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WO2023150389A1 - Compositions and methods for increasing dha availability - Google Patents

Compositions and methods for increasing dha availability Download PDF

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Publication number
WO2023150389A1
WO2023150389A1 PCT/US2023/012526 US2023012526W WO2023150389A1 WO 2023150389 A1 WO2023150389 A1 WO 2023150389A1 US 2023012526 W US2023012526 W US 2023012526W WO 2023150389 A1 WO2023150389 A1 WO 2023150389A1
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WO
WIPO (PCT)
Prior art keywords
dha
choline
supplemental
female
weeks
Prior art date
Application number
PCT/US2023/012526
Other languages
French (fr)
Inventor
Marie CAUDILL
Kevin KLATT
Jonathan Bortz
Original Assignee
Cornell University
Balchem Corporation
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 Cornell University, Balchem Corporation filed Critical Cornell University
Publication of WO2023150389A1 publication Critical patent/WO2023150389A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives 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

Definitions

  • compositions and methods for increasing DHA availability, particularly during gestation are generally directed to compositions and methods for increasing DHA availability, particularly during gestation.
  • choline metabolism i.e., phospholipids, betaine and acetylcholine
  • choline metabolism i.e., phospholipids, betaine and acetylcholine
  • the liver is capable of synthesizing a choline moiety de novo via the activity of the phosphatidylethanolamine /V-methyltransferase (PEMT) enzyme, catalyzing the triple methylation of phosphatidylethanolamine to phosphatidylcholine (PC).
  • PEMT phosphatidylethanolamine /V-methyltransferase
  • the promoter region of the PEMT gene contains an estrogen response element, and thus, the rising estrogen concentration across pregnancy results in increased PEMT activity and de novo choline production.
  • a growing body of data support the notion that such maternal adaptations are inadequate to meet the high choline demands of pregnancy 23 , and that choline intakes exceeding current dietary recommendations are required to support both maternal metabolic needs and fetal neurodevelopment.
  • Described in certain example embodiments herein are methods of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity, the method comprising co-supplementing choline and DHA to the female subject, wherein co- supplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day.
  • DHA docosahexaenoic acid
  • the total amount of choline ranges from about 500 mg to about 600 mg per day, optionally about 550 mg of choline per day.
  • the female subject is pregnant.
  • co-supplementing begin and/occur prior to the female of reproductive maturity being pregnant; prior to the female of reproductive maturity being 16 weeks pregnant; during gestation; after delivery; or any combination thereof.
  • co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant.
  • an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
  • the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
  • DHA bioavailability is increased during pregnancy.
  • the female subject of reproductive age is a mammal, optionally a human.
  • the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
  • the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
  • the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di- phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
  • the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation.
  • the supplemental choline and the supplemental DHA are administered separately.
  • the total amount of supplemental choline is administered in a single dose.
  • the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline.
  • the total amount of supplemental DHA is administered in a single dose.
  • the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA.
  • the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery.
  • the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery.
  • the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semisolid formulation, a gel formulation, or a solid formulation.
  • dietary supplement formulations comprising an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
  • DHA docosahexaenoic acid
  • the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
  • the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg.
  • the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
  • the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
  • the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ diphosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
  • the dietary supplement formulation is a solid, a semi-solid, or a liquid.
  • DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC- DHA or any combination thereof in the female subject of reproductive maturity.
  • the female subject of reproductive maturity is pregnant.
  • the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
  • the female of reproductive maturity is a mammal, optionally a human.
  • supplemental choline at an amount ranging from about 110 mg to about 3,000 mg per day and supplemental docosahexaenoic acid (DHA) at an amount ranging from about 110 mg to about 2500 mg per day in a female of reproductive maturity, optionally a pregnant female, to increase the bioavailability of DHA in the female of reproductive maturity.
  • DHA docosahexaenoic acid
  • FIG. 1 The Impact of Intervention on the Water- and Lipid-Soluble Choline Metabolome Across Pregnancy. Pregnant persons were randomized to 550mg vs 25mg of choline supplementation throughout the 2 nd and 3 rd trimester of pregnancy. Choline, betaine, dimethylglcyine, methionine, TMAO, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were measured via liquid chromatography tandem mass spectrometry in fasting plasma samples taken at Visit 1 (GW 12-16), Visit 2 (GW 20-24), and Visit 3 (GW 28-32), and in Delivery blood samples obtained at parturition. Metabolites were analyzed using mixed linear models to assess the impact of intervention; statistically significant differences between intervention groups at each time point are indicated with a * (P ⁇ 0.05; following Tukey’s Post-Hoc Adjustment).
  • FIG. 2 Study cohort, enrollment, allocation, and criteria flow diagram.
  • FIG. 3A-3D Effect of prenatal choline supplementation on maternal RBC PC-
  • DHA (FIG. 3A), maternal plasma PC-DHA (FIG. 3B), maternal RBC total DHA (FIG. 3C), and maternal plasma total DHA (FIG. 3D) among pregnant participants consuming 200 mg supplemental DHA/d.
  • the effect of prenatal choline supplementation on maternal RBC and plasma DHA outcomes was assessed using mixed linear models. Estimated marginal means and 95% confidence intervals (CI) derived from the baseline adjusted models are shown. Values for all models and p-values are reported in Tables 3 and 5. Statistically significant differences (P ⁇ 0.05) and trends (P ⁇ 0.10) between the choline intervention and control values at a study timepoint are indicated by ‘*’ and - ⁇ ' respectively.
  • FIG 4A-4D Effect of prenatal choline supplementation on maternal plasma total PC (FIG. 4A), d3-PC (FIG. 4B), d9-PC (FIG. 4C), and d3:d9-PC (FIG. 4D).
  • the effect of prenatal choline supplementation on maternal total and labeled PC was assessed using mixed linear models. Estimated marginal means and 95% confidence intervals (CI) are shown; total PC levels represented models adjusted for Visit 1 plasma PC. Statistically significant differences (P ⁇ 0.05) between the choline intervention and control values at a study timepoint are indicated by ‘*’.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure.
  • the upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range.
  • the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’ .
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • a measurable variable such as a parameter, an amount, a temporal duration, and the like
  • a measurable variable such as a parameter, an amount, a temporal duration, and the like
  • variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and/or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention.
  • a given confidence interval e.g. 90%, 95%, or more confidence interval from the mean
  • the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined.
  • an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity.
  • a biologic sample can contain whole cells and/or live cells and/or cell debris, and/or cell products, and/or virus particles.
  • the biological sample can contain (or be derived from) a “bodily fluid”.
  • the biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples.
  • fluid refers to any non-solid excretion, secretion, or other fluid present in an organism and includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g.
  • Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.
  • subject refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • Docosahexaenoic acid is another important fatty acid during fetal development. Hepatic export of DHA into circulation is linked to the synthesis of phosphatidylcholine (PC) by the enzyme phosphatidylethanolamine A'-mcthyltransfcrasc (PEMT). PEMT is important during pregnancy as is demonstrated by biomagnification of PENT-derived PC in fetal cord blood relative to maternal blood at delivery. PEMT activity relies on the universal methyl donor, S-adenosylmethionine (SAM), and thus PMT activity and availably of DHA outside of the liver, is linked to dietary methyl donors, such as choline, that support the synthesis of SAM. The effect of dietary supplementation of methyl donors, such as choline, on downstream PEMT activity, and DHA availability (e.g., via increasing circulating PC-DHA) during gestation, particularly during the last two trimesters, is not fully characterized.
  • SAM S-adenosylme
  • embodiments disclosed herein can provide methods of supplementing dietary DHA and choline during pregnancy and dietary supplemental formulations for the same.
  • Such methods and formulations can without being bound by theory, increase the bioavailability of DHA, particularly during pregnancy.
  • Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
  • dietary supplemental formulations that can contain an amount, effective amount, and/or least effective amount, and/or therapeutically effective amount of one or more compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof (which are also referred to as the primary active agent or ingredient elsewhere herein) described in greater detail elsewhere herein and a pharmaceutically acceptable carrier or excipient.
  • dietary supplemental formulations refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
  • “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
  • the compound can optionally be present in the dietary supplemental formulations as a pharmaceutically acceptable salt.
  • the dietary supplemental formulations can include, such as an active ingredient, an amount of DHA and/or an amount of choline.
  • the active ingredient is present as a pharmaceutically acceptable salt of the active ingredient.
  • pharmaceutically acceptable salt refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
  • Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucoronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzene sulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.
  • Suitable administration routes can include, but are not limited to auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infdtration, interstitial, intra-abdominal, intra- amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intracistemal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavemosum, intradermal, intradiscal, intraductal, intraduoden
  • DHA and/or choline can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a dietary supplemental formulation.
  • a dietary supplemental formulation containing one or more of the compounds and salts thereof, or pharmaceutically acceptable salts thereof described herein.
  • Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucoronate, saccharate, formate, benzoate, glutamate, methane sulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalene sulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.
  • the subject in need thereof is a female of reproductive maturity.
  • the female is pregnant or suspected of being pregnant.
  • the female is 0-45 weeks pregnant.
  • agent refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to.
  • active agent or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to.
  • active agent or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.
  • An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed.
  • An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.
  • the dietary supplemental formulations can include a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.
  • the dietary supplemental formulations can be sterilized, and if desired, mixed with agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active compound.
  • agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active compound.
  • the formulation includes one or more secondary agents that are in addition to the DHA or choline.
  • secondary agents include vitamins, minerals, other fatty acids, and/or the like.
  • the amount of the primary active agent and/or optional secondary agent can be an effective amount, least effective amount, and/or therapeutically effective amount.
  • effective amount refers to the amount of the primary and/or optional secondary agent included in the pharmaceutical formulation that achieve one or more therapeutic effects or desired effect.
  • least effective refers to the lowest amount of the primary and/or optional secondary agent that achieves the one or more therapeutic or other desired effects.
  • therapeutically effective amount refers to the amount of the primary and/or optional secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic effects.
  • the one or more therapeutic effects are increasing the bioavailability of DHA in the subject in need thereof. In some embodiments, the one or more therapeutic effects are increasing the bioavailability of DHA during pregnancy.
  • effective amount, least effective amount, and/or therapeutically effective amount of the choline is about 500 mg, to about 600 mg per day, optionally about 550 mg of choline per day.
  • effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day is about 100 mg to about 300 mg, optionally about 200 mg.
  • effective amount, least effective amount, and/or therapeutically effective amount of the choline per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg,
  • effective amount, least effective amount, and/or therapeutically effective amount of the choline per day ranges from about 110 mg, to about 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg,
  • effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg,
  • effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day ranges from about 110 mg, to 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,
  • the formulation can include the total effective amount, least effective amount, or therapeutically effective amount of the DHA and/or choline or can contain a sub dosage of one or both the DHA or choline.
  • the effective amount of the secondary active agent will vary depending on the secondary agent, the primary agent, the administration route, subject age, disease, stage of disease, among other things, which will be one of ordinary skill in the art.
  • the secondary active agent can be included in the dietary supplemental formulation or can exist as a standalone compound or dietary supplemental formulation that can be administered contemporaneously or sequentially with the compound, derivative thereof, or dietary supplemental formulation thereof.
  • the effective amount of the secondary active agent when optionally present, is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the effective amount of the secondary active agent is any non-zero amount ranging from about O to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
  • the dietary supplemental formulations described herein can be provided in a dosage form.
  • the dosage form can be administered to a subject in need thereof.
  • the dosage form can be effective generate specific concentration, such as an effective concentration, at a given site in the subject in need thereof.
  • dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the primary active agent, and optionally present secondary active ingredient, and/or a dietary supplemental formulation thereof calculated to produce the desired response or responses in association with its administration.
  • the given site is proximal to the administration site. In some embodiments, the given site is distal to the administration site.
  • the dosage form contains a greater amount of one or more of the active ingredients present in the dietary supplemental formulation than the final intended amount needed to reach a specific region or location within the subject to account for loss of the active components such as via first and second pass metabolism.
  • the dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, intemasal, and intradermal. Other appropriate routes are described elsewhere herein.
  • Such formulations can be prepared by any method known in the art.
  • Dosage forms adapted for oral administration can discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • the dietary supplemental formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the dietary supplemental formulation.
  • Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution.
  • the oral dosage form can be administered to a subject in need thereof. Where appropriate, the dosage forms described herein can be microencapsulated.
  • the dosage form can also be prepared to prolong or sustain the release of any ingredient.
  • compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described herein can be the ingredient whose release is delayed.
  • the primary active agent is the ingredient whose release is delayed.
  • an optional secondary agent can be the ingredient whose release is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et. al.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate
  • polyvinyl acetate phthalate acrylic acid polymers and copolymers
  • methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany),
  • Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non-polymeric excipient, to produce the desired release profde.
  • the coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.
  • the dosage forms described herein can be a liposome.
  • primary active ingredient(s), and/or optional secondary active ingredient(s), and/or pharmaceutically acceptable salt thereof where appropriate are incorporated into a liposome.
  • the dietary supplemental formulation is thus a liposomal formulation.
  • the liposomal formulation can be administered to a subject in need thereof.
  • Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the dietary supplemental formulations are applied as a topical ointment or cream.
  • a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be formulated with a paraffinic or water-miscible ointment base.
  • the primary and/or secondary active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
  • Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders.
  • a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be in a dosage form adapted for inhalation is in a particle-size- reduced form that is obtained or obtainable by micronization.
  • the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art.
  • Dosage forms adapted for administration by inhalation also include particle dusts or mists.
  • Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active (primary and/or secondary) ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.
  • the nasal/inhalation formulations can be administered to a subject in need thereof.
  • the dosage forms are aerosol formulations suitable for administration by inhalation.
  • the aerosol formulation contains a solution or fine suspension of a primary active ingredient, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate and a pharmaceutically acceptable aqueous or non-aqueous solvent.
  • Aerosol formulations can be presented in single or multi -dose quantities in sterile form in a sealed container.
  • the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.
  • the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • a suitable propellant under pressure such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • the aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer.
  • the pressurized aerosol formulation can also contain a solution or a suspension of a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof.
  • the aerosol formulation also contains co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation.
  • the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, 3 or more doses are delivered each time.
  • the aerosol formulations can be administered to a subject in need thereof.
  • the dietary supplemental formulation is a dry powder inhalable-formulations.
  • such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and/or starch.
  • a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate is in a particle-size reduced form.
  • a performance modifier such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate.
  • the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the compositions, compounds, vector(s), molecules, cells, and combinations thereof described herein.
  • Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.
  • Dosage forms adapted for rectal administration include suppositories or enemas.
  • the vaginal formulations can be administered to a subject in need thereof.
  • Dosage forms adapted for parenteral administration and/or adapted for injection can include aqueous and/or non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials.
  • the doses can be lyophilized and re-suspended in a sterile carrier to reconstitute the dose prior to administration.
  • Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.
  • the parenteral formulations can be administered to a subject in need thereof.
  • the dosage form contains a predetermined amount of a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate per unit dose.
  • the predetermined amount of primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be an effective amount, a least effect amount, and/or a therapeutically effective amount.
  • the predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate can be an appropriate fraction of the effective amount of the active ingredient.
  • the dietary supplemental formulation(s) described herein are part of a combination treatment or combination therapy.
  • the combination treatment can include the pharmaceutical formulation described herein and an additional treatment modality.
  • the additional treatment modality can be a chemotherapeutic, biological therapeutic, surgery, diet modulation, environmental modulation, a physical activity modulation, radiation treatment and any combination thereof.
  • the co-therapy or combination therapy can additionally include but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.
  • the dietary supplemental formulation and dosage forms thereof described herein can be administered one or more times hourly, daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times hourly, daily, monthly, or yearly).
  • the dietary supplemental formulations or dosage forms thereof described herein can be administered continuously over a period of time ranging from minutes to hours to days.
  • Devices and dosages forms are known in the art and described herein that are effective to provide continuous administration of the dietary supplemental formulations described herein.
  • the first one or a few initial amount(s) administered can be a higher dose than subsequent doses.
  • the dietary supplemental formulations can be administered such that the doses over time are tapered (increased or decreased) overtime so as to wean a subject gradually off of the dietary supplemental formulation or gradually introduce a subject to the dietary supplemental formulation.
  • the dietary supplemental formulation can contain a predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate.
  • the predetermined amount can be an appropriate fraction of the effective amount of the active ingredient.
  • Such unit doses may therefore be administered once or more than once a day, month, or year (e.g., 1, 2, 3, 4, 5, 6, or more times per day, month, or year).
  • Such dietary supplemental formulations may be prepared by any of the methods well known in the art.
  • Sequential administration is administration where an appreciable amount of time occurs between administrations, such as more than about 15, 20, 30, 45, 60 minutes or more.
  • the time between administrations in sequential administration can be on the order of hours, days, months, or even years, depending on the active agent present in each administration.
  • Simultaneous administration refers to administration of two or more formulations at the same time or substantially at the same time (e.g., within seconds or just a few minutes apart), where the intent is that the formulations be administered together at the same time.
  • dietary supplement formulations comprising an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
  • DHA docosahexaenoic acid
  • the increase in DHA bioavailability is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%,
  • the increase in DHA bio availability ranges from about 0.01%, to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%
  • the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
  • the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg.
  • the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
  • the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
  • the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di- phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
  • the dietary supplement formulation is a solid, a semi-solid, or a liquid.
  • DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC- DHA or any combination thereof in the female subject of reproductive maturity.
  • the female subject of reproductive maturity is pregnant.
  • the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
  • the female of reproductive maturity is a mammal, optionally a human.
  • the total amount of choline ranges from about 500 mg to about 600 mg per day, optionally about 550 mg of choline per day.
  • the amount of supplemental DHA per day is about 100 mg to about 300 mg, optionally about 200 mg.
  • the amount of supplemental choline per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg,
  • the supplemental amount of choline per day ranges from about 110 mg, to about 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg,
  • the supplemental amount of DHA per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg,
  • the supplemental amount of DHA per day ranges from about 110 mg, to 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg,
  • the female subject is pregnant. In some embodiments, wherein the female subject of reproductive age is a mammal, optionally a human. In some embodiments, the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less. In some embodiments, the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
  • co-supplementing begin and/occur prior to the female of reproductive maturity being pregnant; prior to the female of reproductive maturity being 16 weeks pregnant; during gestation; after delivery; or any combination thereof.
  • co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant.
  • cosupplementing occurs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or more days, weeks, months, or years.
  • the method increases the bioavailability of DHA.
  • an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
  • the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
  • DHA bioavailability is increased during pregnancy.
  • the increase in DHA bioavailability is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%,
  • the increase in DHA bioavailability ranges from about 0.01%, to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%,
  • the supplemental choline is provided as a choline salt.
  • choline salt refers to any salt that carries choline.
  • Exemplary choline salts include, but are not limited to, choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di -phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof. Additional exemplary salt forms are also discussed below with respect to formulations.
  • Administration of the supplemental DHA and choline can be by any suitable route. Exemplary suitable administration routes are discussed elsewhere herein with respect to formulations.
  • the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation.
  • the supplemental choline and the supplemental DHA are administered separately.
  • the total amount of supplemental choline is administered in a single dose.
  • the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline.
  • the total amount of supplemental DHA is administered in a single dose. In some embodiments, the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA. In some embodiments, the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semi-solid formulation, a gel formulation, or a solid formulation. Exemplary dosage forms are described in greater detail below.
  • the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery. In some embodiments, the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery.
  • KITS KITS
  • any of the compounds, compositions, and/or formulations described herein or a combination thereof can be presented as a combination kit.
  • kit or “kit of parts” refers to the compounds, compositions, formulations, particles, cells and any additional components that are used to package, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein.
  • additional components include, but are not limited to, packaging, syringes, blister packages, bottles, and the like.
  • the combination kit can contain the active agents in a single formulation, such as a pharmaceutical formulation, (e.g., a tablet) or in separate formulations.
  • a pharmaceutical formulation e.g., a tablet
  • the combination kit can contain each agent or other component in separate compositions or formulations.
  • the separate kit components can be contained in a single package or in separate packages within the kit.
  • the combination kit also includes instructions printed on or otherwise contained in a tangible medium of expression.
  • the instructions can provide information regarding the content of the compounds, compositions, or formulations, described herein or a combination thereof contained therein, safety information regarding the content of the compounds, compositions, formulations (e.g., dietary supplemental formulations) described herein or a combination thereof contained therein, information regarding the dosages, indications for use, and/or recommended treatment regimen(s) for the compound(s) and/or dietary supplemental compositions and/or formulations contained therein.
  • the instructions can provide directions for administering the compounds, compositions, formulations, particles, and cells described herein or a combination thereof to a subject in need thereof.
  • the subject in need thereof can be a female of reproductive maturity and/or pregnant, such as any of those described in greater detail elsewhere herein.
  • the kit and/or component thereof can provide that the compositions and/or formulations described herein can increase DHA bioavailability in the subject in need thereof.
  • Docosahexaenoic acid is an omega-3 polyunsaturated fatty acid (PUFA) that plays a critical role in fetal development 1 2 .
  • the hepatic export of DHA into circulation is linked to the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine JV-methyl transferase (PEMT).
  • PC phosphatidylcholine
  • PEMT phosphatidylethanolamine JV-methyl transferase
  • the PEMT pathway in liver generates PC molecules enriched in DHA (PC-DHA) that can subsequently be incorporated into VLDLs for export into plasma and delivery to extrahepatic tissues (including the placenta).
  • Transgenic mice lacking PEMT show substantially reduced DHA concentrations in plasma 3 , and limited accumulation of DHA in fetal brains of pups bom to PEMT deficient dams 4 . Consistent with animal models, previous work has highlighted the importance of PEMT during human pregnancy by demonstrating biomagnification of PEMT-derived PC in fetal cord blood relative to maternal blood at delivery 5 .
  • Dietary methyl donors such as choline, are essential to support the synthesis of the universal methyl donor, S-adenosylmethionine (SAM), utilized by methyltransferases to catalyze the transfer of methyl groups (-CHs) to form their respective products.
  • SAM S-adenosylmethionine
  • -CHs methyl groups
  • An adequate supply of methyl donors for optimal PEMT activity may be particularly important during pregnancy.
  • the promoter region for the PEMT gene contains an estrogen response element 8 , rendering PEMT activity sensitive to the dramatic increase in circulating estrogen during the latter half of pregnancy. Notably, this increase in PEMT activity coincides with the accumulation of DHA and other long chain PUFAs in fetal brain 9 .
  • LPC PEMT-derived lysophosphatidylcholine
  • prenatal choline supplementation would influence circulating PC-DHA if administered prior to maximal PEMT capacity, or for a longer duration, is unknown.
  • Applicant investigated the effect of prenatal choline supplementation, administered throughout the second and third trimesters of pregnancy, on biomarkers of DHA status among pregnant participants consuming supplemental DHA.
  • Pregnant participants were recruited between October 2017 and April 2019 at maternity clinics throughout the Ithaca NY region using flyers. Screening of overall health of the prospective participants was conducted via online questionnaires, which included a short version of the NIH Diet History Questionnaire (DHQ-III; https://www.nal.usda.gov/fhic/dietary-assessment-instruments-research) to estimate usual choline intake, and a validated DSM DHA/EPA food frequency questionnaire (FFQ; kindly provided by DSM Nutritionals Inc) to assess omega-3 fatty acid intake.
  • DHQ-III NIH Diet History Questionnaire
  • FFQ DSM DHA/EPA food frequency questionnaire
  • Eligible participants were 12-16 weeks pregnant at the beginning of the study, between age 21 and 40 years, had a pre-pregnancy body mass index (BMI) ⁇ 32 kg/m 2 , were intending to deliver at Cayuga Medical Center, and were willing to comply with the study protocol. Participants were excluded if they had usual dietary DHA intakes exceeding 400 mg/d (based on FFQ), had usual dietary choline intakes exceeding 450 mg/d (based on DHQ-III), or reported having cardiovascular disease, cancer, type 1 or 2 diabetes mellitus, gastrointestinal disorders, gallbladder disease, kidney disease, liver disease or anemia (based on health questionnaire).
  • BMI body mass index
  • the present study was a single-center, randomized, double-blind, parallel-group choline intervention study, designed to investigate the effects of prenatal choline supplementation on DHA status in healthy free-living pregnant participants consuming supplemental DHA (NCT03194659).
  • the intervention of 550 mg supplemental choline/day or control (25 mg supplemental choline/d) was initiated at gestational age 12 - 16 weeks and continued until delivery; throughout this period, all participants received 200 mg supplemental DHA/d.
  • Data obtained during the study were recorded in a dedicated online secure database developed by Cornell Institute of Social and Economic Research (CISER).
  • Eligible participants were enrolled into the study by MQM on a rolling basis in a parallel arm design.
  • a simple 1 : 1 randomization scheme was generated by KCK using a web-based random number generator assigning participants to either group A or group B; study staff (OVM) not interacting with participants replaced group A or group B with intervention or control.
  • Both groups consumed a grape juice cocktail that provided either 550 mg supplemental choline/d (intervention) or 25 mg supplemental choline/d (control).
  • Preparation of the grape juice cocktail was handled by study staff not interacting with study participants (OVM) to maintain personnel blinding.
  • the grape juice cocktail was served in 15-mL color-coded conical tubes to conceal the intervention assignment from the participant.
  • the 550 mg choline dose for the intervention group was comprised of 500 mg unlabeled choline (dO- choline) and 50 mg methyl -t/9-cholinc (t/9-cholinc).
  • the 25 mg choline dose for the control group was in the form of t/9-cholinc only.
  • the low-dose deuterium-labeled choline tracer (50 mg t/9-cholinc for the intervention group, and 25 mg t/9-cholinc for the control group) was administered to provide insights into the use of choline derived methyl groups for the synthesis of PC by the PEMT pathway (described in the results section).
  • choline supplements were prepared in the Human Metabolic Research Unit (HMRU) at Cornell University.
  • HMRU Human Metabolic Research Unit
  • stock solutions of JO-choline and U9-cholinc were prepared separately by dissolving choline chloride (Balchem Inc.; NY, USA) or U9-cholinc chloride (Cambridge Isotope Uaboratories Inc.; MA, USA) in Milli-Q water to produce 250 mg/mU JO-chol ine and 50 mg/mU U9-cholinc solutions.
  • the stock solutions were fdtered and stored at 4°C for up to one year; tests conducted during this period showed excellent stability with no detectable loss through time.
  • the choline and control supplements were assembled by aliquoting 2 mU of the dO- choline stock solution (intervention group), 1 mU of the U9-cholinc stock solution (intervention group), or 0.5 mU of the U9-cholinc stock solution (control group) into 15-mU sterilized polystyrene tubes.
  • the supplement tubes were filled with grape juice cocktail (Welch’s; MA, USA) and labeled in a manner that was indistinguishable by both the participants and personnel who interacted with the participants.
  • All supplement tubes were packaged in Ziplock bags labeled with the participant ID numbers and kept frozen in a food-only -20°C freezer prior to pick-up. Testing of the supplements that were kept for two months at room temperature, or inside a home refrigerator, showed excellent stability with no detectable loss overtime. Study Protocol
  • Participants were required to visit the HMRU on three separate occasions (FIG. 1). The first visit was during gestational week (GW) 12-16, while the second (GW 20-24) and third (GW 28-32) visits were every 8 weeks thereafter.
  • participants received their daily supplements, which included the grape juice choline cocktail tubes, a 200-mg DHA supplement [Nature's Way EfaGoldNeuromins 200mg DHA (plant source); DSM Nutritional Products; Netherlands], and an over-the-counter prenatal vitamin/mineral supplement (Nature Made Prenatal Tablet; Pharmavite LLC; CA, USA).
  • non-fasting maternal blood, placenta, and fetal cord blood were collected (FIG. 1).
  • maternal and newborn information were obtained from medical charts.
  • Maternal information included due date, complications during pregnancy, and complications during labor or delivery.
  • Newborn information included the date and mode of delivery, gestational age, birth weight, sex and APGAR score.
  • Remaining RBCs were either stored or washed 3x with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the 5-mL EDTA coated tube which provided whole blood for the complete blood counts, was processed within 60 min by research personnel in the Cornell Human Nutritional Chemistry Laboratory.
  • the SST blood which provided serum for the blood chemistry profile, was kept at room temperature, allowed to clot, and centrifuged at 3000 rpm for 15 min at room temperature. All the biological samples were dispensed into 1.8-mL cryostat vials (CryoTube; NUNC) and stored at -80°C.
  • Maternal blood and cord blood samples were collected at delivery. Maternal blood samples were collected into two 10-mL EDTA coated tubes and one 10-mL SST tube at the hospital within 1-h of delivery. Cord blood samples were collected into two 4-mL EDTA- coated tubes at the time of delivery. After collection, maternal and cord blood samples were refrigerated at 4°C, and processed as described previously within 4 h.
  • the placenta was also obtained at delivery, weighed and processed at the hospital within 90 min of delivery. After removal of the amnion, 16 full-thickness tissue biopsies (0.5 x 0.5 x 0.5 cm) were taken from 4 separate locations (i.e., the placenta was visually divided into four quadrants). Samples were rinsed with PBS immediately. The other samples were flash frozen in liquid nitrogen, placed in a cryostat tube, and stored temporarily in a canister containing liquid nitrogen. In the laboratory, all samples were stored at -80°C.
  • a homogenous representation was prepared by taking one piece of placenta from each quadrant and powderizing in liquid nitrogen using a Bessman Tissue Pulverizer (Fisher Scientific, Waltham, MA, USA).
  • RBC lipids were extracted from washed RBCs using a modification of the method from Rose and Oklander 15 , and PC was separated using a Waters 2690 HPLC, a quaternary solvent system of hexane, methanol, acetone and isopropanol, and a Waters YMC-Pack DIOL column.
  • the HPLC column eluant was split 10:90 after which lipids were detected and quantified by a Waters 2424 evaporative light scattering detector and recovered with use of a Waters FCIII fraction collector.
  • fatty acids were converted to methyl esters, separated, and quantified by GLC.
  • RBC PC-DHA is expressed as a percentage of the total fatty acids in PC.
  • the inter-assay CV for RBC PC-DHA (as a percentage of the total fatty acids in PC) was 6.4% based on in-house RBC controls.
  • PC-DHA concentration of plasma was analyzed using stable-isotope dilution LC-MS/MS at Cornell University’s Biotech Proteomics and Metabolomics Facility. Briefly, plasma (20pL) was transferred to 1.5ml Eppendorf vials (VWR20170-038), to which 2.4 nmol J7-PC 15:0-18: 1 (Avanti Polar Lipids, Alabaster, AL) in 0.2 mL methanol: chloroform (2: 1 v/v) was added as an internal standard. The samples were mixed and incubated overnight at -20°C. On the following day, samples were centrifuged, and the supernatant was collected and transferred into another Eppendorf tube without disturbing the solid phase.
  • PC-DHA species were separated under step gradient conditions with a flow rate of 400pL/min. The column temperature was kept at 30°C.
  • the mobile phase consisted of MPA (400mL acetonitrile: 127mL water: 68mL ethanol: 3mL IM ammonium acetate in water: 2mL concentrated glacial acetic acid) and MPB (250 mL acetonitrile: 250 mL water: 42 mL ethanol: 13.5 mL IM ammonium acetate in water: 9 mL of concentrated glacial acetic acid).
  • the following step gradient was employed for the separation and analysis of PC- DHA species: 5% MPB from 0 to 3 min; 30% MPB from 3 to 10 min; 60% MPB from 10 to 14 min; 100% MPB from 16 to 17 min; 5% MPB from 17 to 19 min.
  • the declustering potential (DP) was set to -35V with accumulation time of 0.25s.
  • the MS full scan measurement was done from m/z 100 to m/z 1000 in profde mode followed by MRM HR scan acquired from 0 min to 21 min at collision energies of -40V for each analyte.
  • the acetate adduct negatively charged ion of each analyte was selected as the precursor ion.
  • Calibration curves standard solutions were prepared by serial dilutions of the standards (PC 16:0-DHA, PC 18:0-DHA; Avanti Polar Lipids, Alabaster, AL), mixed with the internal standard (J7-PC 15:0-18: 1) at a concentration of 12 nmol/mL in 6: 1 MeOH:CHCh.
  • the calibration curves ranged from 50 to 5000 pmol for PC 16:0-DHA and PC 18:0-DHA (R 2 >0.99).
  • the lower limit of detection and lower limit of quantitation determined based on S/N in PC DHA standards solutions were 0.15 pmol and 0.5 pmol.
  • PC-DHA in plasma is expressed as the sum of PC 16:0-DHA + PC 18:0-DHA + PC 18: 1-DHA.
  • DHA composition of washed RBCs (% total fatty acids) and concentrations in plasma (pg/mL) were analyzed by gas chromatography (GC) coupled to a flame ionization detector at OmegaQuant® as previously described 16 17 .
  • GC gas chromatography
  • EMD Chemicals, USA were added to RBCs, and BTM (methanol containing 14% boron trifluoride, toluene, methanol; 35:30:35 v/v/v) (Sigma-Aldrich, St. Louis, MO) was added to plasma.
  • Placenta samples were weighed into screw-cap glass vials that contained tritricosanoin as an internal standard (tri-C23:0 TG) (NuCheck Prep, Elysian, MN), homogenized and subsequently subjected to a modified Folch extraction. A portion of the organic layer was transferred to a new screw-cap glass vial and dried in a speed vac. BTM was then added to the dried placenta samples. Next, sample vials (RBCs, plasma and placenta) were briefly vortexed and heated in a hot bath at 100°C for 45 minutes. After cooling, HPLC grade water was added, and vials were recapped, vortexed and centrifuged to separate layers. Aliquots of the hexane layer were subsequently transferred to GC vials.
  • tri-C23:0 TG Tri-C23:0 TG
  • BTM was then added to the dried placenta samples.
  • sample vials (RBCs, plasma and placenta
  • GC was carried out using a GC-2010 Gas Chromatograph (Shimadzu Corporation, Columbia, MD) equipped with a SP-2560, 100-m fused silica capillary column (0.25 mm internal diameter, 0.2 um film thickness; Supelco, Bellefonte, PA). Fatty acids were identified by comparison with a standard mixture of fatty acids (GLC OQ-A, NuCheck Prep, Elysian, MN), which was also used to determine individual fatty acid calibration curves.
  • GLC OQ-A NuCheck Prep, Elysian, MN
  • the following 24 fatty acids were identified: saturated (14:0, 16:0, 18:0, 20:0, 22:0 24:0); cis monounsaturated (16: 1, 18: 1, 20: 1, 24: 1); trans unsaturated [16: 1, 18: 1, 18:2); cis n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4, 22:5); and cis n-3 polyunsaturated (18:3, 20:5, 22:5, 22:6).
  • Fatty acid composition, specifically DHA forthis study (cis C22:6, n-3), was expressed as a percent of total identified fatty acids.
  • the inter-assay CV for total DHA in RBCs, plasma and placenta was ⁇ 5% based on in-house controls.
  • SNPs Single nucleotide polymorphisms in the PEMT gene (P EMT rs7946; PEMT rs4646343), which may influence PEMT activity 18 , were determined as previously described 19,20 so that these variables could be considered in our statistical models if differentially distributed between the intervention and control groups.
  • Stable-isotope dilution LC-MS/MS was used to measure plasma free choline 21 ’ 22 , plasma PC 2223 , and plasma enrichment percentages of t/j-PC and J9-PC 2223 .
  • Intra and inter assay CVs were ⁇ 3% for choline and ⁇ 6% for PC.
  • CBC and blood chemistry profiles were performed at the Human Nutritional Chemistry Service Laboratory at Cornell University. Hematology analysis was conducted using a Beckman-Coulter AcT Diff2 coulter counter; serum albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides were measured using an automated chemistry analyzer (Dimension Xpand Plus; Siemens Healthcare Diagnostics).
  • PC-DHA DHA-containing PC species in circulating maternal RBCs.
  • PC-DHA DHA-containing DHA
  • maternal clinical parameters i.e., WBC counts, RBC counts, and hemoglobin assessed at visit 1, visit 2 and visit 3; serum concentrations of albumin, ALT, AST, glucose, total cholesterol, HDL-C, LDL-C and triglycerides assessed at visit 1, visit 3 and delivery
  • newborn characteristics i.e., infant sex, weight,
  • the study sample size was based on previous results among nonpregnant women 7 , whereby means and standard deviations of erythrocyte PC-DHA for two levels of choline intake yielded a Cohen's d effect size of 1.1, after accounting for statistically significant covariates.
  • Participants were continuously enrolled until 30 participants had completed the trial; all participants who completed the trial (i.e., delivery data were available) were included in all analyses by intention to treat (ITT; a modified ITT excluding participants who were excluded due to the development of pregnancy-related pathologies).
  • ITT intention to treat
  • sensitivity analyses included models without adjustment for baseline (unadjusted model), as well as a model that considered additional covariates hypothesized to influence the intervention effects and/or DHA outcomes, specifically age, pre-pregnancy BMI, and calculated duration of intervention exposure (i.e., gestational age at delivery - gestational age at study entry) (fully adjusted models).
  • a model that considered additional covariates hypothesized to influence the intervention effects and/or DHA outcomes specifically age, pre-pregnancy BMI, and calculated duration of intervention exposure (i.e., gestational age at delivery - gestational age at study entry) (fully adjusted models).
  • linear models that included intervention arm and baseline maternal RBC DHA as fixed effects were used.
  • For self-reported intakes of dietary choline and dietary DHA, /'-values were derived from Hodges- Lehman-Sen estimations for non-parametric comparison of medians.
  • FIG. 2 depicts the flowchart of the study.
  • Table 1 presents baseline demographics and baseline measures of the participants included in the intention-to treat analysis by group allocation.
  • participants’ reported choline intakes were similar to representative samples of choline intake within the North American population 31 .
  • Participant adherence to the study protocol was >99%, based on the number of returned juice containers (containing the choline supplement) and the number of returned DHA capsules. Further, mean concentrations of plasma free choline, which have been shown to be responsive to choline supplementation during pregnancy 10 , were significantly higher in the choline intervention (versus control) group with between group differences detected at with between-group differences detected at visit 2, visit 3, and delivery 32 ..
  • Total DHA in RBCs (% total RBC fatty acids) is a well-validated indicator of tissue DHA levels (i.e., DHA status), consistently linked to health outcomes (including pregnancy-related health outcomes), and thus, the most physiologically relevant outcomes of interest in relation to choline supplementation’s impact.
  • choline as a methyl donor by the PEMT pathway was assessed by measuring JJ-PC enrichment (t/j-PC / total PC) and the ratio of t/j-PCz/9-PC.
  • D5-PC is generated by the PEMT pathway when one of the t/j-labclcd methyl groups is used in the SAM- dependent methylation of phosphatidylethanolamine to PC
  • J9-PC is generated by the CDP-choline pathway when the intact choline molecule (t/9-cholinc) is used to generate PC 5 22 .
  • the below results collectively are consistent with greater use of choline as a methyl donor by the PEMT pathway and greater PEMT activity in response to the choline intervention.
  • Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature. 2014;509(7501):503-506. doi : 10.1038/nature 13241 Innis SM, Dyer RA. Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid. J Lipid Res. 2002;43(9): 1529-1536.
  • Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J. 2007;21(10):2622-2632. doi: 10.1096/fj.07-8227com
  • a method of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity comprising: co-supplementing choline and DHA to the female subject, wherein co- supplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day.
  • DHA docosahexaenoic acid
  • DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC- DHA, or any combination thereof.
  • supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate cytidine 5’ di-phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
  • a dietary supplement formulation comprising: an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
  • DHA docosahexaenoic acid
  • the dietary supplement formulation of any one of aspects 22-25, wherein the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
  • supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di -phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
  • the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
  • supplemental choline at an amount ranging from about 110 mg to about 3,000 mg per day and supplemental docosahexaenoic acid (DHA) at an amount ranging from about 110 mg to about 2500 mg per day in a female of reproductive maturity, optionally a pregnant female, to increase the bioavailability of DHA in the female of reproductive maturity.
  • DHA docosahexaenoic acid

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Abstract

Described in several example embodiments herein are methods and compositions for increasing docosahexaenoic acid (DHA) availability in a subject, particularly a subject in the second or third trimester of gestation. In some example embodiments, the method includes administered to the subject an effective amount of choline and an effective amount of DHA.

Description

COMPOSITIONS AND METHODS FOR INCREASING DHA AVAILABILITY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63/307,306, filed on February 7, 2022, entitled “COMPOSITIONS AND METHODS FOR INCREASING DHA AVAILABILITY,” the contents of which is incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0002] The subject matter disclosed herein is generally directed to compositions and methods for increasing DHA availability, particularly during gestation.
BACKGROUND
[0003] Pregnancy places a unique stress upon choline metabolism, requiring adaptations to support both maternal and fetal requirements. Derivatives of choline metabolism (i.e., phospholipids, betaine and acetylcholine) play a critical role in fetal development, including membrane biosynthesis, tissue expansion, genomic and cellular methylation, and neurotransmission. The liver is capable of synthesizing a choline moiety de novo via the activity of the phosphatidylethanolamine /V-methyltransferase (PEMT) enzyme, catalyzing the triple methylation of phosphatidylethanolamine to phosphatidylcholine (PC). The promoter region of the PEMT gene contains an estrogen response element, and thus, the rising estrogen concentration across pregnancy results in increased PEMT activity and de novo choline production. However, a growing body of data support the notion that such maternal adaptations are inadequate to meet the high choline demands of pregnancy23, and that choline intakes exceeding current dietary recommendations are required to support both maternal metabolic needs and fetal neurodevelopment.
[0004] In rodent models, consumption of choline at intake levels required to prevent deficiency in non-pregnant dams results in a pronounced depletion of maternal hepatic choline levels, similar to those observed in non-pregnant dams consuming choline deficient diets. Such depletion during rodent pregnancy facilitates maintenance of the plasma choline supply to support biomagnification in the fetal compartment, a process actively facilitated by the placenta to meet the high demands of the developing fetus. This decline in maternal hepatic choline stores likely represents a functional inadequacy requiring increased dietary intakes; indeed, a large body of literature demonstrates that supplementing the diet of dams with choline results in increased hepatic choline metabolites, as well as plasma choline, phosphatidylcholine and betaine, and significantly enhances fetal neurodevelopment, resulting in improved offspring cognition.
[0005] Less is known about choline metabolism and requirements during human pregnancy. A limited body of data from prospective studies indicate that plasma choline typically rises across pregnancy with biomagnification in the fetal compartment. In the only controlled feeding choline intervention study2, third trimester pregnant participants randomized to consume either 480 or 930 mg choline/day exhibited a significant rise in choline over time at both intake levels, with plasma choline levels being greater relative to nonpregnant participants consuming equivalent choline intakes. Choline supplementation at the 930 vs 480 mg/d level yielded higher plasma choline and betaine during the third trimester of pregnancy2 and restored the partitioning of choline between betaine synthesis and PC synthesis via the CDP-choline pathway to that observed in non-pregnant state3. Moreover, similar to rodent models, infants bom to participants randomized to the 930 mg/d arm of this trial demonstrated improvements in cognitive function across the first year of life, relative to the 480 mg/d arm. However, the impact of pregnancy and prenatal choline supplementation on longitudinal measures of the choline metabolome prior to the third trimester, and at choline intake levels more closely aligned with habitual intakes, remains relatively uncharacterized, particularly during the second and third trimesters.
[0006] Thus, there is need for not only an improved characterization of the effect of choline supplementation during gestation, particularly during the second and third trimesters, but also improved supplemental choline formulations and treatment regimens appropriate for gestation to improve, inter alia, neonate brain and cognitive function.
[0007] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.
SUMMARY
[0008] Described in certain example embodiments herein are methods of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity, the method comprising co-supplementing choline and DHA to the female subject, wherein co- supplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day.
[0009] In certain example embodiments, the total amount of choline ranges from about 500 mg to about 600 mg per day, optionally about 550 mg of choline per day.
[0010] In certain example embodiments, the female subject is pregnant.
[0011] In certain example embodiments, co-supplementing begin and/occur prior to the female of reproductive maturity being pregnant; prior to the female of reproductive maturity being 16 weeks pregnant; during gestation; after delivery; or any combination thereof.
[0012] In certain example embodiments, co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant.
[0013] In certain example embodiments, an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
[0014] In certain example embodiments, the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
[0015] In certain example embodiments, DHA bioavailability is increased during pregnancy.
[0016] In certain example embodiments, the female subject of reproductive age is a mammal, optionally a human.
[0017] In certain example embodiments, the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
[0018] In certain example embodiments, the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
[0019] In certain example embodiments, the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di- phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
[0020] In certain example embodiments, the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation.
[0021] In certain example embodiments, the supplemental choline and the supplemental DHA are administered separately.
[0022] In certain example embodiments, the total amount of supplemental choline is administered in a single dose.
[0023] In certain example embodiments, the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline.
[0024] In certain example embodiments, the total amount of supplemental DHA is administered in a single dose.
[0025] In certain example embodiments, the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA.
[0026] In certain example embodiments, the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery.
[0027] In certain example embodiments, the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery.
[0028] In certain example embodiments, the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semisolid formulation, a gel formulation, or a solid formulation.
[0029] Described in certain example embodiments herein are dietary supplement formulations comprising an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
[0030] In certain example embodiments, the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
[0031] In certain example embodiments, the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg.
[0032] In certain example embodiments, the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
[0033] In certain example embodiments, the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
[0034] In certain example embodiments, the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ diphosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
[0035] In certain example embodiments, the dietary supplement formulation is a solid, a semi-solid, or a liquid.
[0036] In certain example embodiments, DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC- DHA or any combination thereof in the female subject of reproductive maturity.
[0037] In certain example embodiments, the female subject of reproductive maturity is pregnant.
[0038] In certain example embodiments, the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
[0039] In certain example embodiments, the female of reproductive maturity is a mammal, optionally a human. [0040] Described herein is use of supplemental choline at an amount ranging from about 110 mg to about 3,000 mg per day and supplemental docosahexaenoic acid (DHA) at an amount ranging from about 110 mg to about 2500 mg per day in a female of reproductive maturity, optionally a pregnant female, to increase the bioavailability of DHA in the female of reproductive maturity.
[0041] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:
[0043] FIG. 1 - The Impact of Intervention on the Water- and Lipid-Soluble Choline Metabolome Across Pregnancy. Pregnant persons were randomized to 550mg vs 25mg of choline supplementation throughout the 2nd and 3rd trimester of pregnancy. Choline, betaine, dimethylglcyine, methionine, TMAO, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were measured via liquid chromatography tandem mass spectrometry in fasting plasma samples taken at Visit 1 (GW 12-16), Visit 2 (GW 20-24), and Visit 3 (GW 28-32), and in Delivery blood samples obtained at parturition. Metabolites were analyzed using mixed linear models to assess the impact of intervention; statistically significant differences between intervention groups at each time point are indicated with a * (P<0.05; following Tukey’s Post-Hoc Adjustment).
[0044] FIG. 2 - Study cohort, enrollment, allocation, and criteria flow diagram.
[0045] FIG. 3A-3D - Effect of prenatal choline supplementation on maternal RBC PC-
DHA (FIG. 3A), maternal plasma PC-DHA (FIG. 3B), maternal RBC total DHA (FIG. 3C), and maternal plasma total DHA (FIG. 3D) among pregnant participants consuming 200 mg supplemental DHA/d. The effect of prenatal choline supplementation on maternal RBC and plasma DHA outcomes was assessed using mixed linear models. Estimated marginal means and 95% confidence intervals (CI) derived from the baseline adjusted models are shown. Values for all models and p-values are reported in Tables 3 and 5. Statistically significant differences (P<0.05) and trends (P<0.10) between the choline intervention and control values at a study timepoint are indicated by ‘*’ and -±' respectively.
[0046] FIG 4A-4D - Effect of prenatal choline supplementation on maternal plasma total PC (FIG. 4A), d3-PC (FIG. 4B), d9-PC (FIG. 4C), and d3:d9-PC (FIG. 4D). The effect of prenatal choline supplementation on maternal total and labeled PC was assessed using mixed linear models. Estimated marginal means and 95% confidence intervals (CI) are shown; total PC levels represented models adjusted for Visit 1 plasma PC. Statistically significant differences (P<0.05) between the choline intervention and control values at a study timepoint are indicated by ‘*’.
[0047] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
[0048] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
[0050] All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
[0051] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
[0052] Where a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’ . Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
[0053] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
[0054] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
General Definitions
[0055] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011). [0056] Definitions of common terms and techniques in chemistry and organic chemistry can be found in Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. Physical Chemistry, 5th edition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14th ed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2nd ed. 2012, published by Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5th ed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5th ed. 2010, published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function; 8th ed. (2018) published by W.H. Freeman.
[0057] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
[0058] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and/or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
[0059] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0060] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
[0061] As used herein, a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity. A biologic sample can contain whole cells and/or live cells and/or cell debris, and/or cell products, and/or virus particles. The biological sample can contain (or be derived from) a “bodily fluid”. The biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples. As used herein “bodily fluid” refers to any non-solid excretion, secretion, or other fluid present in an organism and includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g. plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.
[0062] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
[0063] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
[0064] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
OVERVIEW
[0065] Little is known about choline metabolism and requirements during human pregnancy. A limited body of data from prospective studies indicate that plasma choline typically rises across pregnancy with biomagnification in the fetal compartment. In the only controlled feeding choline intervention study2, third trimester pregnant participants randomized to consume either 480 or 930mg choline/day exhibited a significant rise in choline over time at both intake levels, with plasma choline levels being greater relative to nonpregnant participants consuming equivalent choline intakes. Choline supplementation at the 930 vs 480 mg/d level yielded higher plasma choline and betaine during the third trimester of pregnancy2 and restored the partitioning of choline between betaine synthesis and PC synthesis via the CDP-choline pathway to that observed in non-pregnant state3. Moreover, similar to rodent models, infants bom to participants randomized to the 930mg/d arm of this trial demonstrated improvements in cognitive function across the first year of life, relative to the 480mg/d arm. However, the impact of pregnancy and prenatal choline supplementation on longitudinal measures of the choline metabolome prior to the third trimester, and at choline intake levels more closely aligned with habitual intakes, remains relatively uncharacterized, particularly during the second and third trimesters.
[0066] Docosahexaenoic acid (DHA) is another important fatty acid during fetal development. Hepatic export of DHA into circulation is linked to the synthesis of phosphatidylcholine (PC) by the enzyme phosphatidylethanolamine A'-mcthyltransfcrasc (PEMT). PEMT is important during pregnancy as is demonstrated by biomagnification of PENT-derived PC in fetal cord blood relative to maternal blood at delivery. PEMT activity relies on the universal methyl donor, S-adenosylmethionine (SAM), and thus PMT activity and availably of DHA outside of the liver, is linked to dietary methyl donors, such as choline, that support the synthesis of SAM. The effect of dietary supplementation of methyl donors, such as choline, on downstream PEMT activity, and DHA availability (e.g., via increasing circulating PC-DHA) during gestation, particularly during the last two trimesters, is not fully characterized.
[0067] Thus, there is need for not only an improved characterization of the effect of choline supplementation during gestation on DHA availability, particularly during the second and third trimesters, but also improved supplemental choline formulations and treatment regimens appropriate for gestation to improve, inter alia, DHA availability and neonate brain and cognitive function.
[0068] With that said, embodiments disclosed herein can provide methods of supplementing dietary DHA and choline during pregnancy and dietary supplemental formulations for the same. Such methods and formulations, can without being bound by theory, increase the bioavailability of DHA, particularly during pregnancy. Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
DIETARY SUPPLEMENTAL FORMULATIONS
[0069] Also described herein are dietary supplemental formulations that can contain an amount, effective amount, and/or least effective amount, and/or therapeutically effective amount of one or more compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof (which are also referred to as the primary active agent or ingredient elsewhere herein) described in greater detail elsewhere herein and a pharmaceutically acceptable carrier or excipient. As used herein, “dietary supplemental formulations” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient. When present, the compound can optionally be present in the dietary supplemental formulations as a pharmaceutically acceptable salt. In some embodiments, the dietary supplemental formulations can include, such as an active ingredient, an amount of DHA and/or an amount of choline.
[0070] In some embodiments, the active ingredient is present as a pharmaceutically acceptable salt of the active ingredient. As used herein, “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucoronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzene sulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.
[0071] The dietary supplemental formulations described herein can be administered to a subject in need thereof via any suitable method or route to a subject in need thereof. Suitable administration routes can include, but are not limited to auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infdtration, interstitial, intra-abdominal, intra- amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intracistemal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavemosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and/or vaginal administration, and/or any combination of the above administration routes, which typically depends on the disease to be treated and/or the active ingredient(s).
[0072] Where appropriate DHA and/or choline can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a dietary supplemental formulation. As such, also described are dietary supplemental formulations containing one or more of the compounds and salts thereof, or pharmaceutically acceptable salts thereof described herein. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucoronate, saccharate, formate, benzoate, glutamate, methane sulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalene sulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.
[0073] In some embodiments, the subject in need thereof is a female of reproductive maturity. In some embodiments, the female is pregnant or suspected of being pregnant. In some embodiments, the female is 0-45 weeks pregnant. As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to. As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.
Pharmaceutically Acceptable Carriers and Secondary Ingredients and Agents
[0074] The dietary supplemental formulations can include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.
[0075] The dietary supplemental formulations can be sterilized, and if desired, mixed with agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active compound.
[0076] In some embodiments, the formulation includes one or more secondary agents that are in addition to the DHA or choline. Such secondary agents include vitamins, minerals, other fatty acids, and/or the like.
Effective Amounts
[0077] In some embodiments, the amount of the primary active agent and/or optional secondary agent can be an effective amount, least effective amount, and/or therapeutically effective amount. As used herein, “effective amount” refers to the amount of the primary and/or optional secondary agent included in the pharmaceutical formulation that achieve one or more therapeutic effects or desired effect. As used herein, “least effective” amount refers to the lowest amount of the primary and/or optional secondary agent that achieves the one or more therapeutic or other desired effects. As used herein, “therapeutically effective amount” refers to the amount of the primary and/or optional secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic effects. In some embodiments, the one or more therapeutic effects are increasing the bioavailability of DHA in the subject in need thereof. In some embodiments, the one or more therapeutic effects are increasing the bioavailability of DHA during pregnancy.
[0078] In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the choline is about 500 mg, to about 600 mg per day, optionally about 550 mg of choline per day. In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day is about 100 mg to about 300 mg, optionally about 200 mg. In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the choline per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg,
220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg,
430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,
640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg,
850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, 2500 mg, 2510 mg, 2520 mg, 2530 mg, 2540 mg, 2550 mg, 2560 mg, 2570 mg, 2580 mg, 2590 mg, 2600 mg, 2610 mg, 2620 mg, 2630 mg, 2640 mg, 2650 mg, 2660 mg, 2670 mg, 2680 mg, 2690 mg, 2700 mg, 2710 mg, 2720 mg, 2730 mg, 2740 mg, 2750 mg, 2760 mg, 2770 mg, 2780 mg, 2790 mg, 2800 mg, 2810 mg, 2820 mg, 2830 mg, 2840 mg, 2850 mg, 2860 mg, 2870 mg, 2880 mg, 2890 mg, 2900 mg, 2910 mg, 2920 mg, 2930 mg, 2940 mg, 2950 mg, 2960 mg, 2970 mg, 2980 mg, 2990 mg, or about 3000 mg. In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the choline per day ranges from about 110 mg, to about 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, 2500 mg, 2510 mg, 2520 mg, 2530 mg, 2540 mg, 2550 mg, 2560 mg, 2570 mg, 2580 mg, 2590 mg, 2600 mg, 2610 mg, 2620 mg, 2630 mg, 2640 mg, 2650 mg, 2660 mg, 2670 mg, 2680 mg, 2690 mg, 2700 mg, 2710 mg, 2720 mg, 2730 mg, 2740 mg, 2750 mg, 2760 mg, 2770 mg, 2780 mg, 2790 mg, 2800 mg, 2810 mg, 2820 mg, 2830 mg, 2840 mg, 2850 mg, 2860 mg, 2870 mg, 2880 mg, 2890 mg, 2900 mg, 2910 mg, 2920 mg, 2930 mg, 2940 mg, 2950 mg, 2960 mg, 2970 mg, 2980 mg, 2990 mg, or 3000 mg. [0079] In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg,
240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg,
450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg,
660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg,
870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, or about 2500 mg. In some embodiments effective amount, least effective amount, and/or therapeutically effective amount of the DHA per day ranges from about 110 mg, to 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,
340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg,
760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,
970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, or 2500 mg.
[0080] The formulation can include the total effective amount, least effective amount, or therapeutically effective amount of the DHA and/or choline or can contain a sub dosage of one or both the DHA or choline.
[0081] In embodiments where there is a secondary agent contained in the dietary supplemental formulation, the effective amount of the secondary active agent will vary depending on the secondary agent, the primary agent, the administration route, subject age, disease, stage of disease, among other things, which will be one of ordinary skill in the art.
[0082] When optionally present in the dietary supplemental formulation, the secondary active agent can be included in the dietary supplemental formulation or can exist as a standalone compound or dietary supplemental formulation that can be administered contemporaneously or sequentially with the compound, derivative thereof, or dietary supplemental formulation thereof.
[0083] In some embodiments, the effective amount of the secondary active agent, when optionally present, is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w/w, v/v, or w/v of the total active agents present in the dietary supplemental formulation or any numerical value or subrange within these ranges. In additional embodiments, the effective amount of the secondary active agent is any non-zero amount ranging from about O to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w/w, v/v, or w/v of the total dietary supplemental formulation or any numerical value or subrange within these ranges.
Dosage Forms
[0084] In some embodiments, the dietary supplemental formulations described herein can be provided in a dosage form. The dosage form can be administered to a subject in need thereof. The dosage form can be effective generate specific concentration, such as an effective concentration, at a given site in the subject in need thereof. As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the primary active agent, and optionally present secondary active ingredient, and/or a dietary supplemental formulation thereof calculated to produce the desired response or responses in association with its administration. In some embodiments, the given site is proximal to the administration site. In some embodiments, the given site is distal to the administration site. In some cases, the dosage form contains a greater amount of one or more of the active ingredients present in the dietary supplemental formulation than the final intended amount needed to reach a specific region or location within the subject to account for loss of the active components such as via first and second pass metabolism. [0085] The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, intemasal, and intradermal. Other appropriate routes are described elsewhere herein. Such formulations can be prepared by any method known in the art.
[0086] Dosage forms adapted for oral administration can discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the dietary supplemental formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the dietary supplemental formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution. The oral dosage form can be administered to a subject in need thereof. Where appropriate, the dosage forms described herein can be microencapsulated.
[0087] The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described herein can be the ingredient whose release is delayed. In some embodiments the primary active agent is the ingredient whose release is delayed. In some embodiments, an optional secondary agent can be the ingredient whose release is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more. [0088] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
[0089] Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non-polymeric excipient, to produce the desired release profde. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is" formulated as, but not limited to, suspension form or as a sprinkle dosage form.
[0090] Where appropriate, the dosage forms described herein can be a liposome. In these embodiments, primary active ingredient(s), and/or optional secondary active ingredient(s), and/or pharmaceutically acceptable salt thereof where appropriate are incorporated into a liposome. In embodiments where the dosage form is a liposome, the dietary supplemental formulation is thus a liposomal formulation. The liposomal formulation can be administered to a subject in need thereof.
Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the dietary supplemental formulations are applied as a topical ointment or cream. When formulated in an ointment, a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be formulated with a paraffinic or water-miscible ointment base. In other embodiments, the primary and/or secondary active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
[0091] Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be in a dosage form adapted for inhalation is in a particle-size- reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active (primary and/or secondary) ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators. The nasal/inhalation formulations can be administered to a subject in need thereof.
[0092] In some embodiments, the dosage forms are aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains a solution or fine suspension of a primary active ingredient, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multi -dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.
[0093] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof. In further embodiments, the aerosol formulation also contains co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, 3 or more doses are delivered each time. The aerosol formulations can be administered to a subject in need thereof. [0094] For some dosage forms suitable and/or adapted for inhaled administration, the dietary supplemental formulation is a dry powder inhalable-formulations. In addition to a primary active agent, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and/or starch. In some of these embodiments, a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate. In some embodiments, the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the compositions, compounds, vector(s), molecules, cells, and combinations thereof described herein.
[0095] Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas. The vaginal formulations can be administered to a subject in need thereof.
[0096] Dosage forms adapted for parenteral administration and/or adapted for injection can include aqueous and/or non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and re-suspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets. The parenteral formulations can be administered to a subject in need thereof.
[0097] For some embodiments, the dosage form contains a predetermined amount of a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate per unit dose. In an embodiment, the predetermined amount of primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be an effective amount, a least effect amount, and/or a therapeutically effective amount. In other embodiments, the predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate, can be an appropriate fraction of the effective amount of the active ingredient.
Co-Therapies and Combination Therapies
[0098] In some embodiments, the dietary supplemental formulation(s) described herein are part of a combination treatment or combination therapy. The combination treatment can include the pharmaceutical formulation described herein and an additional treatment modality. The additional treatment modality can be a chemotherapeutic, biological therapeutic, surgery, diet modulation, environmental modulation, a physical activity modulation, radiation treatment and any combination thereof.
[0099] In some embodiments, the co-therapy or combination therapy can additionally include but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.
Administration of the Dietary Supplemental Formulations
[0100] The dietary supplemental formulation and dosage forms thereof described herein can be administered one or more times hourly, daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times hourly, daily, monthly, or yearly). In some embodiments, the dietary supplemental formulations or dosage forms thereof described herein can be administered continuously over a period of time ranging from minutes to hours to days. Devices and dosages forms are known in the art and described herein that are effective to provide continuous administration of the dietary supplemental formulations described herein. In some embodiments, the first one or a few initial amount(s) administered can be a higher dose than subsequent doses. This is typically referred to in the art as a loading dose or doses and a maintenance dose, respectively. In some embodiments, the dietary supplemental formulations can be administered such that the doses over time are tapered (increased or decreased) overtime so as to wean a subject gradually off of the dietary supplemental formulation or gradually introduce a subject to the dietary supplemental formulation.
[0101] As previously discussed, the dietary supplemental formulation can contain a predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate. In some of these embodiments, the predetermined amount can be an appropriate fraction of the effective amount of the active ingredient. Such unit doses may therefore be administered once or more than once a day, month, or year (e.g., 1, 2, 3, 4, 5, 6, or more times per day, month, or year). Such dietary supplemental formulations may be prepared by any of the methods well known in the art.
[0102] Where co-therapies or multiple dietary supplemental formulations are to be delivered to a subject, the different therapies or formulations can be administered sequentially or simultaneously. Sequential administration is administration where an appreciable amount of time occurs between administrations, such as more than about 15, 20, 30, 45, 60 minutes or more. The time between administrations in sequential administration can be on the order of hours, days, months, or even years, depending on the active agent present in each administration. Simultaneous administration refers to administration of two or more formulations at the same time or substantially at the same time (e.g., within seconds or just a few minutes apart), where the intent is that the formulations be administered together at the same time.
Exemplary dietary supplemental formulations
[0103] Described in certain example embodiments herein are dietary supplement formulations comprising an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
[0104] In some embodiments, the increase in DHA bioavailability is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%,
0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%,
0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%,
0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%,
0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%,
0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, %, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%1%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%,4%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%,7%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%,0%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%,3%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%,6%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%,9%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 201%,2%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%,5%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%, 227%,8%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%,1%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%, 253%,4%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%,7%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%,0%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%,3%, 294%, 295%, 296%, 297%, 298%, 299%, 300%, 301%, 302%, 303%, 304%, 305%,6%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%, 318%,9%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%, 331%,2%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%, 344%,5%, 346%, 347%, 348%, 349%, 350%, 351%, 352%, 353%, 354%, 355%, 356%, 357%,8%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%, 370%,1%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%, 383%,4%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%, 396%,7%, 398%, 399%, 400%, 401%, 402%, 403%, 404%, 405%, 406%, 407%, 408%, 409%,0%, 411%, 412%, 413%, 414%, 415%, 416%, 417%, 418%, 419%, 420%, 421%, 422%,3%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%, 435%,6%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447%, 448%, 449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%, 461%,
462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%, 474%,
475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%, 487%,
488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 497%, 498%, 499%, 500%, or more. In some embodiments, the increase in DHA bio availability ranges from about 0.01%, to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%,
123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%,
136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%,
149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%,
162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%,
175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%,
188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%,
201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%,
214%, 215%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%,
227%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%,
240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%,
253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%,
279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%,
292%, 293%, 294%, 295%, 296%, 297%, 298%, 299%, 300%, 301%, 302%, 303%, 304%,
305%, 306%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%,
318%, 319%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%,
331%, 332%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%,
344%, 34504 34504 34704 348%, 34904 35904 35104 35204 35304 354%, 35504 35504
357%, 358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%,
370%, 371%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%,
383%, 384%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%,
396%, 397%, 398%, 399%, 400%, 401%, 402%, 403%, 404%, 405%, 406%, 407%, 408%,
409%, 410%, 411%, 412%, 413%, 414%, 415%, 416%, 417%, 418%, 419%, 420%, 421%,
422%, 423%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%,
435%, 436%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447%,
448%, 449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%,
461%, 462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%,
474%, 475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%,
487%, 488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 497%, 498%, 499%,
500%, or more.
[0105] In certain example embodiments, the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
[0106] In certain example embodiments, the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg.
[0107] In certain example embodiments, the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
[0108] In certain example embodiments, the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
[0109] In certain example embodiments, the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di- phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
[0110] In certain example embodiments, the dietary supplement formulation is a solid, a semi-solid, or a liquid.
[oni] In certain example embodiments, DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC- DHA or any combination thereof in the female subject of reproductive maturity.
[0112] In certain example embodiments, the female subject of reproductive maturity is pregnant.
[0113] In certain example embodiments, the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine- DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
[0114] In certain example embodiments, the female of reproductive maturity is a mammal, optionally a human.
METHODS OF USE
[0115] Described herein are methods of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity, the method comprising cosupplementing choline and DHA to the subject in need thereof, wherein co-supplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day. In some embodiments, the total amount of choline ranges from about 500 mg to about 600 mg per day, optionally about 550 mg of choline per day. In some embodiments, the amount of supplemental DHA per day is about 100 mg to about 300 mg, optionally about 200 mg.
[0116] In some embodiments, the amount of supplemental choline per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg,
220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg,
430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,
640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, 2500 mg, 2510 mg, 2520 mg, 2530 mg, 2540 mg, 2550 mg, 2560 mg, 2570 mg, 2580 mg, 2590 mg, 2600 mg, 2610 mg, 2620 mg, 2630 mg, 2640 mg, 2650 mg, 2660 mg, 2670 mg, 2680 mg, 2690 mg, 2700 mg, 2710 mg, 2720 mg, 2730 mg, 2740 mg, 2750 mg, 2760 mg, 2770 mg, 2780 mg, 2790 mg, 2800 mg, 2810 mg, 2820 mg, 2830 mg, 2840 mg, 2850 mg, 2860 mg, 2870 mg, 2880 mg, 2890 mg, 2900 mg, 2910 mg, 2920 mg, 2930 mg, 2940 mg, 2950 mg, 2960 mg, 2970 mg, 2980 mg, 2990 mg, or about 3000 mg. In some embodiments, the supplemental amount of choline per day ranges from about 110 mg, to about 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg,
350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg,
560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg,
770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, 2500 mg, 2510 mg, 2520 mg, 2530 mg, 2540 mg, 2550 mg, 2560 mg, 2570 mg, 2580 mg, 2590 mg, 2600 mg, 2610 mg, 2620 mg, 2630 mg, 2640 mg, 2650 mg, 2660 mg, 2670 mg, 2680 mg, 2690 mg, 2700 mg, 2710 mg, 2720 mg, 2730 mg, 2740 mg, 2750 mg, 2760 mg, 2770 mg, 2780 mg, 2790 mg, 2800 mg, 2810 mg, 2820 mg, 2830 mg, 2840 mg, 2850 mg, 2860 mg, 2870 mg, 2880 mg, 2890 mg, 2900 mg, 2910 mg, 2920 mg, 2930 mg, 2940 mg, 2950 mg, 2960 mg, 2970 mg, 2980 mg, 2990 mg, or 3000 mg.
[0117] In some embodiments, the supplemental amount of DHA per day is about 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg,
330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg,
540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,
750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, or about 2500 mg. In some embodiments, the supplemental amount of DHA per day ranges from about 110 mg, to 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg,
290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg,
500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg,
710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg,
920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1110 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, 1200 mg, 1210 mg, 1220 mg, 1230 mg, 1240 mg, 1250 mg, 1260 mg, 1270 mg, 1280 mg, 1290 mg, 1300 mg, 1310 mg, 1320 mg, 1330 mg, 1340 mg, 1350 mg, 1360 mg, 1370 mg, 1380 mg, 1390 mg, 1400 mg, 1410 mg, 1420 mg, 1430 mg, 1440 mg, 1450 mg, 1460 mg, 1470 mg, 1480 mg, 1490 mg, 1500 mg, 1510 mg, 1520 mg, 1530 mg, 1540 mg, 1550 mg, 1560 mg, 1570 mg, 1580 mg, 1590 mg, 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, 1710 mg, 1720 mg, 1730 mg, 1740 mg, 1750 mg, 1760 mg, 1770 mg, 1780 mg, 1790 mg, 1800 mg, 1810 mg, 1820 mg, 1830 mg, 1840 mg, 1850 mg, 1860 mg, 1870 mg, 1880 mg, 1890 mg, 1900 mg, 1910 mg, 1920 mg, 1930 mg, 1940 mg, 1950 mg, 1960 mg, 1970 mg, 1980 mg, 1990 mg, 2000 mg, 2010 mg, 2020 mg, 2030 mg, 2040 mg, 2050 mg, 2060 mg, 2070 mg, 2080 mg, 2090 mg, 2100 mg, 2110 mg, 2120 mg, 2130 mg, 2140 mg, 2150 mg, 2160 mg, 2170 mg, 2180 mg, 2190 mg, 2200 mg, 2210 mg, 2220 mg, 2230 mg, 2240 mg, 2250 mg, 2260 mg, 2270 mg, 2280 mg, 2290 mg, 2300 mg, 2310 mg, 2320 mg, 2330 mg, 2340 mg, 2350 mg, 2360 mg, 2370 mg, 2380 mg, 2390 mg, 2400 mg, 2410 mg, 2420 mg, 2430 mg, 2440 mg, 2450 mg, 2460 mg, 2470 mg, 2480 mg, 2490 mg, or 2500 mg.
[0118] In some embodiments, the female subject is pregnant. In some embodiments, wherein the female subject of reproductive age is a mammal, optionally a human. In some embodiments, the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less. In some embodiments, the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
[0119] In some embodiments, co-supplementing begin and/occur prior to the female of reproductive maturity being pregnant; prior to the female of reproductive maturity being 16 weeks pregnant; during gestation; after delivery; or any combination thereof. In some embodiments, co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant. In some embodiments, cosupplementing occurs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or more days, weeks, months, or years.
[0120] In some embodiments, the method increases the bioavailability of DHA. In some embodiments, an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity. In some embodiments, the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof. In some embodiments, DHA bioavailability is increased during pregnancy.
[0121] In some embodiments, the increase in DHA bioavailability is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%,
0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%,
0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%,
0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%,
0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%,
0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%,
0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%,
0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%,
124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%,
137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%,
150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%,
163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%,
176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%,
189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 201%,
202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%,
215%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%, 227%,
228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%,
241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%, 253%,
254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%,
267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%,
280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299%, 300%, 301%, 302%, 303%, 304%, 305%,
306%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%, 318%,
319%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%, 331%,
332%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%, 344%,
345%, 346%, 347%, 348%, 349%, 350%, 351%, 352%, 353%, 354%, 355%, 356%, 357%,
358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%, 370%,
371%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%, 383%,
384%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%, 396%,
397%, 398%, 399%, 400%, 401%, 402%, 403%, 404%, 405%, 406%, 407%, 408%, 409%,
410%, 411%, 412%, 413%, 414%, 415%, 416%, 417%, 418%, 419%, 420%, 421%, 422%,
423%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%, 435%,
436%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447%, 448%,
449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%, 461%,
462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%, 474%,
475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%, 487%,
488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 497%, 498%, 499%, 500%, or more. In some embodiments, the increase in DHA bioavailability ranges from about 0.01%, to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, %, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%,%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%,%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%,%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%,%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%,%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%,%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%,%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%,%, 215%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%,%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%,%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%,%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%,%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%,%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%,%, 293%, 294%, 295%, 296%, 297%, 298%, 299%, 300%, 301%, 302%, 303%, 304%,%, 306%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%,%, 319%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%,%, 332%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%,%, 345%, 346%, 347%, 348%, 349%, 350%, 351%, 352%, 353%, 354%, 355%, 356%,%, 358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%,%, 371%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%,%, 384%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%,%, 397%, 398%, 399%, 400%, 401%, 402%, 403%, 404%, 405%, 406%, 407%, 408%,%, 410%, 411%, 412%, 413%, 414%, 415%, 416%, 417%, 418%, 419%, 420%, 421%,%, 423%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%,%, 436%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447%,%, 449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%,%, 462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%,%, 475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%,%, 488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 497%, 498%, 499%,%, or more. [0122] In some embodiments, the supplemental choline is provided as a choline salt. As used herein the term “choline salt” refers to any salt that carries choline. Exemplary choline salts include, but are not limited to, choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di -phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof. Additional exemplary salt forms are also discussed below with respect to formulations.
[0123] Administration of the supplemental DHA and choline can be by any suitable route. Exemplary suitable administration routes are discussed elsewhere herein with respect to formulations. In some embodiments, the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation. In some embodiments, the supplemental choline and the supplemental DHA are administered separately. In some embodiments, the total amount of supplemental choline is administered in a single dose. In some embodiments, the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline. In some embodiments, the total amount of supplemental DHA is administered in a single dose. In some embodiments, the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA. In some embodiments, the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semi-solid formulation, a gel formulation, or a solid formulation. Exemplary dosage forms are described in greater detail below.
[0124] In some embodiments, the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery. In some embodiments, the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery. KITS
[0125] Any of the compounds, compositions, and/or formulations described herein or a combination thereof can be presented as a combination kit. As used herein, the terms "combination kit" or "kit of parts" refers to the compounds, compositions, formulations, particles, cells and any additional components that are used to package, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein. Such additional components include, but are not limited to, packaging, syringes, blister packages, bottles, and the like. When one or more of the compounds, compositions, and/or formulations, described herein or a combination thereof (e.g., agents) contained in the kit are administered simultaneously, the combination kit can contain the active agents in a single formulation, such as a pharmaceutical formulation, (e.g., a tablet) or in separate formulations. When the compounds, compositions, or formulations described herein or a combination thereof and/or kit components are not administered simultaneously, the combination kit can contain each agent or other component in separate compositions or formulations. The separate kit components can be contained in a single package or in separate packages within the kit.
[0126] In some embodiments, the combination kit also includes instructions printed on or otherwise contained in a tangible medium of expression. The instructions can provide information regarding the content of the compounds, compositions, or formulations, described herein or a combination thereof contained therein, safety information regarding the content of the compounds, compositions, formulations (e.g., dietary supplemental formulations) described herein or a combination thereof contained therein, information regarding the dosages, indications for use, and/or recommended treatment regimen(s) for the compound(s) and/or dietary supplemental compositions and/or formulations contained therein. In some embodiments, the instructions can provide directions for administering the compounds, compositions, formulations, particles, and cells described herein or a combination thereof to a subject in need thereof. In some embodiments, the subject in need thereof can be a female of reproductive maturity and/or pregnant, such as any of those described in greater detail elsewhere herein. In some embodiments, the kit and/or component thereof can provide that the compositions and/or formulations described herein can increase DHA bioavailability in the subject in need thereof. EXAMPLES
[0127] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.
Example 1
Introduction
[0128] Docosahexaenoic acid (DHA; 22:6w3) is an omega-3 polyunsaturated fatty acid (PUFA) that plays a critical role in fetal development1 2. The hepatic export of DHA into circulation is linked to the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine JV-methyl transferase (PEMT). The PEMT pathway in liver generates PC molecules enriched in DHA (PC-DHA) that can subsequently be incorporated into VLDLs for export into plasma and delivery to extrahepatic tissues (including the placenta). Transgenic mice lacking PEMT show substantially reduced DHA concentrations in plasma3, and limited accumulation of DHA in fetal brains of pups bom to PEMT deficient dams4. Consistent with animal models, previous work has highlighted the importance of PEMT during human pregnancy by demonstrating biomagnification of PEMT-derived PC in fetal cord blood relative to maternal blood at delivery5.
[0129] Dietary methyl donors, such as choline, are essential to support the synthesis of the universal methyl donor, S-adenosylmethionine (SAM), utilized by methyltransferases to catalyze the transfer of methyl groups (-CHs) to form their respective products. Thus, the activity of the PEMT pathway, and by extension, the availability of DHA to extrahepatic tissues, is intrinsically linked to dietary methyl donor and choline intakes. In women of reproductive age with low choline intake (<50mg/d)-induced organ dysfunction, the proportion of plasma PC containing DHA (PC-DHA/Total PC) is significantly reduced6. Conversely, a high-choline intake (930 vs 480 mg/d), co-administered with 200 mg DHA/d for 12 weeks, upregulated PEMT activity in women of reproductive age5, and led to greater (P < 0.001) erythrocyte PC-DHA enrichment and a more rapid rise in plasma PC-DHA7. An adequate supply of methyl donors for optimal PEMT activity may be particularly important during pregnancy. The promoter region for the PEMT gene contains an estrogen response element8, rendering PEMT activity sensitive to the dramatic increase in circulating estrogen during the latter half of pregnancy. Notably, this increase in PEMT activity coincides with the accumulation of DHA and other long chain PUFAs in fetal brain9.
[0130] As a major consumer of SAM, it is unsurprising that during the timing of this large increase in estrogen and PEMT activity, plasma biomarkers of choline-derived methyl donors are reduced in human pregnancy 10, despite ‘adequate’ choline intakes. A higher choline intake (930 versus 480 mg/d), administered during the third trimester of pregnancy, has been shown to (i) attenuate this decline10, (ii) restore choline partitioning between betaine and CDP-PC pathways to levels observed in nonpregnant women5, and (iii) enhance the generation of PEMT-derived lysophosphatidylcholine (LPC) that is enriched with DHA11, and preferentially taken up by fetal brain12 13. Nonetheless, this higher level of choline intake among third- trimester pregnant women failed to increase PC-DHA7 in circulating erythrocytes, possibly because PEMT is already operating at maximal capacity in the last third of gestation. Whether prenatal choline supplementation would influence circulating PC-DHA if administered prior to maximal PEMT capacity, or for a longer duration, is unknown. To fdl this gap, Applicant investigated the effect of prenatal choline supplementation, administered throughout the second and third trimesters of pregnancy, on biomarkers of DHA status among pregnant participants consuming supplemental DHA.
Subjects Methods
Participants
[0131] Pregnant participants were recruited between October 2017 and April 2019 at maternity clinics throughout the Ithaca NY region using flyers. Screening of overall health of the prospective participants was conducted via online questionnaires, which included a short version of the NIH Diet History Questionnaire (DHQ-III; https://www.nal.usda.gov/fhic/dietary-assessment-instruments-research) to estimate usual choline intake, and a validated DSM DHA/EPA food frequency questionnaire (FFQ; kindly provided by DSM Nutritionals Inc) to assess omega-3 fatty acid intake. Eligible participants were 12-16 weeks pregnant at the beginning of the study, between age 21 and 40 years, had a pre-pregnancy body mass index (BMI) < 32 kg/m2, were intending to deliver at Cayuga Medical Center, and were willing to comply with the study protocol. Participants were excluded if they had usual dietary DHA intakes exceeding 400 mg/d (based on FFQ), had usual dietary choline intakes exceeding 450 mg/d (based on DHQ-III), or reported having cardiovascular disease, cancer, type 1 or 2 diabetes mellitus, gastrointestinal disorders, gallbladder disease, kidney disease, liver disease or anemia (based on health questionnaire). Additional exclusion criteria included: use of prescription medications known to affect liver function; presence of more than one fetus; unwillingness to donate placenta; self-reported tobacco, recreational drug, or alcohol use during gestation; or presence (or development) of pregnancy-associated complications (e.g., preeclampsia, gestational diabetes).
Study Design and Supplements
[0132] The present study was a single-center, randomized, double-blind, parallel-group choline intervention study, designed to investigate the effects of prenatal choline supplementation on DHA status in healthy free-living pregnant participants consuming supplemental DHA (NCT03194659). The intervention of 550 mg supplemental choline/day or control (25 mg supplemental choline/d) was initiated at gestational age 12 - 16 weeks and continued until delivery; throughout this period, all participants received 200 mg supplemental DHA/d. Data obtained during the study were recorded in a dedicated online secure database developed by Cornell Institute of Social and Economic Research (CISER).
Randomization and Masking
[0133] Eligible participants (n=33) were enrolled into the study by MQM on a rolling basis in a parallel arm design. A simple 1 : 1 randomization scheme was generated by KCK using a web-based random number generator assigning participants to either group A or group B; study staff (OVM) not interacting with participants replaced group A or group B with intervention or control. Both groups consumed a grape juice cocktail that provided either 550 mg supplemental choline/d (intervention) or 25 mg supplemental choline/d (control). Preparation of the grape juice cocktail was handled by study staff not interacting with study participants (OVM) to maintain personnel blinding. The grape juice cocktail was served in 15-mL color-coded conical tubes to conceal the intervention assignment from the participant.
Procedures
Choline Supplement
[0134] Participants consumed a grape juice cocktail containing 550 mg choline/d (intervention) or 25 mg choline/d (control) throughout the duration of the study. The 550 mg choline dose for the intervention group was comprised of 500 mg unlabeled choline (dO- choline) and 50 mg methyl -t/9-cholinc (t/9-cholinc). The 25 mg choline dose for the control group was in the form of t/9-cholinc only. The low-dose deuterium-labeled choline tracer (50 mg t/9-cholinc for the intervention group, and 25 mg t/9-cholinc for the control group) was administered to provide insights into the use of choline derived methyl groups for the synthesis of PC by the PEMT pathway (described in the results section).
[0135] The choline supplements were prepared in the Human Metabolic Research Unit (HMRU) at Cornell University. First, stock solutions of JO-choline and U9-cholinc were prepared separately by dissolving choline chloride (Balchem Inc.; NY, USA) or U9-cholinc chloride (Cambridge Isotope Uaboratories Inc.; MA, USA) in Milli-Q water to produce 250 mg/mU JO-chol ine and 50 mg/mU U9-cholinc solutions. The stock solutions were fdtered and stored at 4°C for up to one year; tests conducted during this period showed excellent stability with no detectable loss through time. Next, the choline and control supplements were assembled by aliquoting 2 mU of the dO- choline stock solution (intervention group), 1 mU of the U9-cholinc stock solution (intervention group), or 0.5 mU of the U9-cholinc stock solution (control group) into 15-mU sterilized polystyrene tubes. The supplement tubes were filled with grape juice cocktail (Welch’s; MA, USA) and labeled in a manner that was indistinguishable by both the participants and personnel who interacted with the participants. Finally, all supplement tubes were packaged in Ziplock bags labeled with the participant ID numbers and kept frozen in a food-only -20°C freezer prior to pick-up. Testing of the supplements that were kept for two months at room temperature, or inside a home refrigerator, showed excellent stability with no detectable loss overtime. Study Protocol
[0136] Participants were required to visit the HMRU on three separate occasions (FIG. 1). The first visit was during gestational week (GW) 12-16, while the second (GW 20-24) and third (GW 28-32) visits were every 8 weeks thereafter. At each study visit, participants received their daily supplements, which included the grape juice choline cocktail tubes, a 200-mg DHA supplement [Nature's Way EfaGoldNeuromins 200mg DHA (plant source); DSM Nutritional Products; Netherlands], and an over-the-counter prenatal vitamin/mineral supplement (Nature Made Prenatal Tablet; Pharmavite LLC; CA, USA). Participants were instructed to consume the supplements and grape juice cocktail daily, and at the latter two HMRU visits, to return supplement containers as a means to assess adherence to the study protocol. Participants were also asked to discontinue any supplements outside of those provided by this study. Selfselected diets were consumed by participants throughout the study period.
[0137] At each study visit, participants provided an 8-1 Oh fasting blood sample, and height and weight measurements were recorded. At visits 2 and 3, participants completed a follow-up health questionnaire to assess changes in health status and medication/supplement use. At visit 1 (baseline), participants completed the National Cancer Institute’s Automated Self- Administered 24-hour (ASA24) Dietary Recall, under the supervision of research personnel, to assess usual mean nutrient intakes. Additionally, participants completed two to four selfadministered 24-hr dietary recalls (ASA24) throughout the course of their 2nd and 3rd trimesters, prior to delivery.
[0138] At delivery, non-fasting maternal blood, placenta, and fetal cord blood were collected (FIG. 1). In addition, maternal and newborn information were obtained from medical charts. Maternal information included due date, complications during pregnancy, and complications during labor or delivery. Newborn information included the date and mode of delivery, gestational age, birth weight, sex and APGAR score.
Sample Collection and Processing
[0139] Fasting blood samples (8-10 h) were collected at each of the three HMRU visits in one 10-mL serum separator gel and clot-activator tube (SST Vacutainer; Becton, Dickinson and Company), three 10-mL EDTA-coated tubes (Becton, Dickinson and Company), and one 5-mL EDTA-coated tube (Becton, Dickinson and Company). The three 10-mL EDTA-coated tubes containing whole blood were placed on ice immediately following collection and were centrifuged within 90 min at 2200 rpm for 15 min at 4°C. Plasma was removed for metabolite measurements and buffy coatwas removed and mixed with 50 pL DMSO for DNA extraction. Remaining RBCs were either stored or washed 3x with phosphate-buffered saline (PBS). The 5-mL EDTA coated tube, which provided whole blood for the complete blood counts, was processed within 60 min by research personnel in the Cornell Human Nutritional Chemistry Laboratory. The SST blood, which provided serum for the blood chemistry profile, was kept at room temperature, allowed to clot, and centrifuged at 3000 rpm for 15 min at room temperature. All the biological samples were dispensed into 1.8-mL cryostat vials (CryoTube; NUNC) and stored at -80°C.
[0140] Maternal blood and cord blood samples were collected at delivery. Maternal blood samples were collected into two 10-mL EDTA coated tubes and one 10-mL SST tube at the hospital within 1-h of delivery. Cord blood samples were collected into two 4-mL EDTA- coated tubes at the time of delivery. After collection, maternal and cord blood samples were refrigerated at 4°C, and processed as described previously within 4 h.
[0141] The placenta was also obtained at delivery, weighed and processed at the hospital within 90 min of delivery. After removal of the amnion, 16 full-thickness tissue biopsies (0.5 x 0.5 x 0.5 cm) were taken from 4 separate locations (i.e., the placenta was visually divided into four quadrants). Samples were rinsed with PBS immediately. The other samples were flash frozen in liquid nitrogen, placed in a cryostat tube, and stored temporarily in a canister containing liquid nitrogen. In the laboratory, all samples were stored at -80°C. Prior to using the placental samples for analytical measurements (i.e., total DHA), a homogenous representation was prepared by taking one piece of placenta from each quadrant and powderizing in liquid nitrogen using a Bessman Tissue Pulverizer (Fisher Scientific, Waltham, MA, USA).
Analytical measurements
PCH-DHA Measurements in RBCs
[0142] In a priori planned analysis of RBC PC-DHA (% fatty acids in RBC PC), Applicant proposed utilizing stable -isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS); however, simultaneous measurement of individual PC species containing DHA, and total PC (to facilitate expressing % RBC PC composition) in the RBC sample matrix proved technically challenging and exhibited low reproducibility. As such, samples were sent to the Analytical Core for Metabolomics and Nutrition (BC Children's Hospital Research Institute), which utilizes a we 11 -validated method whereby PC is separated from the other lipids by HPLC followed by analysis of the PC fatty acids using gas-liquid chromatography (GLC)14. Briefly, RBC lipids were extracted from washed RBCs using a modification of the method from Rose and Oklander15, and PC was separated using a Waters 2690 HPLC, a quaternary solvent system of hexane, methanol, acetone and isopropanol, and a Waters YMC-Pack DIOL column. The HPLC column eluant was split 10:90 after which lipids were detected and quantified by a Waters 2424 evaporative light scattering detector and recovered with use of a Waters FCIII fraction collector. Following the evaporation of solvents under nitrogen, fatty acids were converted to methyl esters, separated, and quantified by GLC. RBC PC-DHA is expressed as a percentage of the total fatty acids in PC. The inter-assay CV for RBC PC-DHA (as a percentage of the total fatty acids in PC) was 6.4% based on in-house RBC controls.
PC-DHA Measurements in Plasma
[0143] PC-DHA concentration of plasma (pmol/L) was analyzed using stable-isotope dilution LC-MS/MS at Cornell University’s Biotech Proteomics and Metabolomics Facility. Briefly, plasma (20pL) was transferred to 1.5ml Eppendorf vials (VWR20170-038), to which 2.4 nmol J7-PC 15:0-18: 1 (Avanti Polar Lipids, Alabaster, AL) in 0.2 mL methanol: chloroform (2: 1 v/v) was added as an internal standard. The samples were mixed and incubated overnight at -20°C. On the following day, samples were centrifuged, and the supernatant was collected and transferred into another Eppendorf tube without disturbing the solid phase. To the pellet, 0.25 mL of methanol: chloroform: water (2: 1:0.8) was added, followed by vortexing, and centrifugation. The resulting supernatant was collected and combined with the first supernatant. To the pooled supernatant, chloroform (100 pl) and optima water (100 pL) were added; samples were mixed and then centrifuged. Using a long gel loading pipet tip, the lower chloroform phase was transferred into glass vials, dried in a speed vac, and reconstituted with 200 pl methanol: chloroform (6: 1 v/v) for subsequent LC-MS/MS analysis.
[0144] The LC-MS/MS analysis of PC-DHA species was performed using a Syncronis Silica column (150 mm length x 2.1 mm internal diameter, 5pm particle size; ThermoScientific) in Exion LC system coupled with Sciex X500B QTOF mass spectrometer (Framingham, MA). PC-DHA species were separated under step gradient conditions with a flow rate of 400pL/min. The column temperature was kept at 30°C. The mobile phase (MP) consisted of MPA (400mL acetonitrile: 127mL water: 68mL ethanol: 3mL IM ammonium acetate in water: 2mL concentrated glacial acetic acid) and MPB (250 mL acetonitrile: 250 mL water: 42 mL ethanol: 13.5 mL IM ammonium acetate in water: 9 mL of concentrated glacial acetic acid). The following step gradient was employed for the separation and analysis of PC- DHA species: 5% MPB from 0 to 3 min; 30% MPB from 3 to 10 min; 60% MPB from 10 to 14 min; 100% MPB from 16 to 17 min; 5% MPB from 17 to 19 min. Total run time was 19 min and retention times at 1 ,90min, 1 ,98min, 2.04min and 2. 19min were observed for PC 18:0- DHA, PC 18: 1-DHA, PC 16:0-DHA andt/7-PC 15:0-18: 1 (IS). The injection volume was lOpL for each of the standards and samples. The autosampler temperature was kept constant at 5°C. [0145] The Sciex X500B QTOF mass spectrometer with an ESI Turbo V™ source was operated in the negative ion mode for this analysis. The electrospray voltage was set at 4.5 kV and the temperature of the heated capillary was set at 350°C. It was operated under the Ion Source gasl and 2 at 30 psi, curtain gas at 30 (arbitrary unit), and CAD gas at 7 (arbitrary unit). The declustering potential (DP) was set to -35V with accumulation time of 0.25s. The MS full scan measurement was done from m/z 100 to m/z 1000 in profde mode followed by MRM HR scan acquired from 0 min to 21 min at collision energies of -40V for each analyte. The acetate adduct negatively charged ion of each analyte was selected as the precursor ion. For internal standard (J7-PC 15:0-18: 1), loss of 18: 1 fatty acid from acetate adduct was used as a product ion for an MRM transition pair at m/z 811.62/288.2924. Negative ion MRM transition pairs were monitored at m/z 864.57/327.2340 for PC 16:0-DHA, m/z 892.61/327.2340 for PC 18:0- DHA, and m/z 890.59/327.2340 for PC 18: 1-DHA. Loss of DHA fragments (m/z 327.2340) of acetate adducts from each PC-DHA species was the major fragment used in MRM HR method for quantitation. The data were acquired using Sciex OS 2.0 software and the quantitation ratio PC-DHAs/IS was calculated for each sample by integrating the peak areas by MQ4 Integration Algorithm of each analyte using MRM transitions in the same software.
[0146] Calibration curves standard solutions were prepared by serial dilutions of the standards (PC 16:0-DHA, PC 18:0-DHA; Avanti Polar Lipids, Alabaster, AL), mixed with the internal standard (J7-PC 15:0-18: 1) at a concentration of 12 nmol/mL in 6: 1 MeOH:CHCh. The calibration curves ranged from 50 to 5000 pmol for PC 16:0-DHA and PC 18:0-DHA (R2>0.99). The lower limit of detection and lower limit of quantitation determined based on S/N in PC DHA standards solutions were 0.15 pmol and 0.5 pmol. All area ratios of PC16:0- DHA and PC18:0-DHA quantified and calculated (Peak area of analyte/Peak area of IS) in the plasma samples fell within the linear dynamic range of calibration standards. Since no commercial standard was available for PC 18: 1-DHA, the standard curve for PC 16:0-DHA was used to derive absolute concentrations of PC 18: 1-DHA (which was 1.3% of the total PC- DHA concentration).
[0147] To validate the LC-MS/MS method for quantitation of PC-DHA species in plasma samples, four QA samples were analyzed several times to determine the intra-day and interday variation for assay precision. For plasma samples, which were extracted and analyzed at the same time, intra-assay CVs were 3.1% (PC 16:0-DHA) and 2.1% (PC 18:0-DHA). Recovery yield for PC-DHA extraction method was also determined by spiking the 2.4 nmol IS in 20 pl of plasma samples before and after extraction of PC-DHA species, and it was found to be 92%. Matrix effect (ME) was also calculated as 22% for internal standard. This value represents a loss of 22% of IS peak area (ion suppression) in plasma samples compared to pure solvent due to alteration in ionization efficiency. PC-DHA in plasma is expressed as the sum of PC 16:0-DHA + PC 18:0-DHA + PC 18: 1-DHA.
Total DHA Measurements in RBCs and Plasma
[0148] DHA composition of washed RBCs (% total fatty acids) and concentrations in plasma (pg/mL) were analyzed by gas chromatography (GC) coupled to a flame ionization detector at OmegaQuant® as previously described16 17. Briefly, RBCs and plasma were transferred to screw-cap glass vials, after which 14% boron trifluoride (Sigma-Aldrich, St. Louis, MO) and hexane (EMD Chemicals, USA) were added to RBCs, and BTM (methanol containing 14% boron trifluoride, toluene, methanol; 35:30:35 v/v/v) (Sigma-Aldrich, St. Louis, MO) was added to plasma. Placenta samples were weighed into screw-cap glass vials that contained tritricosanoin as an internal standard (tri-C23:0 TG) (NuCheck Prep, Elysian, MN), homogenized and subsequently subjected to a modified Folch extraction. A portion of the organic layer was transferred to a new screw-cap glass vial and dried in a speed vac. BTM was then added to the dried placenta samples. Next, sample vials (RBCs, plasma and placenta) were briefly vortexed and heated in a hot bath at 100°C for 45 minutes. After cooling, HPLC grade water was added, and vials were recapped, vortexed and centrifuged to separate layers. Aliquots of the hexane layer were subsequently transferred to GC vials.
[0149] GC was carried out using a GC-2010 Gas Chromatograph (Shimadzu Corporation, Columbia, MD) equipped with a SP-2560, 100-m fused silica capillary column (0.25 mm internal diameter, 0.2 um film thickness; Supelco, Bellefonte, PA). Fatty acids were identified by comparison with a standard mixture of fatty acids (GLC OQ-A, NuCheck Prep, Elysian, MN), which was also used to determine individual fatty acid calibration curves. The following 24 fatty acids (by class) were identified: saturated (14:0, 16:0, 18:0, 20:0, 22:0 24:0); cis monounsaturated (16: 1, 18: 1, 20: 1, 24: 1); trans unsaturated [16: 1, 18: 1, 18:2); cis n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4, 22:5); and cis n-3 polyunsaturated (18:3, 20:5, 22:5, 22:6). Fatty acid composition, specifically DHA forthis study (cis C22:6, n-3), was expressed as a percent of total identified fatty acids. The inter-assay CV for total DHA in RBCs, plasma and placenta was <5% based on in-house controls.
Maternal genotypes
[0150] Single nucleotide polymorphisms (SNPs) in the PEMT gene (P EMT rs7946; PEMT rs4646343), which may influence PEMT activity18, were determined as previously described19,20 so that these variables could be considered in our statistical models if differentially distributed between the intervention and control groups.
Choline and Phosphatidylcholine Measurements
[0151] Stable-isotope dilution LC-MS/MS was used to measure plasma free choline21’22, plasma PC2223, and plasma enrichment percentages of t/j-PC and J9-PC 2223. Intra and inter assay CVs were <3% for choline and < 6% for PC.
Hematology and Chemistry Panels
[0152] The CBC and blood chemistry profiles were performed at the Human Nutritional Chemistry Service Laboratory at Cornell University. Hematology analysis was conducted using a Beckman-Coulter AcT Diff2 coulter counter; serum albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides were measured using an automated chemistry analyzer (Dimension Xpand Plus; Siemens Healthcare Diagnostics).
Study Outcomes
[0153] The primary outcome of this study was DHA-containing PC species in circulating maternal RBCs. PC species containing DHA (PC-DHA) at either the sn-1 or sn-2 positions are found circulating in RBCs and have been shown to be responsive to dietary choline intake among women of reproductive age7. Secondary outcomes included total DHA in maternal RBCs, an established surrogate for tissue DHA concentrations24, as well as maternal plasma measures of total DHA, PC-DHA, and PC (unlabeled and isotopically labeled forms). All outcomes were measured at gestational age 12-16 weeks (baseline, study visit 1), gestational age 20-24 weeks (study visit 2), gestational age 28-32 weeks (study visit 3) and at delivery (gestational age 36-42 weeks). In addition, we included measurements of total DHA in fetal RBCs and plasma, as well as the placenta. Exploratory/descriptive outcomes included: (i) selfreported dietary intakes of choline and DHA assessed at baseline (visit 1) and throughout the second and third trimesters of pregnancy; (ii) maternal clinical parameters (i.e., WBC counts, RBC counts, and hemoglobin assessed at visit 1, visit 2 and visit 3; serum concentrations of albumin, ALT, AST, glucose, total cholesterol, HDL-C, LDL-C and triglycerides assessed at visit 1, visit 3 and delivery); and (iii) newborn characteristics (i.e., infant sex, weight, length, head circumference, APGAR scores, and placental weights).
Statistical Analysis
[0154] The study sample size was based on previous results among nonpregnant women7, whereby means and standard deviations of erythrocyte PC-DHA for two levels of choline intake yielded a Cohen's d effect size of 1.1, after accounting for statistically significant covariates. G*Power 3. 1 (Henrich Heine Universitat Dusseldorf) was used to model estimated sample sizes; accordingly, a sample size of 15 for each group (n=30 total) was required to have a power of 80% at an alpha of 0.05 utilizing a t test between the control and intervention groups. Participants were continuously enrolled until 30 participants had completed the trial; all participants who completed the trial (i.e., delivery data were available) were included in all analyses by intention to treat (ITT; a modified ITT excluding participants who were excluded due to the development of pregnancy-related pathologies).
[0155] Differences between intervention and control groups for participant characteristics and baseline measures, as well as infant characteristics, were assessed using Wilcoxon Rank Sum Tests (continuous variables) or chi-square test of independence (categorical variables). Q- Q plots and histograms of DHA-related outcomes were visually assessed for normality; the Shapiro-Wilk test and skewness was used to assess normality and symmetry of the data. Mixed linear models that controlled for baseline measures (with the exception of labeled PC) were used to assess the effect of the choline intervention on maternal outcomes (i.e., RBC PC-DHA, plasma PC-DHA, RBC total DHA, plasma total DHA, plasma free choline, plasma PC, plasma lipids, and clinical parameters) throughout the course of the study. In addition to including baseline values of the respective metabolite, these models included intervention arm, time, and their interaction term (intervention x time) as fixed effects, and participant ID as a random effect. These model estimates are depicted in the graphs and tables, and reported in the primary text. Additional sensitivity analyses were conducted to assess the robustness of the total DHA outcomes, due to their widespread use as a markers of DHA status. These sensitivity analyses included models without adjustment for baseline (unadjusted model), as well as a model that considered additional covariates hypothesized to influence the intervention effects and/or DHA outcomes, specifically age, pre-pregnancy BMI, and calculated duration of intervention exposure (i.e., gestational age at delivery - gestational age at study entry) (fully adjusted models). To assess the impact of choline intake on fetal DHA outcomes, linear models that included intervention arm and baseline maternal RBC DHA as fixed effects were used. For self-reported intakes of dietary choline and dietary DHA, /'-values were derived from Hodges- Lehman-Sen estimations for non-parametric comparison of medians.
[0156] All statistical analyses and graphs were generated using the R statistical program language and environment, version 3.6.1 (R Foundation for Statistical Computing). Graphs were developed using “ggplot2,” plotting estimated marginal means and Cis derived from (mixed) linear models. Statistical significance was set at P < 0.05. For outcomes with a significant interaction term, Tukey’s honestly significant difference post hoc test was used to assess the significance of differences between the subgroups.
RESULTS
Participant Characteristics
[0157] A total of 118 potential participants were prescreened by completing online questionnaires. Of these potential participants, 85 were excluded for the following reasons: did not meet eligibility criteria (n=22), declined to participate (n=34), or were unwilling to comply with study protocol (n=29). The remaining 33 eligible subjectswere randomly assigned to the choline intervention group (n=17) or control group (n=16). Of these 33 participants, three developed gestational diabetes during the intervention trial and were discontinued. Thus, 30 participants (n=15 in the intervention group and n=15 in the control group) were included in the intention-to-treat analyses. FIG. 2 depicts the flowchart of the study. Table 1 presents baseline demographics and baseline measures of the participants included in the intention-to treat analysis by group allocation. Of note, participants’ reported choline intakes were similar to representative samples of choline intake within the North American population31.
[0158] Participant adherence to the study protocol was >99%, based on the number of returned juice containers (containing the choline supplement) and the number of returned DHA capsules. Further, mean concentrations of plasma free choline, which have been shown to be responsive to choline supplementation during pregnancy10, were significantly higher in the choline intervention (versus control) group with between group differences detected at with between-group differences detected at visit 2, visit 3, and delivery32..
[0159] The intervention trial was well-tolerated, and participant-reported in-trial events were similarly distributed between the two groups (Table 2). The choline intervention was found to associate in a statistically significant manner with some of the clinical parameters assessed with the complete blood count and blood chemistry profile (i.e., WBC counts, RBC counts, and hemoglobin; Table 4); however, mean concentrations of the clinical parameters remained within normal range for both the intervention and control groups25’ 27. No differences (P >_0.13) between the groups were observed for serum concentrations of albumin, ALT, AST and glucose at any of the study timepoints (GW 20-24 through delivery). For newborn characteristics, no statistically significant effects of the choline intervention were observed on neonatal weight (P=0.4), length (P=0.8), head circumference (P=0.7), APGAR scores (P=0. 1), or placental weights (P=0.4) (Table 8). Lastly, median intakes of self-reported dietary DHA (17 and 34 mg/d for intervention and control, respectively; P= 0.41) and self-reported dietary choline (353 and 377 mg/d for intervention and control; P=0.15) did not differ between the groups throughout the duration of the study (Table 9).
Study Outcomes
PC-DHA
PC-DHA in Maternal RBCs
[0160] Significant effects of time (P<0.000084) were observed for maternal RBC PC-DHA (FIG. 3A); however, no significant effects of the choline intervention (P = 0.77) or its interaction with time (P = 0.11; choline intervention x time) were detected (FIG. 3A). . Nonetheless, RBC PC-DHA values were non-normally distributed and recalcitrant to various transformations (log; square root; arcsine). Two participants at delivery (1 control, 1 intervention) were significant outliers (>2 SDs) and exhibited atypical responses: the control partic-ipant exhibited a 70% increase and the intervention participant exhibited a 78% decrease in RBC PC-DHA from GW 28-32 to delivery, several times greater than the average 15% decrease between these 2 time points for the cohort. Sensitivity analyses removing these 2 individuals’ data points yielded a significant choline intervention by time interaction (P = 0.010); RBC PC-DHA (as a percentage of RBC PC total fatty acids) was higher in magnitude in the intervention at GW 28-32 [3.2% (95%CI: 2.9, 3.5%) vs. 2.7% (95% CI: 2.4, 3.0%); P = 0.05] and delivery [2.7% (95% CI: 2.4, 3.0%) vs. 2.3% (95% CI: 2.0, 2.6%); P = 0.16] relative to the control group.
[0161] As Applicant’s intervention was powered from a trial without RBC PC-DHA data from delivery, a more variable time point due to its nonfasting nature and the metabolic milieu of parturition, we considered models omitting this time point as well. Significant choline intervention x time interactions were observed in such models (P < 0.00005). RBC PC-DHA tended to be higher in the intervention at GW 28-32 [3.1% (95% CI: 2.9, 3.3%) vs. 2.8%(95% CI: 2.6, 3.0%); P = 0.07] relative to the control group.
PC-DHA in Maternal Plasma
[0162] Significant effects of choline intervention (P=0.0030) and time (P<0.00001) and their interaction (P=0.012; choline intervention x time) were detected for maternal plasma PC- DHA (FIG. 3B). Plasma PC-DHA exhibited an inverted U-shaped response across the study, increasing through the GW28-32 in both the intervention and control groups before declining at delivery; however, a significantly higher plasma PC-DHA concentration (micromoles/liter) was achieved in the intervention group at GW 28-32 [160 (95% CI: 145, 175) v. 121 (95% CI: 106, 136) pmol/L; (P=0.0005) and delivery [130 (95% CI: 115, 145) v. 94 (95% CI: 79, 108) pmol/L (P=0.0012).
Total DHA
[0163] While RBC PC-DHA was the primary outcome of this investigation, due to previous trial evidence that choline supplementation influenced plasma and RBC PC-DHA, choline’s influence on PC-DHA is ultimately physiologically meaningful to the extent that it contributes to total DHA availability in the plasma and total DHA levels in cell membranes (found across all phospholipid species). Indeed, PC-DHA in plasma and in the cell can be remodeled and facilitate DHA entering additional lipid pools (e.g., LPC; cholesterol esters; phosphatidylethanolamine). Total DHA in RBCs (% total RBC fatty acids) is a well-validated indicator of tissue DHA levels (i.e., DHA status), consistently linked to health outcomes (including pregnancy-related health outcomes), and thus, the most physiologically relevant outcomes of interest in relation to choline supplementation’s impact.
Total DHA in Maternal RBCs
[0164] Significant main effects of the choline intervention (P=0.009), time (P<0.001), and their interaction (P=0.002; choline intervention x time) were detected for maternal RBC total DHA (FIG. 3C). Specifically, RBC total DHA (as a percentage of total fatty acids) increased throughout the study in both the intervention and control groups; however, a higher level was achieved in the intervention group at GW 28-32 [7.9% (95% CI: 7.5, 8.2%) vs. 7.3%(95% CI: 6.9, 7.6%); (P=0.008) and delivery [8.0% (95% CI: 7.6, 8.3%) vs. 7.2% (95% CI: 6.8, 7.5%); (P=0.0005) (Table 3). The statistically significant effect of the choline intervention on RBC DHA was robustly observed in both unadjusted models and models adjusting for additional covariates (Table 10).
Total DHA in Maternal Plasma
[0165] Significant effects of the choline intervention (P=0.018), time (P=0.0002) were detected for maternal plasma total DHA; however, the choline intervention x time interaction term did not achieve our threshold for statistical significance (P = 0.068) (FIG. 3D). While both groups exhibited an inverted U-shaped response through time with an initial rise in plasma total DHA until GW 28-32 followed by a fall at delivery, a significantly higher plasma total DHA was observed in the intervention (vs control) group at GW 28-32 (P<0.02) and delivery (P<0.003) (FIG. 3D; Table 3). The statistically significant effect of the choline intervention on plasma DHA was robustly observed in both unadjusted models and models adjusting for additional covariates (Table 7).
Total DHA in Placenta, Cord RBCs and Cord Plasma
[0166] No significant effects of the choline intervention were detected on placenta DHA [as either a percentage of the total fatty acids (P = 0.77) or absolute concentration (micrograms/milligram) (P = 0.42)], cord RBC DHA (P = 0.12), or plasma DHA (P = 0.91) in the model that controlled for baseline concentrations of maternal RBC DHA or in the unadjusted model (Table 5). Unlabeled and Isotopically Labeled PCs in Plasma
[0167] The use of choline as a methyl donor by the PEMT pathway was assessed by measuring JJ-PC enrichment (t/j-PC / total PC) and the ratio of t/j-PCz/9-PC. D5-PC is generated by the PEMT pathway when one of the t/j-labclcd methyl groups is used in the SAM- dependent methylation of phosphatidylethanolamine to PC, whereas J9-PC is generated by the CDP-choline pathway when the intact choline molecule (t/9-cholinc) is used to generate PC5 22. The below results collectively are consistent with greater use of choline as a methyl donor by the PEMT pathway and greater PEMT activity in response to the choline intervention.
Total PC
[0168] A significant choline intervention by time interaction term (P = 0.05) was detected for maternal plasma total PC (FIG. 4A) with higher total plasma PC (micromoles/liter) at delivery (P = 0.006) in the intervention (vs. control) group [2613 (95% CI: 2434, 2792) vs. 2247 (95% CI: 2068, 2426) pmol/L], d3-PC
[0169] Significant main effects of the intervention (P = 0.007) and time (P = 0.045) were detected for maternal plasma d3-PC (FIG. 4B) with higher plasma d3-PC enrichment observed in the intervention (vs. control) group throughout the study period. The choline intervention x time interaction term was not statistically significant (P = 0.27). d9-PC
[0170] Significant main effects of the choline intervention (P = 0.034) were detected for maternal plasma d9-PC with lower plasma d9-PC enrichment observed in the intervention (vs. control) group throughout the study period. The choline intervention x time interaction term was not statistically significant (P = 0.107) (FIG. 4C). d3:d9 PC
[0171] Significant main effects of the intervention (P = 0.000013), time (P < 0.0001), and their interaction (P = 0.04; choline intervention x time) were detected for maternal plasma d3:d9-PC (Figure 3D). A higher ratio of d3:d9-PC enrichment was observed in the intervention (vs. control) group at GW 20-24 [0.79 (95% CI: 0.69, 0.89) vs. 0.53 (95%CI: 0.43, 0.63); P = 0.0005], GW 28-32 [0.92 (95% CI: 0.82, 1.01) vs. 0.54 (95% CI: 0.45, 0.65); P < 0.0001], and delivery [1.05% (95% Maternal Lipid Parameters
[0172] Because of choline’s role in hepatic lipid export (including DHA), we assessed the effect of the choline intervention on lipid parameters in serum (Table 6). Although no differences were detected between groups for HDL cholesterol (P=0.4) and triglycerides (P=0.8), LDL cholesterol (a derivative of VLDL cholesterol) 0.8), LDL cholesterol (a derivative of VLDL cholesterol) tended to be elevated in the choline intervention (vs. control).
Figure imgf000059_0001
Figure imgf000060_0002
Figure imgf000060_0001
Figure imgf000061_0001
Table 4. Maternal Clinical Parameters1
Figure imgf000062_0001
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000064_0002
e
Figure imgf000065_0001
Figure imgf000066_0001
Enrichment % d9-Choline 1.87a 1.85a 0.87 2.73a 2.08a 0.38 2.13a 1.81a 0.06
[1.59,2.19] [1.67,2.05] [1.93, [1.73,2.48] [1.81, [1.19,2.76]
3.85] 2.50] d3-Choline 0.56a 0.74a 0.01 0.74b 0.86b 0.13 0.87c 0.92b 0.60
[0.47, 0.64] [0.63, 0.85] [0.64, [0.73, 0.98] [0.74, [0.75, 1.08]
0.85] 0.99] d9-Betaine 2.45 a 2.77 a 0.14 3.28 a 2.70 a 0.37 2.57 a 3.32 a 0.25
[2.06,2.92] [2.47,3.11] [2.47, [2.20,3.32] [2.18, [1.61,4.14]
4.35] 3.01] d3-Betaine 0.49a 0.62 a 0.11 0.65a-b 0.75 a 0.11 0.76b 0.79 a 0.73
[0.41,0.57] [0.52,0.72] [0.57, [0.66,0.84] [0.66, [0.66,0.92]
0.74] 0.86] d6- 9.63a 11.0a 0.37 10.2a 10.0a 0.87 8.80a 8.68a 0.91
Dimethylglycine [6.71, 12.6] [9.53, 12.5] [8.29, [8.29, 11.7] [7.57, [6.69, 10.7]
12.1] 10.0] d3- 0.45a 0.48a 0.70 0.63b 0.66b 0.69 0.67b 0.75b 0.51
Dimethylglycine [0.33,0.58] [0.37,0.59] [0.48, [0.53,0.80] [0.49, [0.56,0.94]
0.77] 0.84]
Figure imgf000067_0001
d9-TMAO 1.00 a 8.59a <.000 1.05 a 9.87b <000 1.29 a 21.5a-b 0.10
[0.67, 1.50] [6.38, 11.6] 1 [0.78, [4.15,23.5] 1 [0.94, [3.06, 150]
1.42] 1.78] d3-TMAO2 1.10a 2.06a>b 0.42 0.28a 1.96a 0.046 0.36a 0.19b 0.15
[0.00,2.46] [0.00,4.22] [0.17, [0.24,3.68] [0.17, [0.05,0.34]
0.38] 0.55] d9-PC 1.27a 1.20a 0.39 1.45a 1.15a 0.03 1.33a 1.03a 0.06
[1.15, 1.38] [1.06, 1.33] [1.25, [0.95, 1.35] [1.12, [0.81, 1.26]
1.65] 1.53] d3-PC 0.67a 0.95 a 0.002 0.79b 1.00 a 0.07 0.88a-b 1.04 a 0.77
[0.59,0.77] [0.81, 1.13] [0.68, [0.82, 1.23] [0.76, [0.76, 1.44]
0.92] 1.03] d9- 1.40a 1.37a 0.85 1.62a 1.33a 0.04 1.55a 1.20a 0.04
Sphingomyelin [1.21, 1.58] [1.11, 1.62] [1.40, [1.13, 1.53] [1.33, [0.95, 1.46]
1.85] 1.76] d9-LysoPC 1.29a 1.23 a 0.33 1.49a 1.26a 0.046 1.36a 1.18a 0.15
[1.21, 1.39] [1.09, 1.40] [1.37, [0.98, 1.62] [1.16, [0.88, 1.60]
1.63] 1.59]
Enrichment ratios </9-Betaine:</9- 1.33 a 1.50a 0.04 1.27a 1.30b 0.71 1.21 a 1.36ab 0.002
Choline [1.18, 1.47] [1.40, 1.60] [1.17, [1.18, 1.42] [1.17, [1.28, 1.45]
1.38] 1.25] d9-Betaine:d9- 1.90 a 2.35 a 0.001 2.20 a 2.49 a 0.20 1.94 a 3.28 a 0.60
PC [1.72,2.09] [2.16,2.55] [1.79, [2.13,2.91] [1.79, [1.81,5.96]
2.69] 2.11] d9-PC:d9- 0.71a 0.65a 0.11 0.68a 0.58a 0.41 0.64a 0.88a 0.95
Choline [0.64,0.78] [0.61,0.68] [0.52, [0.46,0.74] [0.58, [0.51, 1.51]
0.90] 0.70] d9-SM:d9-PC 1.10a 1.13 a 0.70 1.14a 1.22a 0.43 1.18a 1.15a 0.75
[0.99, 1.21] [1.00, 1.26] [1.04, 1.41] [0.94, 1.36]
Figure imgf000068_0001
Figure imgf000069_0002
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
DISCUSSION
[0173] The findings of this randomized controlled trial reveal that prenatal choline supplementation (administered across the second and third trimesters of pregnancy) significantly improves biomarkers of maternal DHA status among free-living participants consuming supplemental DHA. Higher levels of RBC PC-DHA tended to occur at GW28-32 and higher levels of plasma PC-DHA and plasma total DHA, as well as RBC total DHA were observed by GW 28-32 and were sustained through delivery among pregnant participants consuming habitual diets supplemented with 550 mg choline/d + 200 mg DHA/d, compared to those supplemented with 25 mg choline/d + 200 mg DHA/d. The higher circulating plasma PC- DHA, along with greater enrichments of both plasma t/j-PC and <73-PC:<79-PC in the choline intervention arm (compared to control), strongly support the notion that prenatal choline chloride supplementation, a water-soluble choline salt, facilitates greater PEMT activity (and generation of PC molecules enriched in DHA) by bolstering methyl group supply.
[0174] The finding of higher maternal DHA and PC-DHA in response to choline supplementation is consistent with previous work from our research group conducted in women of reproductive age whereby higher plasma and RBC PC-DHA were reported among those consuming controlled diets containing 930 mg choline/d + 200 mg DHA as compared to 480 mg choline/d + 200 mg DHA/d7. However, the current findings deviate from the pregnant cohort of this study7 whereby administration of 930 mg choline/d + 200 mg DHA/d across the only third trimester of pregnancy did not affect PC-DHA in either RBCs or plasma relative to 480 mg choline/d + 200 mg DHA/d7.
[0175] Two factors distinguish the current and past study — namely, the timing of choline supplementation, with the current intervention earlier in pregnancy, as well as the control group of the current intervention consuming habitual choline intakes, which were substantially lower than 480 mg. While both factors may have influenced PEMT activity, the current investigation observed strong choline supplementation effects on DHA outcomes at GW 28-32, shortly after the initiation of supplementation (~GW 27) in West et al. (7), highlighting the importance of considering key metabolic windows in pregnancy.
[0176] Although no statistically significant effects of prenatal choline on newborn biomarkers of DHA status were detected, the overall pattern in magnitude was comparable to that observed in maternal blood. Indeed, cord blood RBC-DHA levels in the intervention arm were 0.41% higher than in the control arm, a finding that trended towards statistical significance (P=0.12). The lack of significance may be due in part to a slightly lower sample size of the newborn cohort, but also due to differences in metabolic priorities between mother (prioritizes nutrient delivery) and her developing fetus (prioritizes cellular uptake and metabolic use). While Applicant and others have observed a decline in maternal plasma concentrations of PC- DHA and total DHA across the last trimester (7, 33, 34), indicative of significant DHA transport to the fetal compartment, the minimal change in cord RBC-DHA concentrations likely reflects the accretion of DHA in developing tissues, such as the brain [as previously shown in mice (32)], limiting our ability to observe an impact of prenatal choline supplementation on DHA status indicators in newborn RBCs.
[0177] This investigation contains notable strengths and limitations. Strengths included 1) the randomized, double-blind nature of this study spanning a large majority of gestation in women con-suming typical choline intakes; 2) measuring both physiological mediators (i.e., PC-DHA) and validated status indicators (i.e., RBC total DHA); and 3) including stable isotope tracing, which represents a translational approach to the mechanistic physiology of one-carbon and lipid metabolism in pregnancy. Indeed, the overall pattern in DHA outcomes and isotopic labeling scheme present a concordant model by which habitual choline intakes limit, and choline supplementation improves, hepatic metabolism and DHA status across pregnancy. Further, it is a likely strength that these effects were observed despite high (>5%) baseline RBC total DHA status in this cohort, a characteristic typically expected to diminish the effect size of factors influencing additional DHA incorporation into membranes. It is a notable limitation, however, that Applicant observed a nonsignificant (P = 0.105) effect of choline supplementation on our primary outcome of RBC PC-DHA. This may be due to issues of sample size and power, as RBC PC-DHA concentrations were significantly more variable than RBC total DHA (%CV : 18-27% vs. 9- 15% across visits) and 2 significant outliers were present at delivery, a nonfasted time point, that impacted model estimates. However, RBC PC-DHA may be an undesirable outcome for physiological reasons as well. For example, it is notable that, in this cohort, RBC total DHA concentrations were maintained from visit 3 to delivery, whereas RBC PC-DHA exhibited a precipitous decline. The physiological basis for the significant redistribution of DHA from PC to other phospholipid pools in RBCs by the time of delivery, maintaining total DHA concentrations but decreasing the PC-DHA fraction, remains unclear. Applicant’s primary outcome choice was driven by available data at the time; however, our results make evident that plasma concentrations of PC-DHA, as well as RBC total DHA, are more sensitive to the impacts of choline supplementation, and should inform future studies.
[0178]
[0179] These results collectively indicate a significant nutrient-nutrient interaction between supplemental choline and supplemental DHA during pregnancy that warrants reflection on the large body of randomized trial evidence that has attempted to test the hypothesis that prenatal DHA supplementation improves fetal outcomes30. Indeed, despite equivalent DHA intakes in the present investigation, we observed a nearly 75% relative increase in maternal DHA status in the intervention arm compared to the control. These data strongly suggest that prenatal choline supplementation influences the efficiency of hepatic DHA handling, mobilization and ultimately, extra-hepatic utilization, and support the view that existing clinical trials of prenatal DHA supplementation likely achieved non-maximal status, resulting from a limited methyl donor supply. As the field of nutrition pushes towards more ‘precision’ approaches in hopes of identifying factors that influence the physiological response to nutrition exposures, accounting for known determinants of DHA response to supplementation during pregnancy, such as choline intake, will be key for understanding the causal, dose-response relationships between DHA and maternal -infant outcomes.
References for Example 1
1. Brenna JT, Lapillonne A. Background paper on fat and fatty acid requirements during pregnancy and lactation. Ann Nutr Metab. 2009;55(l-3):97-122. doi: 10.1159/000228998
2. Koletzko B, Cetin I, Brenna JT, et al. Dietary fat intakes for pregnant and lactating women. Br JNutr. 2007;98(5): 873-877. doi: 10. 1017/S0007114507764747
3. Watkins SM, Zhu X, Zeisel SH. Phosphatidylethanolamine-N-methyltransferase activity and dietary choline regulate liver-plasma lipid flux and essential fatty acid metabolism in mice. J Nutr. 2003; 133(1 l):3386-3391. doi: 10. 1093/jn/l 33. 11.3386
4. da Costa K-A, Rai KS, Craciunescu CN, et al. Dietary Docosahexaenoic Acid Supplementation Modulates Hippocampal Development in the Pemt-/- Mouse. J Biol Chem. 2010;285(2): 1008-1015. doi: 10.1074/jbc.M109.017137
5. Yan J, Jiang X, West AA, et al. Pregnancy alters choline dynamics: results of a randomized trial using stable isotope methodology in pregnant and nonpregnant women. Am J Clin Nutr. 2013;98(6): 1459-1467. doi: 10.3945/ajcn. 113.066092
6. da Costa K-A, Sanders LM, Fischer LM, Zeisel SH. Docosahexaenoic acid in plasma phosphatidylcholine may be a potential marker for in vivo phosphatidylethanolamine N-methyltransferase activity in humans. Am J Clin Nutr. 2011;93(5):968-974. doi: 10.3945/ajcn. 110.011064
7. West AA, Yan J, Jiang X, Perry CA, Innis SM, Caudill MA. Choline intake influences phosphatidylcholine DHA enrichment in nonpregnant women but not in pregnant women in the third trimester. Am J Clin Nutr. 2013;97(4):718-727. doi: 10.3945/ajcn. 112.050211 Resseguie M, Song J, Niculescu MD, da Costa K-A, Randall TA, Zeisel SH. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J. 2007;21(10):2622-2632. doi: 10.1096/fj.07-8227com Klevebro S, Juul SE, Wood TR. A More Comprehensive Approach to the Neuroprotective Potential of Long-Chain Polyunsaturated Fatty Acids in Preterm Infants Is Needed-Should We Consider Maternal Diet and the n-6:n-3 Fatty Acid Ratio? Front Pediatr. 2019;7:533. doi: 10.3389/fped.2019.00533 Yan J, Jiang X, West AA, et al. Maternal choline intake modulates maternal and fetal biomarkers of choline metabolism in humans. Am J Clin Nutr. 2012;95(5): 1060-1071. doi: 10.3945/ajcn. 111.022772 Klatt KC, McDougall MQ, Malysheva OV, Brenna JT, Roberson MS, Caudill MA. Reproductive state and choline intake influence enrichment of plasma lysophosphatidylcholine-DHA: a post hoc analysis of a controlled feeding trial. British Journal of Nutrition. 2019;122(l 1): 1221-1229. doi: 10. 1017/S0007114519002009 Sugasini D, Thomas R, YalagalaPCR, Tai LM, Subbaiah PV. Dietary docosahexaenoic acid (DHA) as lysophosphatidylcholine, but not as free acid, enriches brain DHA and improves memory in adult mice. Sci Rep. 2017;7(l): 11263. doi: 10.1038/s41598-017- 11766-0 Nguyen LN, Ma D, Shui G, et al. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature. 2014;509(7501):503-506. doi : 10.1038/nature 13241 Innis SM, Dyer RA. Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid. J Lipid Res. 2002;43(9): 1529-1536. doi: 10.1194/jlr.m200120-jlr200 Rose HG, Oklander M. IMPROVED PROCEDURE FOR THE EXTRACTION OF LIPIDS FROM HUMAN ERYTHROCYTES. J Lipid Res. 1965;6:428-431. Harris WS, Pottala JV, Vasan RS, Larson MG, Robins SJ. Changes in erythrocyte membrane trans and marine fatty acids between 1999 and 2006 in older Americans. J Nutr. 2012; 142(7): 1297-1303. doi: 10.3945/jn. 112. 158295 Rudolph MC, Young BE, Jackson KH, Krebs NF, Harris WS, MacLean PS. Human milk faty acid composition: Comparison of novel dried milk spot versus standard liquid extraction methods. J Mammary Gland Biol Neoplasia. 2016;21(3-4): 131- 138. doi: 10.1007/sl0911-016-9365-4 Resseguie ME, da Costa K-A, Galanko JA, Patel M, Davis IJ, Zeisel SH. Aberrant estrogen regulation of PEMT results in choline deficiency-associated liver dysfunction. J Biol Chem. 2011 ;286(2): 1649-1658. doi: 10. 1074/jbc.Ml 10.106922 Ganz AB, Shields K, Fomin VG, et al. Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis. FASEB J. 2016;30(10):3321-3333. doi: 10.1096/fj.201500138RR Ganz AB, Cohen W, Swersky CC, et al. Genetic Variation in Choline-Metabolizing Enzymes Alters Choline Metabolism in Young Women Consuming Choline Intakes Meeting Current Recommendations. Int J Mol Set. 2017118(2): E252. doi: 10.3390/ijmsl8020252 Holm PI, Ueland PM, Kvalheim G, Lien EA. Determination of choline, betaine, and dimethylglycine in plasma by a high-throughput method based on normal-phase chromatography-tandem mass spectrometry. Clin Chem. 2003;49(2):286-294. doi: 10.1373/49.2.286 Yan J, Wang W, Gregory JF, et al. MTHFR C677T genotype influences the isotopic enrichment of one-carbon metabolites in folate-compromised men consuming d9- cholmcMm JClin Nutr. 2011;93(2):348-355. doi: 10.3945/ajcn.l 10.005975 Koc H, Mar M-H, Ranasinghe A, Swenberg JA, Zeisel SH. Quantitation of choline and its metabolites in tissues and foods by liquid chromatography/electrospray ionizationisotope dilution mass spectrometry. Anal Chem. 2002;74(18):4734-4740. doi: 10.1021/ac025624x Harris WS, Sands SA, Windsor SL, et al. Omega-3 faty acids in cardiac biopsies from heart transplantation patients: correlation with erythrocytes and response to supplementation. Circulation. 2004; 110(12): 1645-1649. doi:10.1161/01.CIR.0000142292.10048.B2 Abbassi-Ghanavati M, Greer LG, Cunningham FG. Pregnancy and laboratory studies: a reference table for clinicians. Obstet Gynecol. 2009;l 14(6): 1326-1331. doi: 10.1097/AOG.0b013e3181c2bde8 26. Resseguie M, Song J, Niculescu MD, da Costa K-A, Randall TA, Zeisel SH. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J. 2007;21(10):2622-2632. doi: 10.1096/fj.07-8227com
27. Darmady JM, Postle AD. Lipid metabolism in pregnancy. Br J Obstet Gynaecol. 1982;89(3):211-215. doi: 10.1111/j.1471-0528.1982.tb03616.x
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33. Al MD, van Houwelingen AC, Kester AD, Hasaart TH, de Jong AE, Homstra G. Maternal essential fatty acid patterns during normal pregnancy and their relationship to the neonatal essential fatty acid status. Br J Nutr 1995 ;74( 1): 55- 68.
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* * *
[0180] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.
[0181] Further attributes, features, and embodiments of the present invention can be understood by reference to the following numbered aspects of the disclosed invention. Reference to disclosure in any of the preceding aspects is applicable to any preceding numbered aspect and to any combination of any number of preceding aspects, as recognized by appropriate antecedent disclosure in any combination of preceding aspects that can be made. The following numbered aspects are provided:
1. A method of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity, the method comprising: co-supplementing choline and DHA to the female subject, wherein co- supplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day.
2. The method of aspect 1, wherein the total amount of choline ranges from about 500 mg, to about 600 mg per day, optionally about 550 mg of choline per day.
3. The method of any one aspects 1-2, wherein the female subject is pregnant.
4. The method of aspect 3 wherein co-supplementing begin and/occur a. prior to the female of reproductive maturity being pregnant b. prior to the female of reproductive maturity being 16 weeks pregnant; c. during gestation; d. after delivery; or e. any combination thereof.
5. The method of any one of aspects 1-5, wherein co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant.
6. The method of any one of aspects 1-6, wherein an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine -DHA (PC- DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
7. The method of any one of aspects 3-6, wherein the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC- DHA, or any combination thereof.
8. The method of any one of aspects 1-7, wherein DHA bioavailability is increased during pregnancy.
9. The method of any one of aspects 1-8, wherein the female subject of reproductive age is a mammal, optionally a human.
10. The method of any one of aspects 1-9, wherein the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
11. The method of any one of aspects 1-10, wherein the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
12. The method of any one of aspects 1-11, wherein the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate cytidine 5’ di-phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
13. The method of any one of aspects 1-12, wherein the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation.
14. The method of any one of aspects 1-6, wherein the supplemental choline and the supplemental DHA are administered separately.
15. The method of any one of aspects 1-14, wherein the total amount of supplemental choline is administered in a single dose.
16. The method of any one of aspects 1-14, wherein the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline.
17. The method of any one of aspects 1-16, wherein the total amount of supplemental DHA is administered in a single dose.
18. The method of any one of aspects 1-16, wherein the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA.
19. The method of any one aspects 1-18, wherein the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery.
20. The method of any one of aspects 1-19, wherein the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery.
21. The method of any one of aspects 1-20, wherein the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semi-solid formulation, a gel formulation, or a solid formulation.
22. A dietary supplement formulation comprising: an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
23. The dietary supplement formulation of aspect 22, wherein the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
24. The dietary supplement formulation of any one of aspects 22-23, wherein the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg. 25. The dietary supplement formulation of any one of aspects 22-24, wherein the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
26. The dietary supplement formulation of any one of aspects 22-25, wherein the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
27. The dietary supplement formulation of any one of aspects 22-26, wherein the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di -phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
28. The dietary supplement formulation of any one of aspects 22-27, wherein the dietary supplement formulation is a solid, a semi-solid, or a liquid.
29. The dietary supplemental formulation of any one of aspects 22-28, wherein DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
30. The dietary supplemental formulation of any one of aspects 22-29, wherein the female subject of reproductive maturity is pregnant.
31. The dietary supplemental formulation of aspect 30, wherein the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
32. The dietary supplemental formulation of any one of aspects 22-31, wherein the female of reproductive maturity is a mammal, optionally a human.
33. Use of supplemental choline at an amount ranging from about 110 mg to about 3,000 mg per day and supplemental docosahexaenoic acid (DHA) at an amount ranging from about 110 mg to about 2500 mg per day in a female of reproductive maturity, optionally a pregnant female, to increase the bioavailability of DHA in the female of reproductive maturity.

Claims

CLAIMS What is claimed is:
1. A method of increasing docosahexaenoic acid (DHA) bioavailability in a female subject of reproductive maturity, the method comprising: co-supplementing choline and DHA to the female subject, wherein cosupplementing comprises administering a total amount of supplemental choline to the female subject ranging from about 110 mg per day to about 3000 mg per day; and administering a total amount of supplemental DHA to the female subject ranging from about 110 mg to about 2500 mg per day.
2. The method of claim 1, wherein the total amount of choline ranges from about 500 mg, to about 600 mg per day, optionally about 550 mg of choline per day.
3. The method of claim 2, wherein the female subject is pregnant.
4. The method of claim 3 wherein co-supplementing begin and/occur a. prior to the female of reproductive maturity being pregnant b. prior to the female of reproductive maturity being 16 weeks pregnant; c. during gestation; d. after delivery; or e. any combination thereof.
5. The method of claim 3, wherein co-supplementing begin when the female subject is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks pregnant.
6. The method of claim 1, wherein an increase in DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine-DHA (PC-DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
7. The method of claim 3 , wherein the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC- DHA), maternal red blood cell PC-DHA, fetal cord blood PC-DHA, or any combination thereof.
8. The method of claim 3, wherein DHA bioavailability is increased during pregnancy.
9. The method of claim 1 , wherein the female subj ect of reproductive age is a mammal, optionally a human.
10. The method of claim 1, wherein the female subject has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
11. The method of claim 1, wherein the female subject has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
12. The method of claim 1, wherein the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ diphosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
13. The method of claim 1, wherein the supplemental choline and the supplemental DHA are administered simultaneously, optionally in the same formulation.
14. The method of claim 1, wherein the supplemental choline and the supplemental DHA are administered separately.
15. The method of claim 1, wherein the total amount of supplemental choline is administered in a single dose.
16. The method of claim 1, wherein the total amount of supplemental choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental choline less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental choline.
17. The method of claim 1, wherein the total amount of supplemental DHA is administered in a single dose.
18. The method of claim 1, wherein the total amount of supplemental DHA choline is administered in two or more separate doses per day, wherein each dose contains an amount of supplemental DHA less than the total amount of supplemental choline, and wherein the sum of the amounts of each of the two or more separate doses equals the total amount of the supplemental DHA.
19. The method of claim 1, wherein the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every day, optionally, when the female is pregnant, until delivery.
20. The method of claim 1, wherein the female subject is administered the total amount of supplemental choline and total amount of supplemental DHA every other day, every third day, every fourth day, every fifth day, every sixth day, or one a week, optionally, when the female is pregnant, until delivery.
21. The method of claim 1, wherein the total amount of supplemental choline, the total amount of supplemental DHA, and/or any sub-dosage thereof is in a liquid formulation, a semisolid formulation, a gel formulation, or a solid formulation.
22. A dietary supplement formulation comprising: an effective amount of supplemental choline ranging from about 110 mg to about 3,000 mg; optionally, an effective amount of supplemental docosahexaenoic acid (DHA) of about 110 mg to about 2500 mg of DHA; and optionally a carrier, wherein together, the effective amount of supplemental choline and the effective amount of supplemental DHA are effective increase DHA bioavailability in a female of reproductive maturity.
23. The dietary supplement formulation of claim 22, wherein the amount of supplemental choline is about 500 to about 600 mg; optionally about 550 mg.
24. The dietary supplement formulation of claim 22, wherein the amount of supplemental DHA is about 100 mg to about 300 mg, optionally about 200 mg.
25. The dietary supplement formulation of claim 22, wherein the female of reproductive maturity has about 450, 400, 350, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of choline, optionally 360 mg/d or less.
26. The dietary supplement formulation of claim 22, wherein the female of reproductive maturity about has 400, 300, 200, 100, 50, 40, 30 mg/d or less dietary intake of DHA, optionally about 36 mg/d or less.
27. The dietary supplement formulation of claim 22, wherein the supplemental choline is provided as a choline salt, optionally choline chloride, choline bitartrate, choline dihydrogen citrate, cytidine 5’ di -phosphate, alpha glycerylphosphoryl choline, phosphatidyl choline, or any combination thereof.
28. The dietary supplement formulation of claim 22, wherein the dietary supplement formulation is a solid, a semi-solid, or a liquid.
29. The dietary supplemental formulation of claim 22, wherein DHA bioavailability is as measured by plasma DHA, red blood cell DHA, plasma phosphatidylcholine -DHA (PC- DHA), red blood cell PC-DHA or any combination thereof in the female subject of reproductive maturity.
30. The dietary supplemental formulation of claim 22, wherein the female subject of reproductive maturity is pregnant.
31. The dietary supplemental formulation of claim 30, wherein the DHA bioavailability is as measured by maternal plasma DHA, maternal red blood cell DHA, maternal plasma phosphatidylcholine-DHA (PC-DHA), maternal red blood cell PC-DHA, fetal cord blood PC- DHA, or any combination thereof.
32. The dietary supplemental formulation of claim 22, wherein the female of reproductive maturity is a mammal, optionally a human.
33. Use of supplemental choline at an amount ranging from about 110 mg to about 3,000 mg per day and supplemental docosahexaenoic acid (DHA) at an amount ranging from about 110 mg to about 2500 mg per day in a female of reproductive maturity, optionally a pregnant female, to increase the bioavailability of DHA in the female of reproductive maturity.
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