a number of factors sustaining a certain process once it has started to occur.
  • optically or “optimizing” as used herein refers to an improvement or enhancement.
  • the term "therapeutically effective amount” refers to an amount of the "active" (here, one or more of the above-mentioned mineral nutrients) that gives rise to a therapeutic effect, for example, in terms of one or more of the following effects in an offspring: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; and (vi) learning potential, and (vii) cognitive functioning.
  • a "metabolite” refers to a substance produced during metabolism.
  • a "metabolic precursor” refers to a substance from which another is formed by a metabolic reaction.
  • a nutrient may be comprised in a composition under different forms (as such or in the form of salts, complexes or more complex structures comprising the nutrient) the amounts reported hereafter are intended to make reference to the amount of the nutrient as such.
  • the composition promotes, supports or optimizes the amount and/or spatial distribution of myelinated matter throughout the brain, in particularly the amount and/or spatial temporal distribution of myelinated matter throughout the brain.
  • the composition promotes, supports or optimizes the de novo myelination trajectory.
  • the de novo myelination trajectory measured or observed in breastfed, more particularly exclusively breastfed, subjects of a well-nourished or nutritionally replete mothers may be considered optimal.
  • a composition of the invention may therefore be considered to optimise a subject's myelination trajectory if it brings a subject's de novo myelination trajectory in line or closer to that measured or observed in a breastfed, more particularly exclusively breastfed, subject of a well-nourished or nutritionally replete mother.
  • An offspring's de novo myelination trajectory may be considered to be in line or closer to that measured or observed in a breastfed, more particularly exclusively breastfed subject, in particular of a well-nourished or nutritionally replete mother, if the distance between any equivalent/same measurement points on the offspring's trajectory and said breastfed subject's trajectory is up to 50%, in particular up to 25%, more particularly up to 20%.
  • Non limiting examples within the range of up to 50% include, 50%, 40%, 30%, 25%, 20%, 10%, 5%, 1%, 0.5%, and 0.01%.
  • the trajectories will be considered bioequivalent.
  • the myelination trajectory can be measured at any combination of time points.
  • the time points are within the first 5 years of a human subject's life, more particularly the first 2 or 3 years of a human's life, even more particularly in the first year of a human's life.
  • the de novo myelination trajectory may be determined by measuring the myelin associated water fraction and/or the myelin associated water pool in a subject at different times points, in particular at different time points across the first 5 years of a human subject's life, more particularly the first 2 or 3 years of a human's life, even more particularly the 1 st year of a human's life.
  • the myelin associated water fraction and/or the myelin associated water pool in a subject may be measured using a multicomponent relaxation (MCR) magnetic resonance imaging (MRI) technique and in particular using the mcDESPOT technique (Deoni et al 2008).
  • MCR multicomponent relaxation
  • MRI magnetic resonance imaging
  • mcDESPOT Magnetic elination trajectory
  • the de novo myelination trajectory may be determined by measuring the myelin associated water pool using the mcDESPOT technique (Magn.Reson. Med.2008 60:1372-1387 the subject matter of which is hereby incorporated
  • a composition of the invention may be considered to optimise an offspring's cognitive functioning if it brings one or more offspring's scores in a standardized neurodevelopmental test, for example on the Mullen Scales of Early Learning, in line or closer to that measured or observed in a breastfed, more particularly exclusively breastfed subject, in particular of a well-nourished or nutritionally replete mother.
  • An offspring's cognitive and neurodevelopmental functioning may be considered to be in line or closer to that measured in said breastfed subject, if the difference between one or more of said offspring's standardized neurodevelopmental test scores, for example Mullen's T scores, and that of said breastfed subject is less than one standard deviation, more particularly less than half a standard deviation of a standardized test score, for example less than 10 points, more particularly less than 5 points for the Mullen's T score, in particular less than 2 points.
  • Said standardized neurodevelopmental test scores for example Mullen's T scores, being measured at the same time point in said subject and said breastfed subject.
  • Said Mullen's score can be measured at any appropriate time point and in particular within the first 5 years of a human subject's life, more particularly the first 2 or 3 years of a human's life, even more particularly in the first year of a human's life.
  • the term "female subject” refers to a female, especially prior to or during pregnancy or shortly after childbirth.
  • the female subject is a mammalian subject, more preferably, a cat, dog or human. Even more preferably, the female subject is a human. In all cases the female subject may be deficient or borderline deficient (sub-clinically deficient) in choline, or the female subject does not get a sufficient daily supply of choline through their diet.
  • the term “offspring” encompasses the offspring of the female subject at any stage of development including fetus, neonate, infant, child and adult, or the fetus, infant or adult in the case of other mammals (for example cats and dogs).
  • the term offspring in relation to a human refers to the neonate, infant and child stages, and more preferably the neonate and infant stages.
  • the term offspring in relation to other mammals refers to a neonate or infant stage.
  • the term “neonate” refers to a newborn subject. In humans, the term neonate typically refers to an infant less than 4 weeks old.
  • infant refers to a human infant of up to 12 months of age and includes preterm and very preterm born infants, infants having a low birth weight i.e. a new born having a body weight below 2500g (5.5 pounds) either because of preterm birth or restricted fetal growth, and infants born small for gestational age (SGA) i.e. babies with birth weights below the 10th percentile for babies of the same gestational age.
  • SGA gestational age
  • child refers to a human of 1 to 18 years of age, more preferably a human of 1 to 10 years of age, even more preferably a human of 1 to 5 years of age, and even more preferably a human of 1 to 2 years of age.
  • young child means a child aged between one and five years, (including toddlers).
  • preterm or premature means an infant or young child that was not born at term. Generally it refers to an infant born prior to the completion of 37 weeks of gestation.
  • the expression "term born infant” indicates an infant born after 37 weeks gestation.
  • postnatal period or ""postpartum period” is the period beginning immediately after the birth of a child and extending for about six weeks.
  • the offspring is a formula fed infant or child.
  • formula fed infant or child refers to an infant or child fed either infant formula and/or growing up milk.
  • Exclusive breast feeding / infants or young children exclusively breast fed refers to infants for which the great majority of nutrients and/or energy originates from human breast milk (the "great majority” is preferably at least 90% or at least 95%, or at least 99%).
  • Infants/young children predominantly fed infant formula refers to infants or young children for which nutritional sources of nutrients and/or energy predominantly originates from synthetic infant formula, follow-on milk or growing-up milks. Predominantly refers to at least 50% of those nutrients and/or energy, or at least 75%.
  • de novo myelination refers to the process by which naked axons in the brain of a subject are myelinated during growth and development. The process starts in utero and is most prolific in the first 5 years of a human subject's life, in particular the first 2 or 3 years of a human's life. More preferably, the term refers to the de novo myelination trajectory.
  • de novo myelination trajectory refers to the extent of myelination (as measured for example by the Myelin Water Fraction) as a function of time, and in particular across infancy and childhood, in particular early childhood, and more particularly in the first 5 years of a human subject's life, more particularly the first 2 or 3 years of a human's life, or first year of life.
  • brain structure refers to the structure of grey and white matter within the brain and specific brain regions, and in particular to myelinated white matter within the brain and specific brain regions as determined by de novo myelination and in particular the de novo myelination trajectory i.e. by the de novo structural deposition of myelin. More particularly the term refers to the amount and/or spatial distribution of myelinated matter throughout the brain, and/or in specific brain regions, and even more particularly the amount and/or temporal spatial distribution of myelinated matter throughout the brain and/or in specific brain regions.
  • intellectual potential refers to the possible intellectual ability or capacity attainable by a subject as determined by physiological factors.
  • intellectual potential may refer to fluid intelligence.
  • fluid intelligence refers to a subject's neural potential and/or a subject's novel or abstract problem solving capability as determined by physiological factors. This is distinct from crystallized intelligence which is determined, at least in part by learned or acculturated knowledge.
  • cognitive potential refers to the possible cognitive and/or mental ability or capacity attainable by a subject as determined by physiological factors.
  • the term may refer to one or more of: information processing potential, perception potential, attention potential, thinking potential, reasoning potential, understanding and remembering potential, psychomotor potential, including gross motor and fine motor potential, visual potential, including visual reception potential, language potential, including expressive and receptive language potential, memory and recall potential, concentration potential, executive function potential, including problem-solving, decision-making and inhibition potential.
  • learning potential refers to the possible ability or capacity a subject has to learn e.g. how easily and/or quickly a subject may be able to acquire knowledge or skills through experience, study or being taught, as determined by physiological factors. As well as the possible ability a subject has to adapt in response to environmental factors, as determined by physiological factors.
  • Mineral nutrients such as iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof, may be particularly effective at supporting, promoting or optimizing de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter throughout the brain, in the following brain areas: Cerebellum, brainstem, visual cortex, motor and somatosensory cortices, corpus callosum, frontal cortex, temporal white matter, internal capsule, prefrontal cortex, motor cortex.
  • compositions comprising a mineral may be particularly effective at promoting, supporting or optimising motor function and psychomotor potential (including coordination and execution of movement potential), vision potential, executive functioning potential, working memory potential, problem solving potential, social-emotional functioning potential, language potential, and/or auditory potential.
  • cognitive potential is selected from the group consisting of motor function and psychomotor potential (including coordination and execution of movement potential), vision potential, hemispherical interaction potential, , executive functioning potential, working memory potential, problem solving potential, social-emotional functioning potential, language potential, auditory/listening potential, problem solving potential, and working memory potential.
  • composition of the invention may optimise motor function and psychomotor (including coordination and execution of movement) function, vision, hemispherical interaction, executive functioning, working memory, problem solving, social- emotional functioning, language, auditory/listening functions, problem solving, and working memory.
  • compositions comprise one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof.
  • the mineral nutrient is selected from iron, zinc, calcium, phosphorus, copper and magnesium.
  • compositions according to the present invention comprise iron, iron is a component of a number of proteins including haemoglobin, myoglobin, cytochromes and enzymes involved in redox reactions. Iron can exist in a range of oxidation states, the interconversion of which allows iron to bind reversibly to ligands such as oxygen, nitrogen and sulphur atoms.
  • the mineral nutrient is iron.
  • Iron may be incorporated in the form of a physiologically acceptable salt such as, for example, ferric citrate, ferric phosphate, ferric pyrophosphate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate, ferrous sulfate, or mixtures thereof.
  • Iron may also be incorporated in the form of a physiologically acceptable iron complex (such as for example EDTA ferric sodium salt) or mixtures thereof.
  • Fe2+ is more bioavailable and it may therefore be more beneficial if iron is added into the composition in the form of a ferrous salt or complex e.g. a ferrous salts listed hereinabove.
  • iron is includes all the iron present in the compositions of the invention either in free form, or in the form of a physiologically acceptable salts or complexes thereof.
  • iron may be comprised in the composition of the invention in an amount of about 0.001% to about 99.999% of the composition.
  • the composition according to the present invention comprises levels of iron such that the total daily intake derived from the composition of the invention is from about 2 to about 50 mg, more preferably, from about 2.5 to about 45 mg, even more preferably, from about 2.7 to about 45 mg, more preferably still from about 3 to about 30 mg, or about 5 to about 20 mg.
  • the composition according to the present invention comprises levels of iron such that the total daily intake derived from the nutritional composition of the invention will not exceed about 45 mg.
  • the composition according to the present invention comprises levels of iron such that the total daily intake derived from the nutritional composition of the invention will not exceed 40 mg.
  • the composition according to the present invention comprises zinc.
  • Zinc is a component of various enzymes that help maintain structural integrity of proteins and regulate gene expression.
  • Zinc metalloenzymes include ribonucleic acid polymerases, alcohol dehydrogenase, carbonic anhydrase and alkaline phosphatase.
  • zinc is comprised in the composition of the invention in an amount constituting from about 0.001% to about 99.999% of the composition.
  • zinc is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 1 to about 50 mg, more preferably from about 1 to about 40 mg, more preferably, from about 1.1 to about 40 mg, even more preferably from about 2 to about 20 mg, even more preferably, from about 5 to about 15 mg, even more preferably from about 8 to about 12 mg.
  • the zinc is comprised in the composition in an amount such that the total daily intake derived from the composition is about 10 mg.
  • the composition according to the present invention comprises levels of zinc such that the total daily intake derived from the nutritional composition of the invention will not exceed about 40 mg.
  • Zinc may be incorporated in the compositions of the invention in the form of a physiologically acceptable salt and /or via any source comprising Zinc, more specifically Zn2+, such as, for example, zinc gluconate, zinc sulfate, zinc acetate and mixtures thereof, or in the form of a physiologically acceptable zinc complex (such as, for example, zinc piccolinate) or mixtures thereof.
  • the composition according to the present invention comprises copper.
  • Copper is necessary for the proper growth, development, and maintenance of bone, connective tissue, brain, heart, and many other body organs. Copper is incorporated into a variety of proteins and metalloenzymes which perform essential metabolic functions. Copper is involved in the formation of red blood cells, the absorption and utilization of iron, the metabolism of cholesterol and glucose, and the synthesis and release of life-sustaining proteins and enzymes. These enzymes in turn produce cellular energy and regulate nerve transmission, blood clotting, and oxygen transport.
  • copper is comprised in the composition of the invention in an amount constituting from about 0.001% to about 99.999% of the composition.
  • copper is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 0.1 to about 10 mg, more preferably from about 1 to about 8 mg, more preferably, from about 2 to about 6 mg, even more preferably from about 2 to about 5 mg.
  • the composition according to the present invention comprises levels of copper such that the total daily intake derived from the nutritional composition of the invention will not exceed about 10 mg.
  • Copper more specifically Cu2+, may be incorporated in the compositions of the invention in the form of a physiologically acceptable salt such as, for example, cupric oxide, copper gluconate, copper sulfate, or copper carbonate, or mixtures thereof, or in the form of a physiologically acceptable copper complex or mixtures thereof.
  • the composition according to the present invention comprises magnesium.
  • Magnesium is an essential element in biological systems. Magnesium occurs typically as the Mg 2+ ion. It is an essential mineral nutrient for life and is present in every cell type in every organism. For example, ATP, the main source of energy in cells, must be bound to a magnesium ion in order to be biologically active. As such, magnesium plays a role in the stability of all polyphosphate compounds in the cells, including those associated with the synthesis of DNA and RNA. Over 300 enzymes require the presence of magnesium ions for their catalytic action, including all enzymes utilizing or synthesizing ATP, or those that use other nucleotides to synthesize DNA and RNA.
  • magnesium is comprised in the composition of the invention in an amount constituting from about 0.001% to about 99.999% of the composition.
  • magnesium is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 35 to about 350 mg, more preferably from about 50 to about 250 mg, more preferably, from about 100 to about 200 mg.
  • the composition according to the present invention comprises levels of magnesium such that the total daily intake derived from the nutritional composition of the invention will not exceed 350 mg.
  • Magnesium may be incorporated in the compositions of the invention in the form of a physiologically acceptable salt such as, for example, magnesium oxide, magnesium citrate, magnesium chloride, magnesium aspartate, magnesium lactate and mixtures thereof, or in the form of a physiologically acceptable magnesium complex, or mixtures thereof.
  • the composition according to the present invention comprises calcium.
  • Calcium is required for the normal development and maintenance of the skeleton as well as for the proper functioning of neuromuscular and cardiac function.
  • calcium is comprised in the composition of the invention in an amount constituting from about 0.001% to about 99.999% of the composition.
  • calcium is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 100 to about 2500 mg, more preferably from about 200 to about 2000 mg, more preferably, from about 250 to about 1500 mg, even more preferably from about 500 to about 1000 mg.
  • the composition according to the present invention comprises levels of calcium such that the total daily intake derived from the nutritional composition of the invention will not exceed 2500 mg.
  • Calcium more specifically Ca2+
  • a physiologically acceptable salt such as, for example, calcium carbonate, calcium citrate, calcium phosphate, calcium lactate, calcium gluconate and mixtures thereof, or in the form of a physiologically acceptable calcium complex, or mixtures thereof.
  • the composition according to the present invention comprises phosphorus.
  • Phosphorus in the form of phosphate P0 4 ⁇ is required for all known forms of life and plays a major role in biological molecules such as DNA and RNA, where it forms part of the structural framework of these molecules.
  • Living cells also use phosphate to transport cellular energy in the form of adenosine triphosphate (ATP). Nearly every cellular process that uses energy obtains it in the form of ATP. ATP is also important for phosphorylation, a key regulatory event in cells.
  • phosphorus is comprised in the composition of the invention in an amount constituting from about 0.001% to about 99.999% of the composition.
  • phosphorus is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 70 to about 3500 mg, more preferably from about 100 to about 2500 mg, more preferably, from about 200 to about 2000 mg, even more preferably from about 300 to about 1500 mg, even more preferably, from about 500 to about 1000 mg.
  • the composition according to the present invention comprises levels of phosphorus such that the total daily intake derived from the nutritional composition of the invention will not exceed 3500 mg.
  • Phosphorus may be incorporated in the compositions of the invention in the form of a physiologically acceptable salt such as, for example, dibasic potassium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, monobasic sodium phosphate, tribasic sodium phosphate, calcium phosphate, calcium hydrogen phosphate and mixtures thereof.
  • a physiologically acceptable salt such as, for example, dibasic potassium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, monobasic sodium phosphate, tribasic sodium phosphate, calcium phosphate, calcium hydrogen phosphate and mixtures thereof.
  • compositions of the invention further comprise one or more of the following ingredients: a vitamin, a phospholipid or a metabolite or metabolic precursor thereof, a fatty acid derivative and choline.
  • the amount of each of these additional ingredients is selected depending on whether the composition is intended to be administered/consumed once a day or more frequently.
  • composition of the invention of the invention comprises a mineral nutrient as described above and one or more of these additional ingredients it may have an improved effect in terms of promoting supporting and/or optimizing de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and spatial distribution of myelinated matter throughout the brain and/or brain connectivity, and/or cognitive potential and/or intellectual potential and/or learning potential and/or cognitive function. This may for example be because said ingredients effect de novo myelination in the same and/or separate complementary brain areas.
  • the improved effect may be synergistic.
  • vitamin refers to any vitamin.
  • Non limiting examples of vitamins include: vitamin A, vitamin Bl, vitamin B2, vitamin B6, vitamin K, vitamin C, vitamin D, niacin, biotin, pantothenic acid, folic acid, vitamin B12, and combinations thereof.
  • the composition comprises folic acid.
  • Folic acid may be incorporated in the nutritional compositions of the invention as such or in the form of a physiologically acceptable salt thereof (folate) or mixtures thereof.
  • the term "folic acid” includes all the folic acid present in the compositions of the invention either as such or in the form of a physiologically acceptable salt thereof or mixtures thereof.
  • folic acid is comprised in the composition in an amount constituting about 0.001% to about 99.999% of the composition.
  • the composition according to the present invention comprises levels of folic acid such that the total daily intake derived from the composition of the invention is from about 50 to about 1000 ⁇ g, more preferably from about 60 to about 1000 ⁇ g, even more preferably, from about 70 to about 700 ⁇ g, even more preferably, from about 100 to about 500 ⁇ g, more preferably still from about 200 to about 400 ⁇ g.
  • the composition comprises an amount of folic acid such that the total daily intake derived from the composition of the invention will not exceed about 1000 ⁇ g.
  • the composition of the invention comprises vitamin B12. Vitamin B12 is preferably comprised in the composition in an amount constituting from about 0.001% to about 99.999% of the composition.
  • the composition comprises an amount of vitamin B12 such that the total daily intake derived from the composition of the invention is from about 0.2 to about 250 ⁇ g, more preferably from about 0.26 to about 50 ⁇ g, more preferably from about 0.5 to about 30 ⁇ g, even more preferably from about 1 to about 10 ⁇ g, more preferably still, from about 2 to about 6 ⁇ g.
  • the composition comprises an amount of vitamin B12 such that the total daily intake derived from the composition of the invention is about 250 ⁇ g.
  • the composition comprises an amount of vitamin B12 such that the total daily intake derived from the composition of the invention does not exceed about 50 ⁇ g.
  • Vitamin B12 may be incorporated in the compositions of the invention as such or in the form of one physiologically acceptable salt thereof or mixtures thereof.
  • a composition of the invention comprising a vitamin, in particularly folic acid and/or vitamin B12, may be particularly effective at supporting, promoting or optimising de novo myelination, in particular the de novo myelination trajectory, and/or brain structure in one or more of the following brain areas; cerebellum, brainstem, temporal lobe, frontal lobe, visual cortex, motor and somatosensory cortices. These brain areas are associated with-Motor function (including coordination and execution of movement), visual function and psychomotor function.
  • the composition according to the present invention comprises choline.
  • choline refers to quaternary ammonium salts containing the ⁇ , ⁇ , ⁇ - trimethylethanolammonium cation and having the structure shown below:
  • counterion X- is selected from chloride, hydroxide, citrate, bitartrate and mixtures thereof.
  • choline should be intended to identify all the choline present in the nutritional compositions on the invention, either in free form (or as a salt thereof) such as for example: choline hydroxide.
  • Choline may be incorporated in the composition of the invention as such or in the form of one physiologically acceptable salt such as, for example, choline chloride, choline citrate, choline bitartrate, or mixtures thereof.
  • choline may be comprised in the composition of the invention in an amount from about 0.001% to about 99.999% of the composition.
  • choline is comprised in the composition in an amount such that the total daily intake derived from the composition of the invention is from about 100 to about 1000 mg, more preferably from about 200 to about 600 mg, more preferably, from about 250 to about 550 mg, even more preferably from about 300 to about 500 mg.
  • choline is comprised in the composition in an amount such that the total daily intake of choline derived from the composition of the invention is about 450 mg.
  • a composition of the invention comprising choline may be particularly effective at supporting, promoting or optimising de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in the following brain areas; cerebellum, internal capsule, temporal lobe, frontal cortex, motor cortex, visual cortex, thalamus, parietal cortex, and frontal lobe. These brain areas are associated with motor function (including coordination and execution of movement), vision, working memory and/or executive functioning and/or social-emotional reasoning and/or spatial reasoning.
  • composition of the invention comprises a fatty acid derivative.
  • a fatty acid derivative may be comprised in the composition of the invention in an amount constituting up to 99.999% of the composition.
  • fatty acid derivative includes fatty acids and, in particular, to free fatty acids, and/or a monoacylglycerol (hereinafter MAG), and/or a diacylglycerol (hereinafter DAG), and/or a triacylgylcerol (hereinafter TAG) and/or a cholesterol ester.
  • MAG monoacylglycerol
  • DAG diacylglycerol
  • TAG triacylgylcerol
  • cholesterol ester a cholesterol ester
  • the fatty acid derivative is a free fatty acid.
  • the fatty acid derivative is a MAG, DAG, TAG and/or a cholesterol ester. Even more preferably, the fatty acid derivative is a TAG.
  • MAG refers to a glycerol molecule in which one of the OH groups is modified to form an ester bond with a fatty acid.
  • the MAG is a compound of formula (X)
  • R 18 , R 19 and R 20 are H, and one of R 18 , R 19 and R 20 is a CIO to C24 saturated or unsaturated acyl group, more preferably a C14 to C24 saturated or unsaturated acyl group.
  • DAG refers to glycerol molecule in which two of the OH groups are modified to form ester bonds with fatty acids.
  • the DAG is a compound of formula (X) wherein one of R 18 , R 19 and R 20 are H, and two of R 18 , R 19 and R 20 are each independently a C4 to C44 saturated or unsaturated acyl groups, more preferably a C14 to C24 saturated or unsaturated acyl group.
  • R 18 , R 19 and R 20 are each independently a CIO to C24 saturated or unsaturated acyl group.
  • the two C4 to C44 saturated or unsaturated acyl groups of R 18 , R 19 and R 20 may be the same or different.
  • TAG refers to a glycerol molecule that has formed an ester bond with three fatty acids.
  • the TAG as used herein is a compound of formula (X) wherein R 18 , R 19 and R 20 are each independently a C4 to C44 saturated or unsaturated acyl group, more preferably, a CIO to C24 saturated or unsaturated acyl group, more preferably a C14 to C24 saturated or unsaturated acyl group.
  • the three C4 to C44 saturated or unsaturated acyl groups of R 18 , R 19 and R 20 may all be the same, all different, or two may be the same and one different.
  • cholesterol ester refers to a compound of formula (XI):
  • R 21 is a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 21 is a C9 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a CIO to C44 saturated or unsaturated fatty acid residue, more preferably a CIO to C24 saturated or unsaturated fatty acid residue.
  • fatty acid refers to a compound of formula (XII)
  • R 22 is a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 22 is a C9 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, more preferably a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • Non limiting examples of CIO to C44 saturated or unsaturated fatty acids that may be comprised in the fatty acid derivative i.e. that may be the free fatty acid or fatty acid from which the fatty acid residue(s) of the MAG, DAG, TAG and/or cholesterol ester may stem include; C10:0, C12:0, C14:0, C15:0, C16:0, C16:ln-7, C18:0, C18:ln-7, C18:ln-9, C18:2n-6, 18:3n-3, C20:0, C20:ln-9, C20:2n-6, C20:3n-6, C20:4n-6, 20:5n-3, C21:0, C22:0, C22:ln-9, C22:6n-3 C23:0, C24:l, in particular 24:ln-9, C25:0, C28:l, C30:2, C30:l, C30:0, C32:3, C32:2, C32
  • said fatty acids will be selected from the group consisting of: C10:0, C12:0, C14:0, C16:0, C16:ln-7, C18:0, C18:ln-7, C18:ln-9, C18:2n-6, 18:3n-3, C20:0, C20:ln-9, C20:2n-6, C20:3n-6, C20:4n-6, 20:5n-3, C22:0, C22:ln-9, C22:6n-3, C24:l, 24:ln-9.
  • Any fatty acid derivative suitable for ingestion by a subject for which the composition is intended to be consumed may be used in the invention.
  • the fatty acid derivative will come from natural sources, non limiting examples of which include, eggs, algae, fish oil, mould, yeast, seeds, plants e.g. soy, and animal sources e.g. bovine brains, and/or mammalian milk or extracts thereof.
  • soy sources include soy lecithin-food additive
  • mammalian milk include bovine, camel, sheep, goat milk including skilled milks.
  • Non limiting extracts of milk include protein extracts, milk fat globule membranes (MFGM) and extracts comprising them.
  • Fatty acid derivatives may also come from palm oil, tallow, lard, cotton seed oil, peanut oil.
  • the fatty acid derivative comprises a saturated or unsaturated fatty acid selected from the group consisting of: C20:4n-6, C22:6n-3, C24:ln-9, C16:0, C18:ln-9 and C18.0, in particular C20:4n-6 and/or C22:6n-3 and/or C18:0. More particularly 22:6n-3 and/or C18:0.
  • a composition comprising a phospholipid, in particular sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, more particularly sphingomyelin, may be particularly effective if used in combination with one or more of these fatty acids.
  • C20:4n-6 is arachidonic acid hereinafter ARA.
  • C22:6n-3 is docosahexaenoic acid hereinafter DHA.
  • 24:ln-9 is nervonic acid.
  • C18.0 is stearic acid.
  • C16:0 is palmitic acid.
  • C18:ln-9 is Oleic acid.
  • the fatty acid derivative is DAA and/or ARA and/or Stearic acid.
  • a fatty acid derivative comprising DHA and/ or Stearic acid.
  • DHA may be incorporated in the compositions of the invention as a single species (as a fatty acid, in the form of a physiologically acceptable salt thereof or in the form of a triglyceride comprising it), as an ingredient consisting of a mixture of different DHA species, or by addition of a natural or synthetic ingredient comprising one or more DHA species.
  • DHA includes all the DHA present in the compositions on the invention, either in free form (as a fatty acid or physiologically acceptable salt thereof) or comprised in a triglyceride structure.
  • ARA includes all the ARA present in the compositions on the invention, either in free form (as a fatty acid or a physiologically acceptable salt thereof) or comprised in a triglyceride structure.
  • composition according to the invention comprises a fatty acid derivative comprising DHA and/or ARA and/or nervonic acid and/or stearic acid, in particular a fatty acid derivative comprising DHA and/or ARA.
  • a fatty acid derivative comprising DHA and/or ARA and/or nervonic acid and/or stearic acid may be comprised in the composition of the invention in an amount constituting up to 99.999% of the composition.
  • the composition comprises DHA in an amount such that the total daily intake derived from the composition of the invention is from about 100 to about 1500 mg, more preferably from about 250 to about 1200 mg, even more preferably from about 800 to about 1200 mg.
  • the composition comprises ARA in an amount such that the total daily intake derived from the composition of the invention is from about 100 to about 500 mg, more preferably from about 150 to about 450 mg, even more preferably from about 200 to about 400 mg, more preferably still, from about 250 to about 350 mg, or about 300 mg.
  • the composition comprises nervonic acid in an amount such that the total daily intake derived from the composition of the invention is from about 5 to about 80 mg, more preferably from about 5 to about 50 mg, even more preferably from about 8 to about 32 mg.
  • the composition comprises stearic acid in an amount such that the total daily intake derived from the composition of the invention is from about 5 to about 80 mg, more preferably from about 5 to about 50 mg, even more preferably from about 4 to about 20 mg.
  • Fatty acid derivatives comprising stearic acid are present in natural sources, for example, palm oil, tallow, lard, cotton seed oil, peanut oil.
  • Fatty acid derivatives comprising nervonic acid are present in natural sources, for example, the seed oils of Cardamine gracea, Heliphila longifola, Thlaspi perfoliatum, Tropaeolum speciosum, Lunaria biennis, Lunaria annua and Malania oleifera; the moulds Neocallismastix frontalis, Erysiphe graminis and Sphaerotheca humuli; the bacterium Pseudomonas atlantica; the yeast Saccharomyces cerevisiae and the marine diatom Nitzschia cylindrus.
  • Fatty acid derivatives comprising DHA and/or ARA are present in natural sources such as, for example egg, algae, fungus or fish oil, and plants.
  • Oils comprising fatty acid derivatives comprising DHA and/or ARA and generally other polyunsaturated fatty acids (PUFAs), in particular EPA (eicosapentaenoic acid), may be of various origin.
  • PUFAs polyunsaturated fatty acids
  • EPA eicosapentaenoic acid
  • fatty acid derivatives comprising DHA are provided in the form of a fish oil comprising fatty acid derivatives comprising DHA and/or ARA.
  • Fish oils generally comprise 5wt.% or more, preferably 10wt.% or more of fatty acid derivatives comprising DHA and/or ARA.
  • Oils comprising substantial amounts of fatty acid derivatives comprising DHA and/or ARA, obtained from algae or microorganisms in general are also available.
  • oils harvested from algae comprising 10wt.% or more, for example 20wt.% or more of fatty acid derivatives may be used.
  • the nutritional composition according to the present invention comprises fatty acid derivatives comprising ARA and DHA
  • said ingredients may for example be comprised in the composition of the invention in amounts resulting in a weight ratio of DHA:ARA in the range of 4:1 to 1:4, for example 3:1 to 1:3, for example 2:1 to 1:2, for example 1.5:1 to 1:1.5, in particular 1.1:1 to 1:1.1.
  • composition of the invention comprises a mixture of fatty acid derivatives wherein, the mixture is such that the weight ratio of unsaturated to saturated fatty acids and/or fatty acid residues in the composition of the invention is within the range 1:1 to 1:2; 1:1.2 to 1:1.9, 1:1.25 to 1:1.5; 1:3 to 1:4.
  • fatty acid derivatives comprising DHA and/ or ARA
  • the total amount of fatty acid derivatives comprising saturated long chain fatty acids, in particular C20/24 is increased.
  • saturated long chain fatty acids may be an important component of myelin enabling it to wrap around and enrobe axons.
  • the weight ratio of DHA and/or AA to these unsaturated long fatty acids in the composition of the invention is, for example, within the range 1:1 1:10; 1:2 to 1:9, 1: 3 to 1:4.5, 1:3.5 to 1:4.5.
  • a composition of the invention comprising a fatty acid derivative e.g.
  • a fatty acid derivative comprising DHA and/or AA may be particularly effective at supporting, promoting or optimising de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in one or more of the following brain areas: cerebellum, internal capsule, temporal lobe, parietal lobe, frontal cortex, motor and sensory cortices (including coordination and execution of movement), visual cortex, frontal cortices.
  • This brain areas are associated with vision function, motor function and psychomotor function (including coordination and execution of movement functionl), and/or executive functions, and social-emotional functioning.
  • the composition of the invention comprises a phospholipid or a metabolites or metabolic precursor thereof.
  • phospholipid refers to any phospholipid.
  • phospholipid refers to a molecule that is made up of two fatty acids attached to a glycerol 'head.' The glycerol molecule is also attached to a phosphate group.
  • the phospholipid is a compound of formula (I)
  • R 1 is 0
  • X is NH or O
  • R 2 is a C2-C44 saturated or unsaturated, linear or branched acyl group
  • R 3 is a substituent of formula (II) or formula (III): R 5 O CH 2
  • R 4 is selected from a C5 or C6 substituted or unsubstituted cyclic alkyl or cyclic alkenyl group, and -(CH 2 ) n -R 7 ;
  • R 5 is a C2-C44 saturated or unsaturated, linear or branched acyl group
  • R 6 is a C2-C44 saturated alkyl or alkenyl group
  • R 7 is— N(CH 3 ) 3 + , NH 3 + , or a substituent of formula IV):
  • n is an integer from 1 to 4, preferably 1 or 2.
  • alkyl includes both saturated straight chain and branched alkyl groups, which may be substituted (mono- or poly-) or unsubstituted.
  • cyclic alkyl is to be construed accordingly.
  • the cyclic alkyl group is a C3-8, more preferably, a C3-6 cyclic alkyl group.
  • alkenyl refers to a carbon chain containing one or more double bonds, which may be branched or unbranched, and substituted (mono- or poly-) or unsubstituted.
  • acyclic refers to a group that is not cyclic.
  • R 4 is a C6 cyclic alkyl or cyclic alkenyl group substituted with one or more hydroxy groups.
  • R 4 is derived from inositol (C 6 Hi 2 0 6 ), even more preferably myo-inositol, i.e. R 4 is:
  • Non limiting examples of phospholipids include phosphatidylinositole, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and phosphatidylcholine.
  • the phospholipid is selected from the group consisting of phosphatidyl choline, phosphatidyl inositole, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin and mixtures thereof, and metabolic precursors and metabolites of any of the foregoing, and mixtures thereof.
  • Phosphatidylinositole is a compound of formula (V):
  • R 8 and R 9 are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 8 and R 9 are each independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.
  • R 8 and R 9 are C13 to C23 branched or unbranched acyclic alkyl, or acyclic alkenyl groups which together with their adjacent carbonyl group are C14 to C24 saturated or unsaturated fatty acid residues, wherein the fatty acids from which the fatty acid residues stem are selected from the group consisting of; C14:0, C15:0, C16:0, C18:0, C20:0, C20:3, C20:4, C21:0, C22:0, C23:0, C24:0, C18:ln-9, C18:2n-6, and C24:ln-9. Even more particularly C18:0, C18:ln-9, C18:2, C20:3, and C20:4.
  • Phosphatidylserine refers to Phosphatidyl-L-serine.
  • Phosphatidylserine is a compound of formula (VI):
  • R 10 and R 11 are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 10 and R 11 are each independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.
  • R 10 and R 11 are C13 to C23 branched or unbranched acyclic alkyl, or acyclic alkenyl groups which together with their adjacent carbonyl group are C14 to C24 saturated or unsaturated fatty acid residues, wherein the fatty acids from which the fatty acid residues stem are selected from the group consisting of; C14:0, C15:0, C16:0, C18:0, C20:0, C20:3, C20:4, C21:0, C22:0, C23:0, C24:0, C18:ln-9, C18:2n-6, and C24:ln-9. Even more particularly C18:0, C18:ln-9, C20:4, and C22:6.
  • Phosphatidylethanolamine is a compound of formula (VII):
  • R 12 and R 1 are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group. More preferably, R 12 and R 13 are each independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.
  • sphingomyelin refers to a lipid molecule, or mixture of lipid molecules, wherein a sphingosine backbone is acylated with a fatty acid residue at the amino group (-NH 2 ) and wherein the hydroxyl group at position 1 of the sphingosine backbone is linked to a phospho- choline or phospho-ethanolamine group.
  • the sphingomyelin is a compound of formula (VIII) or a mixture of compounds of formula (VIII):
  • R 14 and R 15 are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 14 is a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue.
  • Non limiting examples of C14 to C44 saturated or unsaturated fatty acids from which the fatty acid residue may stem include; C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:l, C25:0, C28:l, C30:2, C30:l, C30:0, C32:3, C32:2, C32:l, C32:0, C33:l, C34:3, C34:2, C34:l, C34:0, C35:2, C35:0, C36:4, C36:3, C36:2, C36:l, C36:0, C37:l, C37:0, C38:4, C38:3, C38:l, C38:0, C39:l, C39:0, C40:2, C40:l, C40:0, C41:2, C41:l, C41:0, C42:47, C42:3, C42:2, C42:l,
  • R 14 is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue stemmed is selected from the group consisting of; C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:0, C18:ln-9, C18:2n-6, and C24:ln-9.
  • sphingomyelin is a mixture of compounds of formula (VIII) wherein the mixture is such that the total number of fatty acid residues (R 14 together with the adjacent carbonyl group) comprised in the mixture are predominately saturated fatty acids, and the least predominant are unsaturated fatty acids. More preferably, the mixture will be such that that 80% to 96% of said fatty acid residues in the mixture are saturated fatty acids, in particular C14, C15, C16, C18, C20, C22, C23, C24 saturated fatty acids more particularly C16, C18, C20, C22 and C24.
  • Phosphatidylcholine is a compound of formula (IX):
  • R 16 and R 17 are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group. More preferably, R 16 and R 17 are each independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.
  • R 16 and R 17 are C13 to C23 branched or unbranched acyclic alkyl, or acyclic alkenyl groups which together with their adjacent carbonyl group are C14 to C24 saturated or unsaturated fatty acid residues, wherein the fatty acids from which the fatty acid residues stem are selected from the group consisting of; C14:0, C15:0, C16:0, C16:l, C18:0, C20:0, C20:l, C20:3, C20:4, C21:0, C22:0, C22:6, C23:0, C24:0, C18:ln-9, C18:2n-6, and C24:ln-9. Even more particularly C14:0, C16:0, C18:0, C18:ln-9, C18:2n-6, C20:l, C20:3, C20:4, and C22:6.
  • Particularly preferred phospholipids include phosphatidylcholine, phosphatidylinositole, phosphatidylserine, and sphingomyelin, more particularly phosphatidylcholine and/or sphingomyelin.
  • the phospholipid is phosphatidylcholine, phosphatidylinositole, phosphatidylserine, or sphingomyelin, or a metabolic precursor or metabolite of any of the foregoing. More preferably, the phospholipid is phosphatidylcholine or sphingomyelin, or a metabolic precursor or metabolite of either of the foregoing.
  • a phospholipid, a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • sphingomyelin a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • the composition comprises sphingomyelin in an amount 200 to 1000 mg, 400 to 700mg, 650mg.
  • phosphatidylcholine, a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • the composition comprises phosphatidylcholine in an amount 300 to 5000mg, 1000 to 5000mg, 3000 to 5000mg, 4000 to 4500mg.
  • phosphatidylinositole, a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • the composition comprises phosphatidylinositole in an amount 50 to 400, 100 to 250, 200 to 210mg.
  • phosphatidylserine, a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • the composition comprises phosphatidylserine in an amount 50 to 500mg, 200 to 500mg, 400mg.
  • phosphatidylethanolamine, a metabolic precursor and/or metabolite thereof is comprised in the composition in an amount up to 99.999% of the composition.
  • the composition comprises phosphatidylethanolamine in an amount 50 to 500mg, 200 to 500mg, 400mg.
  • a metabolic precursor and/or metabolite of one or more phospholipids is used in a composition in place of or in combination with a phospholipid, said compounds may be used in amounts such that the level of phospholipids physiologically delivered by said composition is in line with those set out hereinabove. It is well within the purview of the skilled person to determine appropriate amounts.
  • metabolic precursor and/or metabolite of one or more phospholipid as used herein does not include choline.
  • Non limiting examples of metabolic precursors and/or metabolites of phospholipids are: galactoceramides, glucoceramides, sphingosine, sphingosine-1- phosphate, ceramide, D-erythro-dihydroceramide and ceramide-l-phosphate, and gangliosides.
  • Particularly effective phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylinositol and/or sphingomyelin, in particular sphingomyelin.
  • the phospholipid is phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin and/or a metabolic precursor and/or metabolite of any of the foregoing and/or combinations of any of the foregoing.
  • the phospholipid is sphingomyelin, a metabolic precursor and/or metabolite thereof.
  • Particularly effective metabolic precursors and/or metabolites of phospholipids, in particular sphingomyelin, include ceramide and gangliosides and gangliosides and ceramide -1-phosphate and d-erythro-dihydroceramide.
  • ceramide indicates a lipid molecule wherein a sphingosine backbone is acylated with a fatty acid residue.
  • ceramide may identify a single ceramide species as well as a mixture of single ceramide species.
  • the ceramide is a compound of formula (IXa), or a mixture of compounds of formula (IXa):
  • R 16a and R 17a are each independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.
  • R 16a is a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue.
  • Non limiting examples of C14 to C44 saturated or unsaturated fatty acids from which the fatty acid residue may stem include; C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:l, C25:0, C28:l, C30:2, C30:l, C30:0, C32:3, C32:2, C32:l, C32:0, C33:l, C34:3, C34:2, C34:l, C34:0, C35:2, C35:0, C36:4, C36:3, C36:2, C36:l, C36:0, C37:l, C37:0, C38:4, C38:3, C38:l, C38:0, C39:l, C39:0, C40:2, C40:l, C40:0, C41:2, C41:l, C41:0, C42:47, C42:3, C42:2, C42:l,
  • R 16a is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group which together with the adjacent carbonyl group corresponds to a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue stemmed is selected from the group consisting of; C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:0, C18:ln-9, C18:2n-6, and C24:ln-9, and more particularly the group consisting of C16:0, C18:0, C20:0, C22:0 and C24:0.
  • the ceramide is a mixture of compounds of formula (IXa) wherein the mixture is such that the total number of fatty acid residues (R 16a together with the adjacent carbonyl group) comprised in the mixture are predominately saturated fatty acids, and the least predominant are unsaturated fatty acids. More preferably, the mixture will be such that that 80% to 96% of said fatty acid residues in the mixture are saturated fatty acids, in particular C14, C15, C16, C18, C20, C22, C23, C24 saturated fatty acids, more particularly C16, C18, C20, C22 and C24.
  • ganglioside indicates an oligoglycosylceramide lipid molecule comprising the residue of a ceramide of formula IXa as defined herein.
  • ganglioside may identify a single ganglioside species as well as a mixture of single ganglioside species comprising the residue of a ceramide of formula IXa as defined herein.
  • Particularly effective gangliosides may be monosialoganglioside-3 (GM3) gangliosides and/or disialogangliosides 3 (GD3) gangliosides.
  • GM3 monosialoganglioside-3
  • GD3 disialogangliosides 3
  • Ceramide -1-phosphate and d-erythro-dihydroceramide with comprise a residue of a ceramide of formula IXa as defined herein.
  • Spingomyelin may be synthesised from ceramide and phosphatidylcholine. Accordingly, it may be particularly beneficial if ceramide and/or one or more ganglioside is used in combination with phosphatidylcholine, a metabolic precursor or metabolite thereof.
  • the phospholipid, metabolic precursor and/or metabolite thereof, comprised in the composition of the invention may be natural, synthetic or a mixture thereof.
  • Said metabolic precursor and/or a metabolite may be used in the composition of the invention in their pure form, or substantially pure form. Alternatively, they may be added in the form of a source comprising them. Any source of a phospholipid metabolic precursors and/or metabolite thereof, suitable for ingestion by a subject for which the composition is intended to be consumed may be used in the invention.
  • the phospholipid, metabolic precursor or metabolite thereof will come from natural sources, non limiting examples of which include, eggs, soy, bovine brains, and/or mammalian milk or extracts thereof.
  • soy sources include soy lecithin-food additive
  • mammalian milk include bovine, camel, sheep, goat milk including skilled milks.
  • Non limiting extracts of milk include protein extracts e.g. whey protein and casein, milk fat globule membranes (MFGM) and extracts comprising them.
  • a particularly useful source of a phospholipid, metabolic precursor or metabolite thereof, in particular sphingomyelin, that may be used in the present invention is a bovine milk whey protein concentrate enriched in alpha-lactalbumin, and/or none pure alpha-lactalbumin which has been extracted from milk whey protein, in particular bovine milk whey protein.
  • Alpha-Lactalbumin is a high-quality, easy-to-digest whey protein and is the primary protein found in HM.
  • Alpha-lactalbumin and/or an alpha-lactalbumin enriched milk fraction is ideal for use in lower protein infant formulas due to its high content of essential amino acids, particularly tryptophan.
  • alpha-Lactalbumin is in itself a protein non pure sources may comprise sphingomyelin.
  • a phospholipid a metabolic precursor or metabolite thereof, in particular sphingomyelin is used in the form of a whey protein concentrate enriched in alpha- lactalbumin or as alpha-lactalbumin.
  • a whey protein concentrate enriched in alpha-lactalbumin or alpha-lactalbumin having a phospholipid content, in particular sphingomyelin content higherthan 500 mg/lOOg dry weight of the composition is used.
  • Another particularly useful source of phospholipid, metabolic precursor, or metabolite thereof may be MFGM or extracts comprising them, in particular MFGM, or extracts comprising them from bovine milk.
  • the MFGM or extracts comprising them comprises at least 1%, 2%, 5%, 10%, 20%, 30%, 40% phospholipids and/or at least 0.1%, 0.2%, 0.5% to 5%, 0.8% to 3%, 1% to 2%, 1.6%, 1.9%, 1.8% of phosphatidylcholine, phosphatidylinositole, phosphatidylserine, phosphatidylethanolamine, and/or sphingomyelin.
  • the MFGM may also further comprise magnesium, phosphorus and or calcium, preferably in concentrations ranging from 0.05% to 2%, 0.1% to 0.4%.
  • a composition of the invention comprising a phospholipid and/or a metabolic precursor and/or a metabolite thereof, in particular sphingomyelin, phosphatidylcholine and/or phosphatidylinositol, may be particularly effective at supporting, promoting or optimising de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in one or more of the following brain areas; cerebellum, visual cortex, corpus callosum, internal capsule, frontal lobe, parietal lobe, temporal lobe, motor cortex, frontal cortex..
  • brain areas are associated with one or more of the following: vision, motor function (including coordination and execution of movement) , hemispherical interaction, language function, auditory function (including listening and attention), working memory, executive functioning including problem solving, social processing, and behaviour interaction, spatial reasoning, and language.
  • the composition according to the invention comprises a mineral nutrient as described above, a phospholipid, preferably phosphatidyl choline and/or sphingomyelin, folic acid, choline and DHA.
  • the composition according to the invention comprises a mineral nutrient as described above, a phospholipid, preferably phosphatidyl choline and/or sphingomyelin, folic acid, choline, DHA, AA, and zinc.
  • the composition according to the invention comprises a mineral nutrient as described above, sphingomyelin and choline.
  • composition according to the invention comprises a mineral nutrient as described above, sphingomyelin and DHA.
  • composition according to the invention comprises a mineral nutrient as described above, sphingomyelin and folic acid.
  • composition according to the invention comprises a mineral nutrient as described above, DHA and choline.
  • composition according to the invention comprises a mineral nutrient as described above, DHA and folic acid.
  • composition according to the invention comprises a mineral nutrient as described above, choline and folic acid.
  • composition according to the invention comprises a mineral nutrient as described above, sphingomyelin, folic acid and DHA.
  • composition according to the invention comprises a mineral nutrient as described above, sphingomyelin, folic acid and choline. In another preferred embodiment, the composition according to the invention comprises a mineral nutrient as described above, folic acid, DHA and choline.
  • composition according to the invention comprises a mineral nutrient as described above, sphingomyelin, DHA and choline.
  • the composition according to the invention comprises a mineral nutrient as described above, sphingomyelin, folic acid, choline and DHA.
  • the composition according to the invention comprises a fatty acid derivative comprising DHA and/or ARA, vitamin B12 and/or folic acid, sphingomyelin and iron.
  • the person skilled in the art may identify appropriate amounts of the above mentioned nutrients, metabolic precursors or metabolites thereof based on the nature, purpose, the target subject and the dosage of the composition e.g. how many times per day the composition is to be ingested by the subject. Typically an effective dose will depend on age, size and health status of the subject, on the subject's lifestyle, the amounts of nutrients in the composition, and maybe on the gender of the subject.
  • composition of the invention may be any type of composition suitable for direct administration to a female subject.
  • compositions according to the present invention are preferably in a solid form.
  • the composition may, for example, be in the form of a chewable tablet, dispersible tablet, capsule, lozenge, pastille, chewing gum, powder (e.g. in a sachet), stickpack sachets, or bottle with powder in the cap.
  • the composition is in the form of a tablet, capsule or powder.
  • the tablet or capsule may be provided as a unit dosage form for, e.g. once or twice daily, preferably once daily, administration.
  • a powder composition may be contained in a sachet.
  • a powder composition according to the present invention may be used to sprinkle onto a food or beverage.
  • a particularly preferred embodiment provides a composition according to the invention in the form of a sachet containing a powder, wherein the powder can be dispersed into a beverage (e.g. water, fruit juice, milk, etc.) to provide a palatable nutrient liquid for oral administration.
  • a beverage e.g. water, fruit juice, milk, etc.
  • the composition is a nutritional composition.
  • the nutritional composition may be a nutritionally complete formula, a nutritional supplement, a food product such as a dairy product, a chilled or shelf stable beverage or a soup, a dietary supplement, a meal replacement, or a nutritional bar for example.
  • the composition is a foodstuff intended for consumption by an adult, in particular a pregnant female.
  • the term "nutritional composition” as used herein refers to a composition that nourishes a subject.
  • the composition may be a nutritionally complete formula, for example including a source of protein, carbohydrate and fat.
  • This nutritional composition may be taken enterally, parenterally or intravenously. In one preferred embodiment, the composition is taken enterally and more preferably, orally.
  • the composition comprises one or more of a protein source, a lipid source and a carbohydrate source.
  • such a composition may comprise protein in the range of about 2 to 6 g/100 kcal, lipids in the range of about 1.5 to 3 g/lOOkcal and/or carbohydrates in the range of about 1.7 to 12 g/100 kcal. If the composition is liquid, its energy density may be between 60 and 75 kcal/lOOml.
  • composition is solid, its energy density may be between 60 and 75 kcal/lOOg.
  • the composition is a synthetic nutritional composition.
  • composition means a mixture obtained by chemical and/or biological means.
  • the composition is a hypoallergenic nutritional composition.
  • hypoallergenic nutritional composition means a nutritional composition which is unlikely to cause allergic reactions.
  • the composition is selected from the group consisting of a pharmaceutical composition, a food product, a food extract, drink, food additive, a pet care product, a nutraceutical, and a nutritional supplement.
  • the composition is a maternal supplement.
  • the supplement is preferably taken throughout pregnancy to build up maternal stores of the various constituent components, although supplementation in the second and more particularly the third trimesters is believed to be particularly advantageous.
  • supplementation may continue after birth either via continued consumption of the composition by the mother if the baby is to be breast fed, or by administering a similar composition directly to the baby, for example by way of an infant formula used to feed the baby.
  • the composition is for use in conjunction with an infant formula and/or starter infant formula/and or growing up milk which is administered to the infant after birth.
  • the composition can also be used in conjunction with baby food and/or a fortifier.
  • an infant formula and/or starter infant formula/and or growing up milk and/or baby food and/or fortifier also comprises a mineral nutrient as described above, which further promotes, supports or optimizes one or more of the following: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv)intellectual potential; (v) cognitive potential; and (vi) learning potential; (vii) cognitive function in the infant.
  • infant formula means a foodstuff intended for particular nutritional use by infants during the first four to six months of life and satisfying by itself the nutritional requirements of this category of person (Article 1.2 of the European Commission Directive 91/321/EEC of May 14, 1991 on infant formulae and follow-on formulae).
  • starter infant formula means a foodstuff intended for particular nutritional use by infants during the first four months of life.
  • follow-on formula means a foodstuff intended for particular nutritional use by infants aged over four months and constituting the principal liquid element in the progressively diversified diet of this category of person.
  • GUM Growing up milk
  • the “growing-up milks” are given from one year onwards. It is generally a milk-based beverage adapted for the specific nutritional needs of young children.
  • baby food means a foodstuff intended for particular nutritional use by infants during the first years of life.
  • fortifier refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.
  • the term “weaning period” means the period during which the mother's milk is substituted by other food in the diet of an infant.
  • compositions of the invention may further comprise any other additional ingredients or excipients known to be employed in the type of composition in question.
  • additional ingredients include: proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, essential fatty acids, nucleotides, nucleosides, other vitamins, minerals and other micronutrients.
  • protein sources based on whey, casein and mixtures thereof may be used, for example.
  • acid whey or sweet whey or mixtures thereof may be used as well as alpha- lactalbumin and betalactoglobulin in whatever proportions are desired.
  • the whey protein may be modified sweet whey.
  • Sweet whey is a readily available by-product of cheese making and is frequently used in the manufacture of infant formulae based on cows' milk.
  • sweet whey includes a component which is undesirably rich in threonine and poor in tryptophan called caseino-glyco-macropeptide (CGMP).
  • CGMP caseino-glyco-macropeptide
  • CGMP from sweet whey results in a protein with a threonine content closer to that of human milk.
  • This modified sweet whey may then be supplemented with those amino acids in respect of which it has a low content (principally histidine and tryptophan).
  • a process for removing CGMP from sweet whey is described in EP 880902 and an infant formula based on this modified sweet whey is described in WO 01/11990.
  • the proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. It may be desirable to supply partially hydrolysed proteins (degree of hydrolysis between 2 and 20%), for example for subjects believed to be at risk of developing cows' milk allergy.
  • a whey protein hydrolysate may be prepared by enzymatically hydrolysing the whey fraction in two steps as described in EP 322589.
  • the whey proteins may be subjected to triple hydrolysis using Alcalase 2.4L (EC 940459), then Neutrase 0.5L (obtainable from Novo Nordisk Ferment AG) and then pancreatin at 55°C.
  • Alcalase 2.4L EC 940459
  • Neutrase 0.5L obtainable from Novo Nordisk Ferment AG
  • pancreatin pancreatin at 55°C.
  • the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockage during the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example about 7% by weight of lysine which greatly improves the nutritional quality of the protein source.
  • Any suitable dietary protein may be used for example animal proteins (such as milk proteins, meat proteins and egg proteins); vegetable proteins (such as soy protein, wheat protein, rice protein, and pea protein); mixtures of free amino acids; or combinations thereof.
  • animal proteins such as milk proteins, meat proteins and egg proteins
  • vegetable proteins such as soy protein, wheat protein, rice protein, and pea protein
  • mixtures of free amino acids or combinations thereof.
  • proteins include casein, alpha-lactalbumin, whey, soy protein, rice protein, corn protein, oat protein, barley protein, wheat protein, rye protein, pea protein, egg protein, sunflower seed protein, potato protein, fish protein, meat protein, lactoferrin, serum albumin, immunoglobins, and combinations thereof.
  • Milk proteins such as casein and whey, and soy proteins are particularly preferred.
  • compositions of the present invention may comprise one or more amino acids.
  • amino acids include leucine, threonine, tyrosine, Isoleucine, arginine, alanine, histidine, isoleucine, proline, valine, cysteine, glutamine, glutamic acid, glycine, serine, arginine, lysine, methionine, phenylalanine, tryptophane, asparagine, aspartic acid, and combinations thereof.
  • compositions of the present invention may contain a carbohydrate source.
  • Any carbohydrate source may be used, such as lactose, saccharose, maltodextrins, fructose, glucose, honey, sucrose, corn syrup solids, starch, and combinations thereof.
  • the compositions of the present invention may contain a lipid source.
  • the lipid source may be any lipid.
  • the lipid source preferably provides 5% to 40% of the energy of the composition, for example 20% to 30% of the energy.
  • a suitable fat profile may be obtained using a blend of canola oil, corn oil and high-oleic acid sunflower oil.
  • Preferred fat sources include milk fat and vegetable oils.
  • the essential fatty acids linoleic and a-linolenic acid may also be added.
  • oils containing high quantities of preformed arachidonic acid (AA) and docosahexaenoic acid (DHA) such as fish oils or microbial oils may be added.
  • the lipid source preferably has a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15:1; for example about 8:1 to about 10:1.
  • Non limiting examples of lipids include: palm oil, palm olein, high oleic sunflower oil, high oleic safflower oil, canola oil, fish oil, coconut oil, bovine milk fat, and combinations thereof.
  • compositions of the present invention may comprise one or more essential fatty acids.
  • essential fatty acids include: linoleic acid (LA), a-linolenic acid (ALA) and polyunsaturated fatty acids (PUFAs).
  • the compositions of the invention may further contain gangliosides monosialoganglioside-3 (GM3) and disialogangliosides 3 (GD3), other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and combinations thereof.
  • compositions may also contain at least one prebiotic, preferably in an amount of about 0.3 to about 10 %.
  • a prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health.
  • Such ingredients are non-digestible in the sense that they are not broken down and absorbed in the stomach or small intestine and thus pass intact to the colon where they are selectively fermented by the beneficial bacteria.
  • prebiotics include certain oligosaccharides.
  • Non-limiting examples of prebiotics include: oligosaccharides optionally containing fructose, galactose, mannose; dietary fibres, in particular soluble fibres, soy fibres; inulin; and combinations thereof.
  • Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS), mannan-oligosaccharides (MOS), oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), malto- oligosaccharides, gums and/or hydrolysates thereof, pectins and/or hydrolysates thereof, and combinations of the foregoing
  • oligosaccharides are described in Wrodnigg, T. M.; Stutz, A.E. (1999) Angew. Chem. Int. Ed. 38:827-828 and in WO 2012/069416 which is incorporated herein by reference.
  • compositions may also comprise at least one probiotic bacterial strain.
  • a probiotic is a microbial cell preparation or components of microbial cells with a beneficial effect on the health or well-being of the host.
  • probiotics include: Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, in particular selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus planta
  • the amount of probiotic if present, preferably varies as a function of the age of the person or animal.
  • the probiotic may be present in amounts of: about 5 million to about 2500 million, about 10 million to about 2500 million, about 30 million to about 2500 million, about 50 million to about 2500 million, about 50 million to about 1000 million, about 75 million to about 2500 million, about 75 million to about 1000 million, about 100 million to about 2500 million, about 100 million to about 1000 million, about 250 million to about 2500 million, about 250 million to about 1000 million, about 500 million to about 2500 million, about 500 million to about 1000 million, about 750 million to about 2500 million or about 750 million to about 1000 million, about 1 billion to about 2.5 billion, about 1.5 to about 2.5 billion bacteria per dosage form.
  • compositions of the invention may further comprise dietary fibre.
  • Dietary fibre passes through the small intestine undigested by enzymes and functions as a natural bulking agent and laxative. Dietary fibre may be soluble or insoluble and in general a blend of the two types is preferred. Suitable sources of dietary fibre include soy, pea, oat, pectin, guar gum, gum Arabic, fructooligosaccharides, galacto-oligosaccharides, sialyl-lactose and oligosaccharides derived from animal milks.
  • a preferred fibre blend is a mixture of inulin with shorter chain fructooligosaccharides.
  • the fibre content is between 10 and 40 g/1 of the formula as consumed.
  • the composition may also contain minerals and micronutrients such as trace elements and vitamins in accordance with the recommendations of Government bodies such as the USRDA.
  • the compositions may also contain one or more vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals.
  • vitamins and minerals include: vitamin A, vitamin Bl, vitamin B2, vitamin B6, vitamin K, vitamin C, vitamin D, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, magnesium, copper, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine, L- carnitine, and combinations thereof. Minerals are usually added in salt form.
  • composition of the invention comprises B12 and/or folic acid, it is particularly beneficial if it further comprises B6.
  • B6 The presence and amounts of specific minerals and other vitamins will vary depending on the numerous factors, such as age weight and condition of the person or animal the composition is administered to.
  • the composition may contain per daily dose one or more of the following micronutrients in the ranges given:- 300 to 500 mg calcium, 50 to 100 mg magnesium, 150 to 250 mg phosphorus, 5 to 20 mg iron, 1 to 7 mg zinc, 0.1 to 0.3 mg copper, 50 to 200 [mu]g iodine, 5 to 15 [mu]g selenium, 1000 to 3000 [mu]g beta carotene, 10 to 80 mg Vitamin C, 1 to 2 mg Vitamin Bl, 0.5 to 1.5 mg Vitamin B6, 0.5 to 2 mg Vitamin B2, 5 to 18 mg niacin, 0.5 to 2.0 [mu]g Vitamin B 12, 100 to 800 [mu]g folic acid, 30 to 70 [mu]g biotin, 1 to 5 [mu]g Vitamin D, 3 to 10 IU Vitamin E.
  • micronutrients in the ranges given:- 300 to 500 mg calcium, 50 to 100 mg magnesium, 150 to 250 mg phosphorus, 5 to 20 mg iron, 1 to 7 mg zinc, 0.1 to 0.3 mg copper
  • One or more food grade emulsifiers may be incorporated into the formula if desired; for example diacetyl tartaric acid esters of mono- and di- glycerides, lecithin and mono- and di-glycerides. Similarly suitable salts and stabilisers may be included.
  • the formula is preferably enterally administrable; for example in the form of a powder or a liquid concentrate for re-constitution with milk or water, a solid product or a ready-to-drink beverage.
  • compositions may optionally contain other substances which may have a beneficial effect such as nucleotides, nucleosides, and the like.
  • nucleotides include: cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), and combinations thereof.
  • compositions for use in the invention may be prepared in any suitable manner.
  • the composition may be prepared by blending together the protein source, the carbohydrate source, and the fat source in appropriate proportions.
  • the emulsifiers may be included in the blend.
  • the vitamins and minerals may be added at this point but are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending.
  • Water preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The liquid mixture may then be thermally treated to reduce bacterial loads.
  • the liquid mixture may be rapid ly heated to a temperature in the range of about 80°C to about 110°C for about 5 seconds to about 5 minutes. This may be carried out by steam injection or by heat exchanger; for example a plate heat exchanger.
  • the liquid mixture may then be cooled to about 60°C to about 85°C; for example by flash cooling.
  • the liquid mixture may then be homogenised; for example in two stages at about 7 M Pa to about 40 M Pa in the first stage and about 2 M Pa to about 14 M Pa in the second stage.
  • the homogenised mixture may then be fu rther cooled to add any heat sensitive components; such as vitamins and minerals.
  • the pH and solids content of the homogenised mixture is conveniently standardised at this point.
  • the homogenised mixture is transferred to a suitable drying apparatus such as a spray d rier or freeze drier and converted to powder.
  • the powder should have a moistu re content of less than about 5% by weight.
  • probiotic(s) they may be cultured according to any suitable method and prepared for addition to the formula freeze-d rying or spray-drying for example.
  • bacterial preparations can be bought from specialist suppliers such as Ch ristian Hansen and Morinaga already prepared in a suitable form for addition to food products. Such bacterial preparations may be added to the formula by dry mixing.
  • the phospholipid may be added at any stage during this procedure, but is preferably added after the heating step.
  • the composition comprises triglycerides with high sn-2 palmitate, preferably triglycerides having more than 33% of the palmitic acids in sn-2 position.
  • palmitic acid comprises from about 15 to about 25%, such as from about 15 to about 20%, of the total fatty acids content of the formula, by weight, and at least from about 30%, for example, from about 35 to about 43% of the total palmitic acid content is in the sn-2 position.
  • a commercially available composition sold by Lipid Nutrition is BetapolTM B-55, which is a triglyceride mixture derived from vegetable oil in which at least 54% of the palmitic acid is in the sn-2 position of the glycerol molecule.
  • the fat content of the composition of the invention is about 40-50% BetapolTM B-55 by weight, for example, from about 43% to about 45% by weight.
  • a conventional food product such as a yoghurt, or a breakfast cereal may be enriched with one or more mineral nutrients as described above.
  • a composition containing a mineral nutrient as described above in an amount sufficient to achieve the desired effect in an individual can be prepared.
  • This composition may be in the form of tablets, capsules, pastilles or a liquid for example.
  • the supplement may further contain protective hydrocolloids (such as gu ms, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), sweeteners, texturizers, adsorbents, carriers, chelating agents, fillers, co- compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobials.
  • protective hydrocolloids such as gu ms, proteins, modified starches
  • binders film forming agents
  • composition may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, ta lc, sugars, starch, gu m arabic, vegetable oils, polyalkylene glycols, flavouring agents, thickeners, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.
  • conventional pharmaceutical additives and adjuvants, excipients and diluents including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, ta lc, sugars, starch, gu m arabic, vegetable oils, polyalkylene glycols, flavouring agents, thickeners, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers
  • composition of the invention has a positive effect on denovo myelination, in particular the denovo myelination trajectory, in the brain of the infants or young children whose mothers are administered such compositions, before or during pregnancy, or during postpartum lactation.
  • Such positive effects can comprise the promotion and/or support of an optimal denovo myelination, in particular de novo myelination trajectory, which may determine appropriate cognitive, intellectual and learning potential, the development of cognitive skills and abilities and learning in the infant or young children.
  • An optimal de novo myelination trajectory may also prevent development of cognitive impairment or delay.
  • the health effect can be observed after a few days, weeks, months or years of use of the composition comprising choline.
  • the effect of the invention can be preventive (for example, avoiding a suboptimal de novo myelination, in particular de novo myelination trajectory in the brain, brain structure, brain connectivity, cognitive, intellectual and/or learning potential or cognitive function) or curative (for example, restoring an optimal de novo myelination, in particular de novo myelination trajectory in the brain, brain structure, brain connectivity, cognitive, intellectual and/or learning potential or cognitive function).
  • the health effect related to the infant can be measured by various methods as illustrated in the example below.
  • the composition of the invention may be used to promote, support or optimize de novo myelination, in particular the de novo myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter in the brain, and/or brain connectivity and/or intellectual potential, and/or cognitive potential and/or learning potential and/or cognitive functioning in a subject whose mother receives such a composition, before or during pregnancy, or during postpartum lactation.
  • composition of the invention described herein may have long term health benefits.
  • Dementia e.g. Alzheimer's disease
  • the risk of a subject suffering from dementia, in particular Alzheimer's disease is often associated with a person's intellectual ability or intelligence. Accordingly, by optimizing a subject's intellectual, cognitive and or learning potential the risk of a subject developing dementia in particular Alzheimer's disease may be reduced.
  • a variety of other psychiatric and/or neurological disorders e.g. autism and schizophrenia are also linked to brain structure, and in particular to the amount and/or spatial distribution of white matter throughout the brain.
  • psychiatric and/or neurological disorders e.g. autism are prevented or that the risk of them developing is reduced, or that the severity of said condition(s) is reduced.
  • choline may be employed directly in place of the composition of the present invention in any method or use set out herein.
  • choline is administered separately, sequentially and/or simultaneously to one or more of the following ingredients: a vitamin, a mineral, a fatty acid derivative and a phospholipid or metabolite thereof, or a metabolic precursor thereof.
  • the additional ingredient is administered concurrently with the choline.
  • the additional ingredient is administered simultaneously as part of the composition according to the invention.
  • subsequentially means administering the additional ingredient within a certain time period before or after the choline.
  • the time delay will vary depending on the nature of the additional ingredient and/or the choline.
  • the additional ingredient is administered at least 2 hours, or at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours, before the choline.
  • the additional ingredient is administered at least 2 hours, or at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours, after the choline. All particulars of the invention apply equally to the composition comprising choline, and to the direct use of choline and/or any other ingredient.
  • the invention provides a means of promoting, supporting or optimizing de novo myelination in infants or young children showing a sub-optimal de novo myelination trajectory in the brain which may result in cognitive deficits, impaired cognitive abilities and/or sub-optimal cognitive development.
  • infants can be preterm or low birth weight infants or infants born small for gestational age.
  • the invention is also suitable for promoting, supporting or optimizing myelination in infants or young children that are born at term. All infants can benefit from the invention as all infants are or can be susceptible to develop a sub-optimal myelination trajectory in the brain.
  • the infants and young children are 0-3 months, 0-6 months, or 0-12 months or 0-36 months of age or 0-60 months of age.
  • composition of the invention can be administered to the female subject pre-pregnancy, during pregnancy, or during lactation, or a combination thereof.
  • the compositions of the invention are administered prenatally to a female subject and thereby indirectly transmitted to the developing embryo or fetus e.g. via the placenta or amniotic fluid.
  • the exposure of the offspring to the compositions of the invention is in utero when the composition is administered to the mother during pregnancy.
  • Prenatal administration of the compositions may prevent the onset of, or reduce the risk, of sub- optimal myelination in the offspring, and the effects associated therewith.
  • compositions of the invention are administered postnatally to a lactating female, and are thereby indirectly transmitted to the neonate or infant via the ingestion of maternal milk, i.e. the exposure of the offspring to the compound is solely via the mother's milk.
  • the composition is for administration to the female subject pre-pregnancy, for example, in female subjects desiring to get pregnant.
  • Pre-pregnancy administration of compositions according to the invention may impact the intrauterine environment.
  • pre-pregnancy supplementation or administration refers to administration from about 1-24 months, 1-18 months, 1-12 months, 1-6 months, or 1-3 months prior to pregnancy.
  • the composition is administered for at least 1 week, or for at least 2 weeks, or for at least 3 weeks, or for at least 1 month, or for at least 2 months, or for at least 3 months, or for at least 4 months, or for at least 5 months, or for at least 6 months, or for at least 12 months, or at least 18 months or 24 months prior to conception, for example, in women who are intending to, or trying to, become pregnant.
  • the composition is for administration to the female subject during pregnancy, e.g. prenatally.
  • prenatally refers to the period before birth, during or relating to pregnancy.
  • a reference to administration of a composition according to the invention during pregnancy i.e. prenatal administration
  • administration of the composition is initiated as soon as possible following conception until at least the end of the period of embryogenesis.
  • the embryogenesis period encompasses the first 8 weeks of development (10 weeks of gestation).
  • the composition is administered throughout the whole gestation period.
  • compositions of the invention are administered during a substantial part of the gestation period.
  • administration can be within: the first week, the first two weeks, the first month, the first trimester, the second trimester or the third trimester of pregnancy.
  • the administration may be continued until at least the birth of the offspring.
  • the composition is administered as soon as possible from conception until birth, i.e. during the full gestation period.
  • the administration is preferably for a period of from: about 1 week to birth, about 2 weeks to birth, about 4 weeks to birth, about 8 weeks to birth, about 12 weeks to birth, about 18 weeks to birth, about 24 weeks to birth.
  • composition of the invention may additionally be administered pre-pregnancy to the female subject as well as during pregnancy and/or during lactation.
  • the composition is for administration to the female subject after birth, for example, during postpartum lactation. During this period, the infant can receive the beneficial effects of the composition via breast milk from the mother. After birth, the composition can be administered for part or all of the lactation period.
  • the present invention relates to a composition for a lactating postpartum female subject, the composition comprising one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper and magnesium, for promoting, supporting or optimizing one or more of the following:
  • Reference to a lactating female subject refers to a female subject who is exclusively or partially breast feeding her offspring.
  • a reference to administration of a composition according to the invention during lactation includes administration of the compound postnatally at any time during which the offspring is exclusively or partially ingesting the maternal milk (the subject's breast milk).
  • administration during lactation may be for the period starting from onset of lactation until the end of the weaning process, i.e. when the offspring has ceased to ingest the maternal milk. During this period, the offspring may be exclusively or partially ingesting the maternal milk.
  • the administration of the composition during lactation includes administration for a period of 1-24 months, 2-20 months, 3-18 months, 6-12 months, 4-12 months or 4-8 months following the onset of lactation during which the offspring is exclusively or partially ingesting the maternal milk.
  • the composition of the invention may be particu larly beneficial when administered to infants just after birth (0-4 weeks, 0-8, 0-12, 0-24 weeks) because it is at that time that myelination process has started and significantly develops.
  • the composition of the invention is administered during lactation for two weeks following the onset of lactation when the offspring is exclusively or partially ingesting the maternal milk.
  • administration of the compositions to the female subject is initiated postpartum, i.e. not during pregnancy, but instead from the onset of lactation.
  • composition can be advantageously used for promoting, supporting or optimizing one or more of the above aspects in the infant wherein the composition is administered to the mother of the infant during lactation postpartum and the advantageous effect in the infant can be observed later in life, e.g. during childhood and/or adolescence.
  • the composition of the invention is administered both prenatally, i.e. at any period from conception to birth, as well as postnatally during lactation, i.e. at any period from birth until the end of the weaning process, i.e. when the offspring has ceased to ingest the maternal milk.
  • the composition may be administered both prenatally for any period as defined above in relation to prenatal administration, as well as postnatally for any period as defined above in relation to the lactation period, and any combination of these periods as described above.
  • compositions of the invention are administered as a pre-pregnancy, pregnancy or lactation dietary supplement in a female subject to promote myelination. Further, when employed as a pre-pregnancy supplement alone, or in combination as a pregnancy and/or lactation supplement, the compositions of the invention may provide health benefits to future offspring.
  • the composition of the invention is administered to the female subject for a period of from 2 to 52 weeks. In one preferred embodiment, it is administered to the female subject for a period of from 2 to 24 weeks, or 2 to 12 weeks. In a more preferred embodiment, the composition of the invention is administered to the female subject for 2 to 52 weeks and started shortly after the infant or young children are born or breastfeeding is interrupted. In one embodiment, composition of the invention is fed to the mother for 2 to 24 weeks, or 2 to 12 weeks, and started shortly after the infants or young children are born or breastfeeding is interrupted.
  • the maternal supplement is used in conjunction with an infant formula and/or starter infant formula/and or growing up milk which is administered to the infant after birth.
  • the composition can also be used in conjunction with baby food and/or a fortifier.
  • an infant formula and/or starter infant formula/and or growing up milk and/or baby food and/or fortifier also comprises one or more mineral nutrients selected from iron, zinc, calcium, phosphorus, copper and magnesium, which further promotes, supports or optimizes one or more of the following: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv)intellectual potential; (v) cognitive potential; and (vi) learning potential; (vii) cognitive function in the infant.
  • MRI Magnetic Resonance Imaging: MRI brain scans of infants and children between 0 and 5 years were acquired using a white matter imaging technique. This technique provides a quantitative measure, the Myelin Water Fraction (MWF), which is a surrogate marker of myelin content in the brain. When mapped as a function of time across early childhood, myelination trajectories can be generated.
  • MMF Myelin Water Fraction
  • Infant formula composition six infant formulas fed to infants participating in a study were analyzed for their composition/level of myelin-relevant nutrients.
  • Nutritional compositions were tested in standard, commercially-available infant formulas of different brands/suppliers and showing variable levels on the nutrients therein contained.
  • T Age-standardized (T)- scores of gross motor, visual reception and language (expressive and receptive) derived from the Mullen Scales of Early Learning, a standardized and validated measurement tool of early cognitive development for infants and children 6 years of age or younger.
  • Maternal nutritional intake during pregnancy was assessed at 34 weeks of gestation using the Automated Self-Administered 24-hour (ASA24) dietary assessment tool, a web-based tool that enables multiple, automatically coded, self-administered 24-hour recalls.
  • ASA24 Automated Self-Administered 24-hour
  • a combination of retrospective and prospective data were acquired from parents via detailed medical histories and parental interview on the type of infant formula used, percentage of breastfeeding to formula feeding, and length of exclusive breastfeeding. This information was updated at each study visit, which occurred approximately every 6 months for children under 2 years, and yearly for older children. Using this information, children were categorized into one of 2 groups: #1. Exclusively formula-fed; and #2. Exclusively breastfed for at least 90 days (3 months). Children who were fed a combination of breastmilk and formula within 3 months were excluded from our analysis. Infants within the exclusively formula-fed group were further subdivided based on parental reports of the main infant formula used throughout the first 3 months. Main formula was defined as that given 90% of the time or more (in the case were parents used an alternate brand during vacation, for example).
  • mcDESPOT multicomponent Driven Equilibrium Single Pulse Observation of Ti and T 2 white matter imaging technique Deoni et al. (Magn. Reson. Med. 2008, 60:1372-1387), which provides a quantitative measure of the myelin water fraction (MWF)- a measure of myelin content - at each point throughout the brain. All infants were scanned during natural (i.e. non-sedated) sleep using acoustically-muffled mcDESPOT imaging protocols. Total imaging times ranged from 19 minutes for the youngest toddlers to 24 minutes for the older 4 year-old children.
  • a three-pool signal model (comprising the myelin- associated water; intra-extra axonal water; and a non-exchanging free-water pool) was fit to the mcDESPOT data to derive voxel-wise MWF maps.
  • Each child's map was then non-linearly aligned to a study specific template.
  • White matter masks corresponding to 5 bilateral regions (frontal, temporal, occipital, parietal, and cerebellar WM) as well as the body, genu, and splenium of the corpus callosum were created from common databases, registered to the common template, and superimposed onto each child's MWF map. Mean values for each region were then determined for each child and used for subsequent developmental analysis and trajectory modeling.
  • MSEL Mullen Scales of Early Learning
  • P ⁇ 0.05 iron, sphingomyelin, folic acid, choline, DHA.
  • P ⁇ 0.15 zinc, and phosphatidylcholine.
  • Nutritional components that were found to be highly correlated with each other were:
  • Zinc, calcium, magnesium, copper, and phosphorus Zinc, calcium, magnesium, copper, and phosphorus.
  • myelination myelin water fraction
  • cerebellum visual cortex
  • internal capsule motor & somatosensory cortices
  • corpus callosum frontal cortex
  • temporal white matter results are reported in Fig.le
  • myelination For calcium, an association with myelination (myelin water fraction) was observed over time in the brain, in particular in the cerebellum, visual cortex, Motor & Somatosensory Cortices, Corpus Callosum, Frontal Cortex, Temporal White Matter. Results are reported in Fig. If
  • myelination For phosphorus, an association with myelination (myelin water fraction) was observed over time in the brain, in particular in the cerebellum, visual cortex, motor & somatosensory cortices, prefrontal cortex. Results are reported in Fig.lg
  • myelin water fraction For phosphatidylinositol, an association with myelination (myelin water fraction) was observed over time in the brain, in particular in the cerebellum, visual cortex, motor cortex, frontal cortex. Results are reported in Fig.lL.
  • myelination For phosphatidylcholine, an association with myelination (myelin water fraction) was observed over time in the brain, in particular in the cerebellum, visual cortex, internal capsule, frontal lobe, parietal lobe, temporal lobe. Results are reported in Fig.lM.
  • myelination For choline, an association with myelination (myelin water fraction) was observed over time in the brain, in particular in the cerebellum, visual cortex, thalamus, parietal cortex, and frontal lobe. Results are reported in Fig. In.
  • trajectories of longitudinal myelin development were calculated using repeated MWF data from children for whose infant formulas contained a differing amount of zinc, calcium, magnesium, phosphorus, and copper (composition of such formulas is reported below in Table 2b). Trajectories were calculated using a longitudinal nonlinear mixed effects approach. Modified Gompertz growth models were fitted to the data of children for each formula group. Results are reported in Fig.lc.
  • the available data set of 21 mother-child pairs were taken from a hybrid cross- sectional/longitudinal cohort at the Children's Hospital Colorado, US.
  • Pregnant women were drawn from the local community with the following inclusion criteria: At least 18 years of age, English-speaking (primary language), singleton pregnancy, no reports of smoking, alcohol or illicit drug use during pregnancy, no abnormalities on fetal ultrasound, no history of high blood pressure or diabetes before or during pregnancy, no personal history of major psychiatric or learning disorder (including major depressive disorder) or use of psychiatric medication, no reports of major psychiatric or learning disorders in other children, suspicion of metal in body or other MRI contraindications. These criteria are meant to provide a generally healthy cohort devoid of major risk factors for abnormal or altered neurodevelopment.
  • B12 Cerebellum, temporal lobes, frontal lobe
  • Folic Acid Cerebellum, brainstem
  • DHA Cerebellum, Internal Capsule, Temporal lobe, Frontal cortex, Motor Cortex Results are reported in Figures.50a to 50e.
  • NPCs neural progenitor cell
  • E14 pup brains were harvested and placed in ice cold DPBS(IX) + 10% Pen/Strep, then they were dissected using a dissecting microscope. From each pup, one brain hemisphere was placed in 2 ml of Neurobasal media/ 10% P/S/ 10X Hepes and another brain hemisphere was placed in another tube.
  • tissue from each tube was aseptically and manually dissociate into single cells, neurobasal complete medium was added and centrifuged at 130G for 5 min. The tissue was then re- suspended in neurobasal complete media with GF and placed in a corning suspension culture dish 100mm X 20mm (# 430591). Cells were passage twice using a 1:3 ratio, after what they were centrifuged (130g 5min), resuspended in freezing media (10%DMSO and neurobasal complete media, no GF) and frozen in liquid nitrogen (LN2).
  • Vials were remove from LN2, quickly defrosted, and cells were transferred, dropwise, to a 15mL conical.10 mis of complete neurobasal media was added. Cells were transferred to a suspension culture dish, and placed in an incubator for 2 hours. At 1.5 hours, cells were examined. Based on the health and number, the number of plates needed was estimated and the appropriate amount of complete neurobasal media was warmed. After 2 hours, cells were put in a 15mL conical tube and spun at 130G, 5
  • 3-4 mLs of cells were taken out of the tilted plate and add to a 15 ml conical. Some of the remaining media was used to rinse down the plate. All remaining media was drawn up and put into a 15 ml conical tube, and Spun at 130G for 5 min. All media was removed. The cells were gently resuspended in 5mls of warm PBS, spun again. PBS was then removed and the cells were then gently resuspended in 500 ⁇ of Accutase(CorningTM AccutaseTM Cell Detachment Solution, # 25058CI). The cells were then Pipetted gently with a ⁇ tip to break up pellet, and then they were placed in a shaking water bath for 5-10 minutes, after which time they were swirled by hand frequently.
  • Control and compound media was made with #2 media and contain 29uM Choline,
  • the media was Pipetted GENTLY using a 1000 ⁇ tip and a then a 200ul tip to further disperse cells.
  • Clumps were no bigger than ⁇ 3-5 cells. 5-10 mis of warm media (GF) was added to dilute enzyme. 2 mis of media was added. This was pipetted with a 1000 ul pipette, then 3 mis with added with a serological pipette. Cells were strained through a cell culture approved 40uM strainer before they were plated.
  • GF warm media
  • AB staining solution 1% Goat serum, IxPBS, and 0.1% triton X
  • AB staining solution 1% Goat serum, IxPBS, and 0.1% triton X
  • the cells were then Stained with Dapi 1:5000 in AB staining solution, lOOul per well, the cells were then incubated at room temp forl5 min in the dark. The cells were then washed 2 times
  • Imaging was carried out using a GE Cytell imager or LSM 710, Zeiss confocal microscope and the diameters of neurospheres with ImageJ software (National Institutes of Health) was analysed.
  • Control and compound media were made with #2 media and contain 29uM Choline, Low (5uM) and medium (70uM) choline media was made with #3 media.
  • the cells were culture for 9 d in media plus 2% Nu Serum, the medium was changed every 2nd day. For fixation and subsequent immunohistochemical analysis the medium was removed, the cells were rinsed once with IX PBS for 5 min, and fixed with 4% PFA in IX PBS for 15min at 4°C. The cells were then washed twice with IX PBS for 5 min, left in IX PBS, wrapped in foil and left overnight at 4C, or they were immediately primary antibody staining was carried out.
  • AB staining solution 250 ul per well (the antibodies were only kept out on ice for a short time, mouse anti-MAP2 or TUJ 1 1:500 (neuron marker),rabbit anti-GFAP( glial marker) 1:1000, chicken anti-Nestin CFP (EGFP antibody (progenitor cell marker)) 1: 1000.
  • AB staining solution was removed and a solution of primary antibodies was added to each chamber. The cells were wrapped in foil and kept overnight 4°C. The cells were then washed with 400ul of
  • the cells were incubated at room temp fori hour in the dark and washed 2 time in AB staining solution for five mins. They were then kept at 4°C or Imaged using a GE Cytell imager or LSM 710, Zeiss confocal microscope and analyze with ImageJ software(National Institutes of Health).
  • MAP2 Microtubule-associated protein 2
  • GFAP glial fibrillary acidic protein
  • EGFP antibody Nestin CFP
  • Control and compound media was made with #2 media and contains 29uM Choline, Low (5uM) and medium (70uM) choline media was made with #3 media.
  • LSM 710 Zeiss confocal microscope (with ImageJ software (National Institutes of Health) analysis).
  • Results are shown in Tables 3 -7 and figures 2 to 5.
  • Ingredient C2 a whey protein concentrate enriched in alpha lactalbumin(Sample Manager ID: K2Q-00030); first infant milk containing whey protein concentrate enriched in alpha Lactalbumin (Sample Manager ID: K2Q-00032); cow's milk (whole milk); human breast milk (quality control pool of 6 individual samples, collected after week 4 following child birth; Lee Biosolutions, St Louis, Ml, USA).
  • Cow milk and Human milk A quantity of 500 ⁇ of homogenized liquid was aliquoted to a 10-mL glass tube.
  • Analytes were extracted following the MP on quantification of human breast milk by UPLC-MS/MS (RDLS-MP-80138-Rev01) in triplicate using 9.5 mL of a mixture of chloroform/methanol (2+1). Briefly, the tubes were shaken and placed in an ultrasonic bath at 40 °C for 15 min, followed by centrifugation for 10 min at 2500 rpm. A volume of 2 mL of potassium chloride solution (0.88%, m/m) was added to the liquid phase then shaken and centrifuged for 10 min at 2500 rpm. The lower organic phase was transferred into a glass vial, evaporated to dryness under gentle N 2 stream and reconstituted in 500 ⁇ of chloroform/methanol (9+1) before injection into the LC-MS.
  • Q Exactive Plus Orbitrap was equipped with an atmospheric pressure chemical ionization (APCI) probe operated in the positive ion mode.
  • APCI and MS parameters were as follows: corona discharge current 4.0 ⁇ , sheath gas and auxiliary gas 24 and 5 arbitrary units, respectively; capillary and vaporizer temperatures 320 and 390 °C, respectively, sweep gas flow rate was 0 arbitrary unit and s-lens RF level was 80.
  • Automatic gain control (AGC) target value was set at 1 ⁇ 10 6 charges and maximum injection time at 100 ms with resolution of 35 ⁇ 00 and 1 microscan per full MS.
  • AGC was set to 1 ⁇ 10 6 charges and maximum injection time of 250 ms with resolution of 17'500 with 1 microscan in the data independent fragmentation mode.
  • An inclusion list of selected parent ions was used with normalized collision energy of 30%. Data were acquired over the mass range 133-2000 Da in profile mode. External mass calibration was applied. The system was controlled by Xcalibur 3.0 (Ther
  • SM species were extracted from total ion chromatogram using accurate mass.
  • Parent ions corresponded to in-source loss of phosphatidylcholine into ceramide.
  • An inclusion list was used for specific fragmentation of 57 SM regioisomers built on parent ions corresponding to m/z of ceramide with loss of water [Cer-hhO+hT], based on LipidView database and literature (Trenerry V.C., Akbaridoust G., Plozza T., Rochfort S., Wales W.J., Auldist M ., Ajilouni S. Ultra-high- performance liquid chromatography-ion trap mass spectrometry characterisation of milk polar lipids from dairy cows fed different diets.
  • SM fractions were collected between 8.5 and 10 min into glass tubes 5 times for each sample.
  • FAM E analyses were conducted in triplicate following the MP for quantification of fatty acid in human milk by gas chromatography (RDLS-M P- 8980-00030-Rev01-FAME_Human milk fat 2012, Vers. 1.0).
  • Hydrophilic interaction liquid chromatography was used to separate PL classes (i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and SM).
  • PL classes i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and SM.
  • PI phosphatidylinositol
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • SM Hydrophilic interaction liquid chromatography
  • Table 8 SM species detected (indicated by x) in ingredient, infant formula, cow's milk and human milk samples. SM species that were only detected in human milk are indicated in bold.
  • the species SM 24: 1, SM 38:4, SM 38:3 and SM 42:4 were only found at trace levels in human milk.
  • Relative abundance of SM species The relative abundance (%) of SM within different milk products was estimated based on the peak area divided by the sum of all peak area corresponding to SM species in the chromatogram per each sample.
  • Figure 6 shows the relative abundance of the main SM species in ingredient, infant formula, cow's milk and human milk.
  • SM 32:1, SM 32:0, SM 33:1; SM 34:1, SM 38:0, SM 39:1, SM 39:0 and SM 41:1) were lower in human milk than in ingredient, infant formula and cow's milk.
  • SM 36:2, SM 36:1, SM 36:0, SM 37:1, SM 38:2, SM 38:1, SM 40:1, SM 42:2 and SM 42:1 had higher relative abundance in human milk compared to the other milk products.
  • Human milk sample consisted of a quality control pool of 6 individual samples collected at or later than 4 weeks after child birth. Knowing that SM abundance in human milk varies in function of the diet and lactation time, this can partly explain the observed differences. Nevertheless, despite the variations in the relative abundance of some SM species, >85 % of the SM species that were detected in human milk were also identified in infant formula and in cow's milk.
  • Table 9 Percentage of SFA, MUFA and PUFA detected in SM fraction from different milk products.
  • MUFAs Monounsaturated FAs
  • Oleic acid 18:ln-9 and nervonic acid 24:ln-9 were the 2 MUFAs detected.
  • 24:ln-9 was found in relative higher proportion in human milk compared to the other milk products and this is in agreement with the literature.
  • the only PUFA linoleic acid 18:2n-6 was found relatively higher in the tested infant formula and human milk compared to the other products.
  • omega-3 PUFAs were not detected in SM fraction.
  • Example 6 An examples composition in accordance with the invention is set out in Table 10. Said composition could be a pre-pregnancy, pregnancy or lactation supplement.
  • Neurons / Oligodendrocytes were cultured as previously described by Charles et al., 2000. Pregnant female rats of 17 days gestation were killed by cervical dislocation (Rats Wistar) and the foetuses removed from the uterus. The Forebrains were removed and placed in ice-cold medium of Leibovitz (L15) containing 2% of Penicillin-Streptomycin (PS) and 1% of bovine serum albumin (BSA). Forebrains were dissociated by trypsinisation for 20 min at 37 Q C (Trypsin EDTA IX).
  • PS Penicillin-Streptomycin
  • BSA bovine serum albumin
  • DM EM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • the supernatant was discarded and the cells of pellet were re-suspended in a culture medium consisting of Neurobasal supplemented with 2% of B27, 2 mM of L- glutamine (L Gl u), 2% of PS solution, 1 % of FCS and 10 ng/m l of platelet-derived growth factor (PDGF-AA).
  • Viable cells were cou nted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 20000 cells/well in 96 well-plates pre- coated with poly-L-lysine and laminin .
  • estradiol was used as positive control. Estradiol is known to induce O PC proliferation.
  • the positive effect of estradiol on OL d ifferentiation has also been demonstrated, as has its effect on the early myelination process.
  • the positive effect of estradiol on neurite outgrowth was also published (for review see Alevaro et al., 2010).
  • the plates were maintained at 37°C in a humidified incubator, in an atmosphere of air (95%)-C02 (5%).
  • the total number of OPC (number of A2B5 positive cells) was quantified (to evaluate the proliferation), the axonal network was measured (total axonal length (N F)) to assess the effect of the compound on the neuronal network (the quality of the myelination is directly linked to the quality of the axonal network).
  • the total number of OL was quantified (number and area of MAG positive cells) (to evaluate the differentiation process), as well as the wrapping of OPC around axons (overlap MAG/NF wrapping) (myelination process).
  • the axonal network was measured (total axonal length (NF) to assess the effect of the compounds on the neuronal network.
  • the total number of OL was assessed (number and area of M BP positive cells) (to evaluate the OL maturation) as well as the wrapping of myelin around axon (overlap M BP/NF(wrapping)).
  • the axonal network was measured (Total axonal length (NF)) to assess the effect of the compounds on the neuronal network. For all measurements, once the cu lture was done (6 wells per conditions). For each condition tested, 30 pictures (each picture representing a field) per well were taken and analyzed using ImageXpress (Molecular devices) with 20x magn ification equ ipped with LED lamp (excitation 360/480/565 and emission 460/535/620). The 30 pictu res were automatically taken and represented 80% of the total surface of the culture well.
  • Results were expressed in terms of cumulated mean length in ⁇ of neurite network, or myelin sheath labeled for a given marker (MAG or M BP) per field.
  • the overlapping area between N F and MAG or M BP was measured to evaluate the wrapping.
  • PLATE 1 (A2B5 / NF)
  • Feeder layer preparation Dissociation of neonatal cortices and maintenance of mixed glial cultures
  • Tissue were subsequently triturated using a sterile flame-polished glass Pasteur pipette, then 4 mL of mixed glial culture media per brain was added. Cells were centrifuged at 1200 rpm ( ⁇ 300 g) for 5 min, then cells were resuspended in warm mixed glial culture media and plated into PLL- coated flask.
  • Hippocampal neurons were isolated from embryonic (E18) pups of Sprague Dawley rats. Briefly, following animal sacrifice, brains were isolated, meninges removed from the medial aspect of the cerebral hemispheres, then hippocampi dissected out and kept at 4°C until process completion.
  • Tissue were then incubated with 2.5% trypsin for 15 min in a water bath at 37°C, then gently washed and kept in culturing media.
  • Hippocampal dissociation was performed by repeatedly pipetting them up and down with a functionalized sterile Pasteur pipette. Following mechanical dissociation, cells were plated at desired density in neuronal plating medium, let recover for 4 hours, then put in compete neuronal culturing medium.
  • Flasks were then repositioned onto the shaker, equilibrated for approximately 3 hours, then shaken for approximately 16 hours at 220 rpm (overnight).
  • mixed glia culture medium containing microglia and OPCs cells were collected and pre-plated on P100 petri dish (not treated for culture) for 30 minutes in order to purify OPCs cells; microglia cells start immediately to adhere to petri while OPCs cells remained in the surnatant medium.
  • Pen/Strep (0.33% from stock) 33 units/mL Penicillin and 33 ⁇ g/mL Streptomycin
  • Bovine insulin from 1 mg/mL stock

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