Compositions and methods for improving brain function

Abstract

Methods for improving brain function and increasing brain activity by oral administration of choline are provided. Compositions including unit doses of choline sufficient to provide improved brain function and increasing brain activity are also provided. In some embodiments, it includes a method for improving brain function and / or increasing brain efficiency in a subject in need thereof, the methods comprising administering a pharmaceutical composition comprising 550 mg or greater of choline. The increases in brain efficiency are measured by functional Magnetic Resonance Imaging, as compared to placebo, when the subject is performing an N-Back task.

Description

COMPOSITIONS AND METHODS FOR IMPROVING BRAIN FUNCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 565,364, filed March 14, 2024, the entirety of which is incorporated by reference herein.FIELD OF THE DISCLOSURE

[0002] The disclosure is directed to methods for improving brain function in a subject by administration of a composition comprising choline.BACKGROUND

[0003] The brain is a major target for circulating gonadal steroids and the change in hormone levels after menopause is likely to have implications for cognitive functioning. Clinical and preclinical studies have linked gonadal steroids and cognition and it has been hypothesized that menopause has detrimental effects on cognition that are over and above the expected effects of normal. However, evidence for changes in cognition after menopause is equivocal. Some studies found decreased cognitive performance after menopause in domains such as memory, attention, problem solving, and motor skills. Other studies have not found changes in cognition after menopause. Additionally, not all women experience negative effects of menopause on cognition. The neurobiological processes underlying individual differences in cognition after menopause are not fully understood.

[0004] Choline is an essential nutrient that, in addition to its role in the brain, has a number of critical structural and physiologic roles throughout the body, including providing structural integrity and signaling function for cell membranes, facilitating lipid transport from the liver, and acting as the major source of methyl groups through diet. Aside from dietary intake, the only source of choline in the body is de novo synthesis of phosphatidylcholine, catalyzed by phosphatidylethanolamine-ZV-methyltransferase (PEMT). The PEMT gene has several estrogen-responsive components in its promoter region and is induced by estrogen. Postmenopausal (hypo-estrogenic) women with loss of function mutations in PEMT have been found to exhibit end-organ damage typical of choline deficiency. A number of other genes are implicated in the end-organ effects of choline metabolism and processing, including choline kinase alpha (CHKA), methylenetetrahydrofolate dehydrogenase 1 (MTHFDT), methylenetetrahydrofolate reductase (MTHFR) and choline dehydrogenase (CHDH).SUMMARY OF THE INVENTION

[0005] Disclosed herein is a method for improving brain function and / or increasing brain efficiency in a subject in need thereof, the methods comprising administering a pharmaceutical composition comprising 550 mg or greater of choline. The increases in brain efficiency are measured by functional Magnetic Resonance Imaging, as compared to placebo, when the subject is performing an N-Back task.BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 A is a schematic of the timeline for each study day.

[0007] FIGs. 2A shows Magnetic Resonance Spectroscopy (MRS) from one subject on Day 1 from a voxel in the left posterior parietal cortex.

[0008] FIG. 2B shows Magnetic Resonance Spectroscopy (MRS) from one subject on Day 2 from a voxel in the left posterior parietal cortex.

[0009] FIG. 3 shows single dose choline effect on postmenopausal brain as detected by fMRI during an N-back task. Central opercular (Right), insular cortex (Upper Left) and postcentral gyrus (Lower Left) showed differential activity. Red regions indicate greater activation for choline compared to placebo and blue regions indicate decreased activation for choline compared to placebo. Choline reduced frontal working memory-related activation and increased posterior activation compared to placebo.

[0010] FIG. 4 shows a consolidated fMRI image across all task conditions in 19 postmenopausal women given 1650 mg choline orally 3 hrs prior to imaging.

[0011] FIG. 5 shows connectivity data between different parts of the brain after administration of choline.

[0012] FIG. 6A shows results described in Example 6. The 1650 mg choline dose increased posterior activation and decreased frontal activation (temporal), as compared to placebo. Red indicates increased activation for choline versus placebo. Blue indicates decreased activation for choline versus placebo. This pattern is consistent with a “younger looking” brain.

[0013] FIG. 6B shows results described in Example 6. The 1100 mg choline dose increased posterior and occipital activation (very strongly), as compared to placebo. Red indicates increased activation for choline versus placebo. Blue indicates decreased activation for choline versus placebo. This pattern is consistent with a “younger looking” brain.

[0014] FIG. 6C shows results described in Example 6. The 550 mg choline dose shows frontal activation only, as compared to placebo. This demonstrates the functional presence of choline in the brain after 3.5 hours.DETAILED DESCRIPTION OF THE INVENTION

[0015] Choline is an essential nutrient that is involved in the synthesis of acetylcholine, a major neurotransmitter in the central and peripheral nervous systems. The relationship between brain effects of the normal functioning of the cholinergic system and hormone changes after menopause have been demonstrated in preclinical studies in rat models and experimentally in human studies. While the preclinical and clinical experiment studies have many similar findings regarding the influence of estrogen on cholinergic functioning, what is not clear from this research is how individual differences arise in the effects of the hormone withdrawal after menopause on brain functioning in middle aged women. It is also not clear how this information can be used in therapeutic interventions.

[0016] As described in Lippelt et al. (“No Acute Effects of Choline Bitartrate Food Supplements on Memory in Healthy, Young, Human Adults” PLoS ONE, 2016, 11 (6):e0157714), 800 mg of choline supplementation in young adults produced no acute effects on brain function. Specifically, Lippelt et al. found that there was no improvement in visuospatial working memory, declarative memory, replication and verbal working memory one hour after choline supplementation. Therefore, the state of the art teaches away from choline supplementation to improve brain function.

[0017] However, the inventors surprisingly found that choline supplementation can acutely improve brain function and impact connectivity between multiple areas of the brain in postmenopausal women. This indicates that, despite the teachings of Lippelt and others, choline supplementation can improve brain function, including short term memory, working memory, executive function, and cognitive complexity.I. Methods

[0018] The methods described herein are directed to, among other things, increasing brain efficiency in a subject, in particular, a human subject, in need of increased brain efficiency. As used herein, “increased brain efficiency” is an increase in brain activity, a decrease in brain activity, or a combination thereof, as measured by functional Magnetic Resonance Imaging (MRI), as compared to placebo, when the subject is performing a task.

[0019] The disclosure encompasses methods for improving brain function in a subject in need thereof, the methods comprising administering to the subject any one or more of the pharmaceutical compositions disclosed herein.

[0020] In some aspects, the choline in the pharmaceutical composition may be provided in any one or more of the forms of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof. In an aspect, the choline in the combination therapy may be provided in any one or more of the forms of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof. Non-limiting examples of choline salts include choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof. In an aspect, the choline salt or precursor may comprise a commercially available choline salt or precursors, non-limiting examples of which include choline dihydrogen citrate (CDHC), choline bitartrate (CBT), choline DL-bitartrate (CBT-DL), crocodile choline (CCL), citicoline, L-alpha-glycerylphosphorylcholine (Alfa GPC), choline orotate, phosphatidyl choline, choline ascorbate, choline hydroxide, choline succinate and choline monoxide. In an aspect, the phospholipid-bound choline may comprise phosphatidylcholine, soy lecithin, sphingomyelin, platelet-activating factor, synthetic phospholipid bound choline compounds, or derivatives thereof. In an aspect, the choline metabolite may comprise cytidine diphosphatecholine.

[0021] In an aspect, the pharmaceutical composition may comprise 550 mg of choline. In other aspects, the pharmaceutical composition comprises more than 550 mg of choline. In some aspects, the pharmaceutical compositions comprises 550 mg or greater of choline or greater than 800 mg of choline or greater than 1000 mg of choline, or greater than 1200 mg of choline, or greater than 1500 mg of choline, or greater than 2000 mg of choline. In an aspect, the pharmaceutical composition comprises from 600 to 3500 mg of choline, from 1000 to 2500 mg of choline, from 1500 to 2250 mg of choline, from 1200 to 1800 mg of choline, or from 1400 to 1700 mg of choline. In an aspect, the pharmaceutical composition may comprise 1650 mg of choline.

[0022] In some aspects, the subject is administered 550 mg of choline. In some aspects, the subject is administered greater than 550 mg of choline. In some aspects, the subject is administered 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, or 1650 mg of choline. The choline can be administered in a single unit dose or in multiple unit doses.

[0023] In an aspect, the combination comprises greater than 800 mg of choline, or greater than 1000 mg of choline, or greater than 1200 mg of choline, or greater than 1500 mg of choline, or greater than 2000 mg of choline. In an aspect, the combination comprises from 600to 3500 mg of choline, from 1000 to 2500 mg of choline, from 1500 to 2250 mg of choline, from 1200 to 1800 mg of choline, or from 1400 to 1700 mg of choline. In an aspect, the combination may comprise 1650 mg of choline.

[0024] In an aspect of the method, the choline in the pharmaceutical composition may be provided in any one or more of the forms of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof. In an aspect of the method, the pharmaceutical composition may comprise greater than 800 mg of choline or greater than 1000 mg of choline, or greater than 1200 mg of choline, or greater than 1500 mg of choline, or greater than 2000 mg of choline. In an aspect, the pharmaceutical composition comprises from 600 to 3500 mg of choline, from 1000 to 2500 mg of choline, from 1500 to 2250 mg of choline, from 1200 to 1800 mg of choline, or from 1400 to 1700 mg of choline. In an aspect, the pharmaceutical composition may comprise 1650 mg of choline. In an aspect, the pharmaceutical composition may be administered in a single unit dose. In an aspect, pharmaceutical composition may be administered in multiple unit doses, wherein each of the unit dose may be the same or different. In an aspect, the administration may occur once per day, or twice per day, or three times per day, or once per day for at least one week, or once per day for at least two weeks, or once per day for at least four weeks, or once per day for at least eight weeks, or once per day for at least twelve weeks. In an aspect, the subject is female with low estrogen levels. In an aspect, the subject is female with an estradiol level <50 pg / mL. In an aspect, the subject is a postmenopausal female. In an aspect, the subject is a male with normal estrogen levels. In an aspect, the male has an estradiol level of <50 pg / mL. In an aspect, the subject is an estrogen-deficient subject. In an aspect, the subject has low estrogen levels as measured in serum.

[0025] In an aspect, the current disclosure also encompasses a method for improving task efficiency in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose. In an aspect, the improvement in task efficiency is determined by measuring activity of the subject’s postcentral cortex and occipital lobe during a task challenge. In an aspect, the task challenge comprises an N-back test. In an aspect, the activity is measured by a functional MRI scan after administration of the pharmaceutical composition. In an aspect, the activity is measured about 3 hours after the administration of the pharmaceutical composition. In an aspect, the activity is measured about 3.5 hours after the administration of the pharmaceutical composition.

[0026] In an aspect, the current disclosure also encompasses a method for improving working memory in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose. Improvement inworking memory efficiency is determined by measuring activity of the subject’s frontal lobe, and more specifically right frontal lobe during a task challenge. In an aspect, the task challenge comprises an N-back test, letter number sequencing or a combination thereof. In an aspect, the activity is measured by a functional MRI scan after administration of the pharmaceutical composition. In an aspect, the activity is measured about 3 hours after the administration of the pharmaceutical composition. In an aspect, the activity is measured about 3.5 hours after the administration of the pharmaceutical composition.

[0027] As used herein, the term “improving brain function” may refer to an improvement in any brain related ability of the subject. For example, the improvement in brain function may comprise one or more of enhancement in cognitive function and alertness, better memory processing, improved performance and regulation, increase motivation and drive, improved focus, clarity, and concentration, increased speed of cognition and information processing, improved wakefulness and alertness, reduced anxiety and stress, increase energy levels, improved reaction times, enhanced creativity, balanced mood, enhanced clarity, increase mental stamina, eliminate mental gridlock, boost speed of thought, regulate the synthesis of a number of neurotransmitters, improve brain cell function and production, increased antioxidant activity and brain blood flow, modulation of pain receptors, enhance neural growth and neuroplasticity, or any combination thereof. In some aspects, the improvement comprises positive enhancement of any function that is under cholinergic regulation. In some aspects, the improvement comprises enhanced adaptive behavioral responses to threatening stimuli, for example learned responses to threats. In some aspects, the improvement comprises improved response to harm and fear triggers that induce disabling behavior, anxiety, and emotional disequilibrium, as may occur in Post Traumatic Stress Disorder (PTSD). In some embodiments, the improvement comprises improved working memory, improved attention, improved short term memory, improved executive function, and improved memory overall.

[0028] Methods for detecting improvement in brain function are well known in the art, some of which are also disclosed herein and in the examples. These methods allow researchers to observe, measure, and manipulate brain activity and may include brain visualization tools, and / or one or more task efficiency tests as disclosed below. In some aspects, the improvement in brain function may be assessed by neural activity, by neural function by using one or more of the tasks provided below to study task efficiency, by linguistic function by using one or more of the tasks provided below to study task efficiency, by assessing the subject’s ability to communicate by using one or more of the tasks provided below to study task efficiency, by measurement of levels of acetylcholine or other neurotransmitters or brain chemicals correlated with cognitive function, by the number of capillaries in the subject's brain or in aspecific region of the subject's brain, or by visualizing the brain using computational methods and measurements. Some commonly used brain visualization techniques include functional magnetic resonance imaging (fMRI), electroencephalography (EEG), magnetoencephalography (MEG), positron emission tomography (PET), single-photon emission computed tomography (SPECT), transcranial magnetic stimulation (TMS), functional near-infrared spectroscopy (fNIRS) and two-photon microscopy, and electrocorticography (ECoG). In an example, the visualization technique includes fMRI.

[0029] In an aspect, the subject has an increased parietal activation as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject has a functional increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition. In an aspect, the subject has an increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject has an increase in activity in the occipital lobe as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject has decreased connectivity between regions of the brain associated with working memory as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject has decreased connectivity between the brain regions involved in complex cognition as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to placebo. In an aspect, the functional MRI measurement is taken about 3 hours after the administration of the pharmaceutical composition. In an aspect, the task challenge comprises an N-back test. In an aspect, the functional MRI measurement is taken about 3.5 hours after the administration of the pharmaceutical composition. In an aspect, the task challenge comprises an N-back test. See, e.g., Gary Gilmour, et al., Relating constructs of attention and working memory to social withdrawal in Alzheimer’s disease and schizophrenia: issues regarding paradigm selection, Neuroscience & Biobehavioral Reviews, Volume 97, 2019, Pages 47-69. https: / / doi.Org / 10.1016 / j.neubiorev.2018.09.025.

[0030] The improvement in brain function may be evidenced by a change in the connectivity between regions of the brain. The connectivity may be determined by the correlation between observed activity in different parts of the brain in response to a particular task within the time frame of the task. In an example, the subject may show decreased connectivity between thebrain regions, which are involved in complex cognition, as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to placebo.

[0031] The improvement in brain function may also be measured by a change in the location of brain activity in the subject. Such changes may be related to various brain regions. For example, as shown in FIG. 3 and FIG. 4, activity in the brain may shift from frontal regions of the brain to posterior regions of the brain after administration of the pharmaceutical composition as compared to placebo.

[0032] The present disclosure further encompasses methods for improving task efficiency in a subject in need thereof, the method comprising administering to the subject any one or more of the pharmaceutical compositions disclosed herein. As used herein, “improving task efficiency” may comprise discernable improvements in one or more brain related tasks. Methods for determining improved task efficiency are known in the art and are also described herein. Some exemplary tests to determine task efficiency include the Mattis Dementia Rating Scale-2 (DRS), Letter Number Sequencing (LNS), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Delis-Kaplan Executive Function System (D-KEFS), Trail Making and Verbal Fluency subtests, the Stroop test, the Digit Span Test, Rey Auditory Verbal Learning Test (RAVLT), the Clock Drawing Test (CDT), the Wisconsin Card Sorting Test, the California Verbal Learning Test (CVLT), and combinations thereof. DRS includes battery-style assessment of overall level of cognitive functioning. The tasks are grouped into five subscales, each one evaluating different cognitive areas, including: attention, initiation / perseveration (i / p), construction, conceptualization, and memory. LNS is a measure of working memory. The examinee is read a sequence of numbers and letters and asked to recall the numbers in ascending order and the letters in alphabetical order. The task involves attention, concentration, mental manipulation, sequential processing, memory span, and short-term auditory memory. RBANS is a brief test designed to evaluate neuropsychological status of adults, ages 12-89. The test assesses five cognitive domains including immediate memory, language, visuospatial / constructional ability, attention, and delayed memory and provides a global total measure. D-KEFS is used to measure a variety of verbal and nonverbal executive functions. The trail making test is a visual-motor sequencing task that measures cognitive flexibility. The verbal fluency test measures letter and category fluency. The Stroop test assesses cognitive flexibility and response inhibition. It typically involves naming the color of ink used to print words that spell out color names, with incongruent colorword pairs causing interference. The digit span test assesses working memory by requiring participants to repeat a sequence of numbers forward or backward. RAVLT assesses verballearning and memory by presenting a list of words for immediate recall and subsequent recall after a delay. CDT assesses visuospatial skills, executive function, and abstract thinking by asking participants to draw a clock face and set the time to a specified hour. WCST assesses executive function, cognitive flexibility, and problem-solving by requiring participants to sort cards based on changing rules without explicit instruction. CVLT assesses verbal learning and memory through the presentation and recall of word lists under various conditions. Those having ordinary skill in the art are capable of administering and interpreting results of these tests.

[0033] The present disclosure also encompasses a method of improving working memory in a subject in need thereof, the method comprising administering to the subject any one or more of the pharmaceutical compositions disclosed herein. Methods to assess improvement in working memory are well known in the art and include some of the tests detailed above. In an aspect, tasks discerning short term memory may be used as an indicator of working memory. In an aspect, tasks that help to determine executive function and cognitive complexity may be used as an indicator of working memory. Non-limiting examples of methods used to determine working memory include the digit span, digit span-running, letter numbering sequence, spatial span test, N-back task, delayed match-to-sample task and non-word repetition tasks. In an aspect, visualization methods measuring activity of the subject’s frontal lobe, and more specifically right frontal lobe during a task challenge may be used to determine improvement in working memory.

[0034] In an aspect, the disclosed method comprises administering to the subject a pharmaceutical composition as described in Section I above. In an aspect of the disclosed method, the method may comprise administering 600 mg of choline or greater to a subject in need thereof. In an aspect, the choline may be administered in a single dose. In another aspect, choline may be administered in multiple doses. In another aspect, choline may be administered in a single unit dosage form. In another aspect, choline may be administered as multiple unit dosage form.

[0035] Suitable routes of administration may, for example, include oral, intravenous, intracranial, intrathecal, subcutaneous, intranasal route, cranial, transmucosal, trans-nasal, transcranial, intracerebroventricular, intestinal, and / or parenteral delivery. In some aspects, a suitable mode of administration is oral delivery. In an aspect, suitable dosage forms for use in the method disclosed herein are those that are orally administered. In an aspect, the administering of the pharmaceutical composition may comprise oral administration. Nonlimiting examples of suitable dosage forms for oral delivery include capsules, tablets, pills,lozenges, soluble films, elixirs, sachets, stick pack, granules, syrups, solutions, suspensions, emulsions, semisolids and gels. In other aspects, non-limiting examples of suitable forms for oral administration include a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or any combination thereof. Capsules may be a one-piece or two-piece capsule and have a soft or hard shell. Non-limiting examples of tablets include a suspension tablet, a chewable tablet, an effervescent tablet, and an orally disintegrating tablet. Semisolids may include, but are not limited to, gel-filled chews and gelatinous chews. In an aspect, the dosage form may be such that administration of a single unit dosage form is sufficient to provide a therapeutically effective amount of choline to the subject in need thereof. In an aspect, the dosage form may be such that administration of multiple unit dosage forms may be needed to provide the therapeutically effective amount of choline to the subject in need thereof. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In an aspect, the multiple unit dose forms may need to be administered to deliver a therapeutically effective amount of active ingredient.

[0036] One or more unit dosage forms may be administered to provide the dose of choline desired. For example, to administer a dose of 1650 mg of choline, six capsules containing 275 mg of choline each may be administered to the subject to provide the 1650 mg dose. In some aspects, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more unit dosage forms may be administered to provide the dose of choline desired.

[0037] In some embodiments, one or more of the unit dosage forms may be administered concurrently or over a period. Alternatively, one or more of the unit dosage forms may be administered only once during an individual’s lifetime (acute dose). In some aspects, one or more of the unit dosage forms may be administered at least yearly, monthly, fortnightly, weekly, or daily for a length of time. The administration may be stopped after one or more days or weeks, for example after 1 day to after about 1 week, after about 1 week to after about 2 weeks, after about 2 weeks to after about 4 weeks, after about 4 weeks to after about 6 weeks, after about 6 weeks to after about 8 weeks, after about 8 weeks to after about 10 weeks, after about 12 weeks to after about after 14 weeks, after about 14 weeks to after about 16 weeks, after about 16 weeks to after about 20 weeks, after about 20 weeks to after about 22 weeks, or after 24 weeks. In an aspect, one or more unit dosage forms may be administered at least once every day, once every week, once every 4 weeks, once every 6 weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks, once every 14 weeks, once every 16 weeks, once every 20 weeks, once every 22 weeks, or once every 24 weeks. In an aspect,one or more of the unit dosage forms may be administered as needed or at a certain predetermined cadence throughout the post-menopausal lifetime of a female subject. The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. Concentrations and dosage values may also vary. For any subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0038] In an aspect, the administration may occur once per day, or twice per day, or three times per day, or once per day for at least one week, or once per day for at least two weeks, or once per day for at least four weeks, or once per day for at least eight weeks, or once per day for at least twelve weeks.

[0039] The methods may further comprise administering at least one additional therapeutic to the subject in need thereof. In an aspect, the therapeutic is an estrogen replacement agent. In an aspect, the method further comprises administering an estrogen replacement agent to the subject. In an aspect, the current disclosure also encompasses a combination therapy comprising a combination of greater than 600 mg of choline in a single unit dose and a therapeutically effective amount of an estrogen replacement agent. In an aspect, the estrogen replacement agent may comprise medroxyprogesterone acetate, megestrol acetate, clonidine, norethindrone acetate, ethinyl estradiol, conjugated estrogen, natural estrogen, synthetic estrogen, estradiol, progesterone, clomiphene, clomiphene citrate, zuclomiphene, zuclomiphene citrate, enclomiphene, enclomiphene citrate, aspirin, calcitonin, alendronate, etidronate, pamidronate, clodronate, tiludronate, residronate, ibandronate, or any combination thereof. The estrogen replacement agent may include one or more phytoestrogens, such as isoflavones (such as daidzein, genistein, glycitein, formononetin, biochanin A, and the like), coumestan, lignan, stibene, and prenylflavonoids. In an aspect, the phytoestrogen comprises an isoflavone, coumestan, lignan, stibene, a prenylflavonoid, or a combination thereof. In an aspect, the estrogen replacement agent comprises a phytoestrogen. Non-limiting examples of isoflavone include daidzein, genistein, glycitein, formononetin, biochanin A, or any combination thereof. In an aspect, the estrogen replacement agent may be administered concurrently with the pharmaceutical composition. In an aspect, the estrogen replacement agent may be administered separately from the pharmaceutical composition. In an aspect, the pharmaceutical composition may further comprise an estrogen replacement agent. In an aspect, the choline and the estrogen replacement agent are formulated in separatecompositions. In an aspect, the choline and the estrogen replacement agent are formulated in a single composition.

[0040] In some embodiments, the subject is an adult mammal. The subject may be an adult human 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older.

[0041] The subject may be an adult male having normal male estrogen status. In some aspects, the subject is an adult female having an estrogen status of 50 pg / ml or greater. In some aspects, the subject is a pre-menopausal female.

[0042] The subject may be a postmenopausal female. As used herein, the term “postmenopausal female” refers to an adult woman who has not had a period in the last 12 months, has levels of FSH>30 lll / L, and / or levels of estradiol (E2) <50 pg / ml. In an aspect, the subject is female with low estrogen levels. In an aspect, the woman has serum levels of estradiol (E2) of <5 pg / ml, or <10 pg / ml, or <15 pg / ml, or <20 pg / ml, or <25 pg / ml, or <30 pg / ml, or <35 pg / ml, or <40 pg / ml, or <45 pg / ml, or <50 pg / ml. In an aspect, the subject is an estrogen-deficient female, for example a woman who has had an ovariectomy. In an aspect, the subject is female with an estradiol level <50 pg / mL.

[0043] In an aspect, the subject is a male with low estrogen status. A male with low estrogen status is a healthy male with <50 pg / ml, <45 pg / ml, <40 pg / ml, <35 pg / ml, <20 pg / ml, or <15 pg / ml of estradiol in the blood-serum. In an aspect, the male has an estradiol level of <50 pg / mL. In an aspect, the subject is an estrogen-deficient male. A male with low levels of estrogen is a male with <10 pg / ml, <8 pg / ml, <6 pg / ml, <4 pg / ml, <2 pg / ml, <1 pg / ml of estradiol in the blood-serum. In an aspect, the subject is a male with normal estrogen levels.

[0044] The subject may be a pre-menopausal woman with low levels of estrogen. In an aspect, the pre-menopausal women has an estrogen serum levels of Estradiol of <5 pg / ml, or <10 pg / ml, or <15 pg / ml, or <20 pg / ml, or <25 pg / ml, or <30 pg / ml, or <35 pg / ml, or <40 pg / ml, or <45 pg / ml, or <50 pg / ml, or <55 pg / ml, or <60 pg / ml, or <65 pg / ml, or <70 pg / ml, or <75 pg / ml, or <80 pg / ml, or <85 pg / ml, or <90 pg / ml, or <95 pg / ml, or <100 pg / ml.

[0045] The subject may be experiencing at least one symptom associated with decreased brain function for example decreased cognitive abilities, memory loss, difficulty concentrating, language difficulties, decreased problem solving skills, confusion, learning difficulties,impaired judgement or any combination thereof prior to administration of the pharmaceutical composition. Methods of detecting decreased brain function are well known in the art and exemplary methods are disclosed herein above.

[0046] In some embodiments, the subject may be an estrogen-deficient subject. As defined herein, an “estrogen-deficient” subject is one who, as a consequence of a disease, disorder, or condition, does not produce estrogen in an amount that an otherwise healthy subject would normally produce. For example, an estrogen-deficient subject may include a premenopausal female who has had an oophorectomy.

[0047] In some embodiments, the subject may be a subject having low estrogen status. As defined herein, a subject having “low estrogen status” is one who naturally has low levels of estrogen. In an aspect, the subject has low estrogen levels as measured in serum. For example, a postmenopausal female, a female having a serum estradiol level of <50 pg / mL, and a male having a serum estradiol level of <50 pg / mL are all subjects having low estrogen status.

[0048] The subject may have an increased parietal activation as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject may have a functional increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject may have a functional increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject may have an increase in activity in the occipital lobe as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. In an aspect, the subject may exhibit decreased connectivity between certain regions of the brain as provided in FIG. 5, as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control. The MRI scan may be taken at least about 2 hrs, or about 3 hrs, or about 4 hrs, or about 5 hrs, or about 6 hrs, or about 7 hrs, or about 8 hrs, or about 9 hrs, or about 10 hrs, or about 11 hrs, or about 12 hrs, or more after the administration of the pharmaceutical composition or the placebo.

[0049] According to the described methods, the subject’s increased brain efficiency is an increase in parietal activation, as measured by a functional MRI scan, as compared to placebo,when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0050] According to the described methods, the subject’s increased brain efficiency is an increase in posterior activation including but not limited to parietal and occipital regions, as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3 hours after administration of the choline. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0051] In some aspects, the subject’s increased brain efficiency is an increase in activity in the postcentral cortex, as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3 hours after administration of the choline. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0052] In some aspects, the subject’s increased brain efficiency is an increase in activity in the postcentral cortex including but not limited to the parietal and occipital regions, as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0053] In some aspects, the subject’s increased brain efficiency is an increase in activity in the occipital lobe, as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3 hours after administration of the choline. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0054] In some aspects, the subject’s increased brain efficiency is a decrease in activity in the frontal and temporal lobes as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0055] In some aspects, the subject has decreased connectivity between regions of the brain associated with working memory, as measured by a functional MRI scan, as compared to placebo, when the subject is performing an N-back task. In these aspects, the increase in brain efficiency is measured about 3 hours after administration of the choline. In these aspects, the increase in brain efficiency is measured about 3.5 hours after administration of the choline.

[0056] In some aspects, the choline administration results in a younger pattern of cerebral activity in the subject, as measured by a functional MRI scan after about 3.5 hours afteradministration of the choline. In some aspects, the administration of at least 1100 mg of choline results in a younger pattern of cerebral activity in the subject, as measured by a functional MRI scan after about 3.5 hours after administration of the choline. According to the disclosure, the administration of choline results in cerebral activity that is consistent with the cerebral activity observed in humans who are of a younger chronological age, as measured by functional MRI at 3.5 hours. According to the disclosure, the administration of at least 1100 mg of choline results in cerebral activity that is consistent with the cerebral activity observed in humans who are of a younger chronological age, as measured by functional MRI at 3.5 hours.I. Pharmaceutical Compositions

[0057] Provided herein are pharmaceutical compositions for use in improving brain function in a subject in need thereof, the pharmaceutical composition comprising choline or at least one choline compound.

[0058] Choline (hydroxyethyl trimethyl ammonium hydroxide) is considered to be an essential nutrient and is derivable from many foods. The term choline, as used herein, refers not only to the isolated choline molecule (i.e. , free choline), but also choline compounds, for example any biologically compatible salt of choline (e.g., choline bitartrate), phospholipid bound choline, choline precursors and choline metabolites, wherein the choline precursors or choline metabolites are capable of being converted into choline, or any derivatives thereof. The salt of choline comprises the chemical formula (CH3)3N+(CH2)2OH+X_, wherein X" is a negative counter ion. Non-limiting examples of choline salts for use in the current disclosure include choline bitartrate, choline bitartrate DL, choline orotate, choline chloride, choline dihydrogen citrate, choline salicylate, choline ascorbate, choline phosphate, choline bicarbonate, or choline magnesium trisalicylate, choline hydroxide, choline succinate and derivatives or combinations thereof. Non-limiting examples of choline salts include choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.

[0059] Choline also exists in phospholipid bound form in nature or can also be synthesized to be bound to phospholipids. Non-limiting examples of phospholipid-bound choline include phosphatidylcholine, sphingomyelin, platelet-activating factor, various synthetic phospholipid bound choline compounds, and derivatives or combinations thereof. Phosphatidylcholines (PCs) include lecithin and are one of the most abundant phospholipids in cell membranes. PC consists of a glycerol backbone, two fatty acid chains, a phosphate group, and a choline molecule. It is found in various vegetable, animal and microbial sources such as egg yolks,soybeans (soy lecithin), and some plant oils. Soybean, sunflower and rapeseed are the major plant sources of commercial lecithin. Soybean is the most common source. Phosphatidylcholine is also the major delivery form of the essential nutrient choline. Sphingomyelins are non-traditional glycerophospholipid with a sphingosine backbone, a fatty acid chain, a phosphate group, and a choline molecule. It is abundant in myelin sheaths of nerve cells and also present in smaller amounts in other cell membranes. PI ate let- activating factor (PAF) contains a fatty acid chain, a glycerol backbone, a phosphate group, and a choline molecule. Synthetic choline-containing phospholipids are often designed for various pharmaceutical and industrial applications. These compounds can mimic natural phospholipids and may offer specific properties tailored for particular uses. Non limiting examples include dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), and lipidoids. DOPC is a synthetic phospholipid often used in liposomal formulations and drug delivery systems. It consists of two oleic acid chains attached to a glycerol backbone, along with a phosphate group and a choline molecule. DPPC is commonly used in liposomal formulations and as a component of lung surfactant. It contains two palmitic acid chains attached to a glycerol backbone, along with a phosphate group and a choline molecule. EPC, although also naturally occurring, can also be synthesized for various applications. It is commonly used in liposomal formulations, drug delivery systems, and as an emulsifier in food and pharmaceutical products. EPC contains various fatty acid chains (depending on the source) attached to a glycerol backbone, along with a phosphate group and a choline molecule. Lipidoids are a class of synthetic lipids that can be designed to contain choline groups among other functional moieties. Lipidoids are often used in gene delivery systems and as components of lipid nanoparticles for drug delivery. They are designed to mimic the structure and properties of natural phospholipids while offering improved stability and functionality for specific applications.

[0060] In addition, choline precursors and choline metabolites such as CDP-choline (also known as citicoline, cytidine diphosphate-choline or cytidine 5'-diphosphocholine), betaine, phosphocholine, alpha-glycerophosphocholine may be used in a pharmaceutical composition of the disclosure.

[0061] The pharmaceutical compositions include choline in a therapeutically effective amount to achieve the intended purpose, i.e., to improve brain function. In some aspects, a therapeutically effective amount means an amount of active ingredient(s) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder or condition, or to prolong the survival of the subject being treated.

[0062] The pharmaceutical compositions of the present disclosure may comprise about 600 mg or greater of free choline (i.e. , the choline cation). Thus, as a non-limiting example, a pharmaceutical composition comprising 1463 mg of choline bitartrate would comprise about 600 mg of free choline. The pharmaceutical composition may be provided in multiple dosage forms or may be provided as a single unit dose as described further herein. In an aspect, the pharmaceutical composition may be administered in a single unit dose. In an aspect, pharmaceutical composition may be administered in multiple unit doses, wherein each of the unit dose may be the same or different. In some embodiments, the pharmaceutical compositions of the present disclosure may comprise about 700 mg of choline or greater, about 800 mg of choline or greater, about 900 mg of choline or greater, about 1000 mg of choline or greater, about 1100 mg of choline or greater, about 1200 mg of choline or greater, about 1300 mg of choline or greater, about 1400 mg of choline or greater, about 1500 mg of choline or greater, about 1600 mg of choline or greater, about 1700 mg of choline or greater, about 1800 mg of choline or greater, about 1900 mg of choline or greater, about 2000 mg of choline or about 2200 mg or greater. In an example, the composition comprises about 1650 mg of choline.

[0063] The pharmaceutical compositions of the present disclosure may comprise from about 600 mg choline to about 3500 mg of free choline. In some embodiments, the pharmaceutical compositions of the present disclosure may comprise from about 600 mg choline to about 700 mg choline, about 600 mg choline to about 800 mg choline, about 600 mg choline to about 900 mg choline, about 600 mg choline to about 1000 mg choline, about 600 mg choline to about 1100 mg choline, about 600 mg choline to about 1200 mg choline, about 600 mg choline to about 1300 mg choline, about 600 mg choline to about 1400 mg choline, about 600 mg choline to about 1500 mg choline, about 600 mg choline to about 1600 mg choline, about 600 mg choline to about 1700 mg choline, about 600 mg choline to about 1800 mg choline, about 600 mg choline to about 1900 mg choline, about 600 mg choline to about 2000 mg choline, about 600 mg choline to about 2100 mg choline, about 600 mg choline to about 2200 mg choline, about 600 mg choline to about 2300 mg choline, about 600 mg choline to about 2400 mg choline, about 600 mg choline to about 2500 mg choline, about 600 mg choline to about 2600 mg choline, about 600 mg choline to about 2700 mg choline, about 600 mg choline to about 2800 mg choline, about 600 mg choline to about 2900 mg choline, about 600 mg choline to about 3000 mg choline, about 600 mg choline to about 3100 mg choline, about 600 mg choline to about 3200 mg choline, about 600 mg choline to about 3300 mg choline, about 600 mg choline to about 3400 mg choline, about 600 mg choline to about 3500 mg choline, about 700 mg choline to about 3500 mg choline, about 800 mg choline to about 3500 mg choline, about 900 mg choline to about 3500 mg choline, about 1000 mg choline to about 3500 mgcholine, about 1100 mg choline to about 3500 mg choline, about 1200 mg choline to about 3500 mg choline, about 1300 mg choline to about 3500 mg choline, about 1400 mg choline to about 3500 mg choline, about 1500 mg choline to about 3500 mg choline, about 1600 mg choline to about 3500 mg choline, about 1700 mg choline to about 3500 mg choline, about 1800 mg choline to about 3500 mg choline, about 1900 mg choline to about 3500 mg choline, about 2000 mg choline to about 3500 mg choline, about 2100 mg choline to about 3500 mg choline, about 2200 mg choline to about 3500 mg choline, about 2300 mg choline to about 3500 mg choline, about 2400 mg choline to about 3500 mg choline, about 2500 mg choline to about 3500 mg choline, about 2600 mg choline to about 3500 mg choline, about 2700 mg choline to about 3500 mg choline, about 2800 mg choline to about 3500 mg choline, about 2900 mg choline to about 3500 mg choline, about 3000 mg choline to about 3500 mg choline, about 3100 mg choline to about 3500 mg choline, about 3200 mg choline to about 3500 mg choline, about 3300 mg choline to about 3500 mg choline, or from about 3400 mg choline to about 3500 mg choline. In some aspects, the pharmaceutical composition may include from about 600 mg to about 3500 mg of choline, about 1000 mg choline to about 2500 mg choline, about 1500 mg choline to about 2250 mg choline, about 1200 mg choline to about 1800 mg choline, or from about 1400 mg choline to about 1700 mg choline.

[0064] In an aspect, the current disclosure also encompasses a pharmaceutical composition for use in improving brain function in a subject in need thereof, the pharmaceutical composition comprising 550 mg or greater of choline in a single unit dose suitable for oral administration. In an aspect, the current disclosure also encompasses a pharmaceutical composition for use in improving brain function in a subject in need thereof, the pharmaceutical composition comprising greater than 550 mg of choline in a single unit dose suitable for oral administration.

[0065] In an aspect, the current disclosure also encompasses a pharmaceutical composition for use in improving brain function in a subject in need thereof, the pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose suitable for oral administration.

[0066] The pharmaceutical compositions of the present disclosure may be formulated as a unit dosage form. In some aspects, about 1 to 3500 mg of the choline compound or a mixture of one or more choline compounds disclosed herein (for example, a choline salt, a phospholipid-bound choline, a choline precursor, a choline metabolite, or a combination thereof) or derivatives thereof, may be compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, and so forth, in a unit dosage form as called for by accepted pharmaceutical practice. It is to be noted that the unit dosage form may vary depending on the manufacturer, the compounding requirements, the suitability of intake, the determination of effective amounts etc. The amount of active substance in thosecompositions or preparations is such that a suitable dosage in the range indicated is obtained. The compositions may be formulated in a unit dosage form, each dosage containing from about 1-3000, 1-1800 mg, 2-800 mg, 5-500 mg, 10-400 mg, 50-200 mg, e.g., about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg or 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, 3000 mg, 3100 mg, 3200 mg, 3300 mg, 3400 mg, or 3500 mg of choline. In an example, the unit dose form comprises about 275 mg of choline. The concentration and / or amount of choline in the composition will depend on absorption, inactivation, and excretion rates of choline compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[0067] The pharmaceutical composition of the present disclosure may comprise from about 5 to 85 wt.% of free choline, such as from about 10 to 75 wt.%, or about 12to 50 wt.% or about20to 45wt.% based upon the total weight of the pharmaceutical composition. For example, the pharmaceutical composition of the present disclosure may comprise from about 5 wt% to about 10 wt% of free choline, about 5 wt% to about 15 wt% of free choline, about 5 wt% to about 20 wt% of free choline, about 5 wt% to about 25 wt% of free choline, about 5 wt% to about 30 wt% of free choline, about 5 wt% to about 35 wt% of free choline, about 5 wt% to about 40 wt% of free choline, about 5 wt% to about 45 wt% of free choline, about 5 wt% to about 50 wt% of free choline, about 5 wt% to about 55 wt% of free choline, about 5 wt% to about 60 wt% of free choline, about 5 wt% to about 65 wt% of free choline, about 5 wt% to about 70 wt% of free choline, about 5 wt% to about 75 wt% of free choline, about 5 wt% to about 80 wt% of free choline, about 5 wt% to about 85 wt% of free choline, about 10 wt% to about 85 wt% of free choline, about 15 wt% to about 85 wt% of free choline, about 20 wt% to about 85 wt% of free choline, about 25 wt% to about 85 wt% of free choline, about 30 wt% to about 85 wt% of free choline, about 35 wt% to about 85 wt% of free choline, about 40 wt% to about 85 wt% of free choline, about 45 wt% to about 85 wt% of free choline, about 50 wt% to about 85 wt% of free choline, about 55 wt% to about 85 wt% of free choline, about 60 wt% to about 85 wt% of free choline, about 65 wt% to about 85 wt% of free choline, about 70 wt% to about 85 wt% of free choline, about 75 wt% to about 85 wt% of free choline, about 80 wt% to about 85 wt% of free choline, about 20 wt% to about 60 wt% of free choline, or about 30 wt% to about 50 wt% of free choline.

[0068] The pharmaceutical compositions of the present disclosure may further comprise at least one pharmaceutically acceptable diluent(s), excipient(s), and / or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitablefor administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0069] The pharmaceutical compositions of the present disclosure may comprise stabilizers, anti-oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0070] To prepare the disclosed pharmaceutical compositions, the choline salt, phospholipidbound choline, a choline precursor, a choline metabolite, or a combination thereof may be mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. Pharmaceutical carriers or vehicles suitable for administration of the compositions provided herein include any such carriers known to be suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action (e.g. improvement of working memory in an individual). The choline compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. The amount of the choline in the pharmaceutical composition is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the choline compound is administered (e.g. postmenopausal cognitive decline) and / or that is effective in a prophylactic context. The pharmaceutical composition may be formulated as a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.

[0071] The pharmaceutical compositions disclosed herein may comprise pharmaceutically acceptable excipients selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, or at least 50% of one or more pharmaceutically acceptable excipients by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more pharmaceutically acceptable excipients by total weight of the pharmaceutical composition. In some aspects, one or more pharmaceutically acceptable excipients may be added to improve the performance, efficacy, safety, shelf-life and / or other property of the pharmaceutical composition of the present disclosure. In some aspects, the one or more pharmaceutically acceptable excipients may be biocompatible, without being harsh, abrasive, and / or allergenic.

[0072] The pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acids, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, nitric acid, and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more acidifying agents by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more acidifying agents by total weight of the pharmaceutical composition.

[0073] The pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, trolamine, and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more alkalizing agents by total weight of the pharmaceutical composition. In some aspects, pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more alkalizing agents by total weight of the pharmaceutical composition.

[0074] The pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and other materials known to one of ordinary skill in the art. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more antioxidants by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more antioxidants by total weight of the pharmaceutical composition.

[0075] The pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate, sodium citrate anhydrous and dihydrate, and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more buffering agents by total weight of the pharmaceutical composition. In some aspects, pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more buffering agents by total weight of the pharmaceutical composition.

[0076] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition when formulated as a solution. In some aspects, pharmaceutical compositions disclosed herein may have a pH from about 6 to about 9. In some aspects, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0077] The pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limitingexamples of preservatives include benzoic acid, benzyl alcohol, sorbic acid, salicylic acid, methylparabens, propylparabens, isopropyl alcohol, or combinations thereof and other preservatives known to those having ordinary skill in the art. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more preservatives by total weight of the pharmaceutical composition. In some aspects, pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more preservatives by total weight of the pharmaceutical composition.

[0078] The pharmaceutical compositions disclosed herein may comprise one or more surface active agents or detergents. In some aspects, surface active agents or detergents may be synthetic, natural, or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more surface active agents or detergents by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more surface active agents or detergents by total weight of the composition.

[0079] The pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more stabilizers by total weight of the pharmaceutical composition. In some aspects, pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more stabilizers by total weight of the pharmaceutical composition.

[0080] The pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agent” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and others known to those of ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of one or more tonicity modifiers by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of one or more tonicity modifiers by total weight of the pharmaceutical composition.

[0081] The pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In some aspects, the pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of polymers as suspending agent(s) by total weight of the pharmaceutical composition. In some aspects, the pharmaceutical compositions disclosed herein may comprise from about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% of polymers as suspending agent(s) by total weight of the pharmaceutical composition.

[0082] The pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0083] The pharmaceutical compositions of the present disclosure may be formulated for one or more routes of administration. Suitable routes of administration may, for example, include oral, intravenous, intracranial, intrathecal, cranial, transcranial, enteral, and / or parenteral delivery. In some aspects, compositions herein formulated may be formulated for oral delivery.

[0084] When oral administration is desired, the composition may be provided in a formulation that protects it from the acidic environment of the stomach. For example, the composition maybe formulated in an enteric coating that maintains its integrity in the stomach and releases the choline (and other active ingredients, when present) in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient. Oral compositions generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral administration, the compositions may be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials may be included as part of the composition. In various aspects, the tablets, pills, capsules, troches, and the like may contain any of the following ingredients or equivalents thereof: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[0085] Non-limiting examples of suitable dosage forms for oral administration include capsules, tablets, pills, lozenges, soluble films, elixirs, syrups, solutions, suspensions, emulsions, semisolids and gels. Capsules may be a one-piece or two-piece capsule, and have a soft or hard shell. Non-limiting examples of tablets include a suspension tablet, a chewable tablet, an effervescent tablet, and an orally disintegrating tablet. Semisolids may include, but are not limited to, gel-filled chews and gelatinous chews. The pharmaceutical compositions may also be administered as a component of an elixir, suspension, syrup, wafer, medicated chewing gum or the like. A syrup may contain, in addition to the active compositions, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.

[0086] The pharmaceutical compositions of the present disclosure may further include a therapeutically effective amount of one or more additional therapeutic agents. The one or more additional therapeutic agents may be an estrogen replacement agent. In an aspect, the estrogen replacement agent may comprise medroxyprogesterone acetate, megestrol acetate, clonidine, norethindrone acetate, ethinyl estradiol, conjugated estrogen, natural estrogen, synthetic estrogen, estradiol, progesterone, clomiphene, clomiphene citrate, zuclomiphene, zuclomiphene citrate, enclomiphene, enclomiphene citrate, aspirin, calcitonin, alendronate, etidronate, pamidronate, clodronate, tiludronate, residronate, ibandronate, or any combination thereof.

[0087] The pharmaceutical compositions disclosed herein may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Controlled release is a mechanism of formulation to release a drug over an extended time. Use of controlled release formulation may reduce the frequency of administration, reduce fluctuations in blood concentration and protect the gastrointestinal tract from side effects. The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coating. Such carriers include controlled release formulations (also known as modified, delayed, extended or sustained release or gastric retention dosage forms, such as the Depomed GR™ system in which agents are encapsulated by polymers that swell in the stomach and are retained for about eight hours, sufficient for daily dosing of many drugs). Controlled release systems include microencapsulated delivery systems, implants and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, matrix-controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion-exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof. The release rate of the active ingredient can also be modified by varying the particle size of the active ingredient(s).

[0088] In some aspects, the pharmaceutical compositions herein may comprise the active ingredient in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0089] The disclosed pharmaceutical compositions may be provided in kits, for example, including component parts that can be assembled for use. A kit may include a choline compound and a second therapeutic agent for co-administration. In an aspect, the second therapeutic may be a estrogen replacement agent. In an aspect, the estrogen replace agent may comprise the estrogen replacement agent comprises medroxyprogesterone acetate, megestrol acetate, clonidine, norethindrone acetate, ethinyl estradiol, conjugated estrogen, natural estrogen, synthetic estrogen, estradiol, progesterone, clomiphene, clomiphene citrate, zuclomiphene, zuclomiphene citrate, enclomiphene, enclomiphene citrate, aspirin, calcitonin, alendronate, etidronate, pamidronate, clodronate, tiludronate, residronate, ibandronate, or any combination thereof. The choline compound and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the one or more active agents. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, prefilled syringes, ampules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical or transdermal administration.

[0090] III. Definitions

[0091] It is to be understood that this disclosure is not limited to the particular methods, compositions, or materials specified herein, but is extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0092] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0093] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1 %, such as less than or equal to ± 0.05%.

[0094] The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

[0095] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0097] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0098] Further, as the present inventive concept is susceptible to “aspects” or “embodiments” used interchangeably, of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific aspects shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

[0099] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly 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 subrange is explicitly recited. As an illustration, a numerical range of “about 2 to about 50” should be interpreted to include not only the explicitly recited values of 2 to 50, but also include all individual values and sub-ranges within the indicated range. Thus, included in this numericalrange are individual values such as 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1 , 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1 , 38.0, 40, 44, 44.6, 45, 48, and sub-ranges such as from 1-3, from 2-4, from 5-10, from 5-20, from 5-25, from 5-30, from 5-35, from 5-40, from 5-50, from 2-10, from 2-20, from 2-30, from 2-40, from 2-50, etc. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0100] As disclosed herein above, the term “dosage form” refers to a formulation of the components in physical form designed to allow the accurate and efficient administration to a subject, preferably to a human. The term “unit dosage from” refers to physically discrete units suitable as unitary (i.e., single) dosages or multiple unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of the compounds, in association with one or more suitable pharmaceutical excipients.AspectsAspect 1. A method for improving brain function in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising 600 mg or greater of choline.Aspect 2. The method of Aspect 1 , wherein the administering comprises oral administration.Aspect 3. The method of Aspect 1 , wherein the pharmaceutical composition is a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.Aspect 4. The method of Aspect 1 , wherein the choline is provided in the form of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof.Aspect 5. The method of Aspect 4, wherein the choline salt comprises choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.Aspect 6. The method of Aspect 4, wherein the phospholipid-bound choline comprises phosphatidylcholine, soy lecithin, sphingomyelin, platelet-activating factor, synthetic phospholipid bound choline compounds, or derivatives thereof.Aspect 7. The method of Aspect 4, wherein the choline metabolite comprises cytidine diphosphate-choline.Aspect s. The method of Aspect 1, wherein the pharmaceutical composition comprises greater than 800 mg of choline.Aspect 9. The method of Aspect 1, wherein the pharmaceutical composition comprises greater than 1000 mg of choline.Aspect 10. The method of Aspect 1, wherein the pharmaceutical composition comprises greater than 1200 mg of choline.Aspect 11. The method of Aspect 1, wherein the pharmaceutical composition comprises greater than 1500 mg of choline.Aspect 12. The method of Aspect 1, wherein the pharmaceutical composition comprises greater than 2000 mg of choline.Aspect 13. The method of Aspect 1, wherein the pharmaceutical composition comprises from 600 to 3500 mg of choline.Aspect 14. The method of Aspect 1, wherein the pharmaceutical composition comprises from 1000 to 2500 mg of choline.Aspect 15. The method of Aspect 1, wherein the pharmaceutical composition comprises from 1500 to 2250 mg of choline.Aspect 16. The method of Aspect 1, wherein the pharmaceutical composition comprises from 1200 to 1800 mg of choline.Aspect 17. The method of Aspect 1, wherein the pharmaceutical composition comprises from 1400 to 1700 mg of choline.Aspect 18. The method of Aspect 1, wherein the pharmaceutical composition comprises 1650 mg of choline.Aspect 19. The method of Aspect 1 , wherein the pharmaceutical composition is administered in a single unit dose.Aspect 20. The method of Aspect 1 , wherein the pharmaceutical composition is administered in multiple unit doses, wherein each of the unit dose may be the same or different.Aspect 21 . The method of Aspect 1 , wherein the administration occurs once per day.Aspect 22. The method of Aspect 1 , wherein the administration occurs twice per day.Aspect 23. The method of Aspect 1 , wherein the administration occurs three times per day.Aspect 24. The method of Aspect 1 , wherein the administration occurs once per day for at least one week.Aspect 25. The method of Aspect 1 , wherein the administration occurs once per day for at least two weeks.Aspect 26. The method of Aspect 1 , wherein the administration occurs once per day for at least four weeks.Aspect 27. The method of Aspect 1 , wherein the administration occurs once per day for at least eight weeks.Aspect 28. The method of Aspect 1 , wherein the administration occurs once per day for at least twelve weeks.Aspect 29. The method of Aspect 1 , wherein the subject is female with low estrogen levels.Aspect 30. The method of Aspect 29, wherein the subject is female with an estradiol level <50 pg / mL.Aspect 31 . The method of Aspect 29, wherein the subject is a postmenopausal female.Aspect 32. The method of Aspect 1 , wherein the subject is a male with normal estrogen levels.Aspect 33. The method of Aspect 32, wherein the male has an estradiol level of <50 pg / mL.Aspect 34. The method of Aspect 1 , wherein the subject is an estrogen-deficient subject.Aspect 35. The method of Aspect 1 , wherein the subject has low estrogen levels as measured in serum.Aspect 36. The method of Aspect 1 , wherein the subject has increased parietal activation as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control.Aspect 37. The method of Aspect 1 , wherein the subject has a functional increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition.Aspect 38. The method of Aspect 1 , wherein the subject has an increase in activity in the postcentral cortex as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control.Aspect 39. The method of Aspect 38, wherein the measurement is taken about 3 hours after the administration of the pharmaceutical composition.Aspect 40. The method of Aspect 1 , wherein the subject has an increase in activity in the occipital lobe as measured by functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control.Aspect 41 . The method of Aspect 40 wherein the measurement is taken about 3 hours after the administration of the pharmaceutical composition.Aspect 42. The method of Aspect 1 , wherein the subject has decreased connectivity between regions of the brain associated with working memory as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to after administration of a placebo control.Aspect 43. The method of Aspect 1 , wherein the subject has decreased connectivity between the brain regions involved in complex cognition as measured by a functional MRI scan during a task challenge after administration of the pharmaceutical composition as compared to placebo.Aspect 44. The method of Aspect 43 wherein the measurement is taken about 3 hours after the administration of the pharmaceutical composition.Aspect 45. The method of any one of Aspects 36-38, 40, 41 , 43, and 44, wherein the task challenge comprises an N-back test.Aspect 46. The method of Aspect 1 , further comprising administering an estrogen replacement agent to the subject.Aspect 47. The method of Aspect 46, wherein the estrogen replacement agent is administered concurrently with the pharmaceutical composition.Aspect 48. The method of Aspect 46, wherein the estrogen replacement agent is administered separately from the pharmaceutical composition.Aspect 49. The method of Aspect 46, wherein the pharmaceutical composition further comprises the estrogen replacement agent.Aspect 50. A method for improving task efficiency in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose.Aspect 51 . The method of Aspect 50, wherein improvement in task efficiency is determined by measuring activity of the subject’s postcentral cortex and occipital lobe during a task challenge.Aspect 52. The method of Aspect 51 , wherein the task challenge comprises an N-back test.Aspect 53. The method of Aspect 52, wherein the activity is measured by a functional MRI scan after administration of the pharmaceutical composition.Aspect 54. The method of Aspect 53, wherein the activity is measured about 3 hours after the administration of the pharmaceutical composition.Aspect 55. The method of Aspect 51 , wherein the administering comprises oral administration.Aspect 56. The method of Aspect 51 , wherein the pharmaceutical composition is a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.Aspect 57. The method of Aspect 51 , wherein the choline is provided in the form of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof.Aspect 58. The method of Aspect 57, wherein the choline salt comprises choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.Aspect 59. The method of Aspect 57, wherein the phospholipid-bound choline comprises phosphatidylcholine, soy lecithin, sphingomyelin, platelet-activating factor, synthetic phospholipid bound choline compounds, or derivatives thereof.Aspect 60. The method of Aspect 57, wherein the choline metabolite comprises cytidine diphosphate-choline.Aspect 61. The method of Aspect 51 , wherein the pharmaceutical composition comprises greater than 800 mg of choline.Aspect 62. The method of Aspect 51 , wherein the pharmaceutical composition comprises from 600 to 3500 mg of choline.Aspect 63. The method of Aspect 51 , wherein the pharmaceutical composition comprises 1650 mg of choline.Aspect 64. The method of Aspect 51 , wherein the pharmaceutical composition is administered in a single unit dose.Aspect 65. The method of Aspect 51 , wherein the pharmaceutical composition is administered in multiple unit doses, wherein each of the unit dose may be the same or different.Aspect 66. The method of Aspect 51 , wherein the subject is female with low estrogen levels.Aspect 67. The method of Aspect 66, wherein the subject is female with an estradiol level <50 pg / mL.Aspect 68. The method of Aspect 66, wherein the subject is a postmenopausal female.Aspect 69. The method of Aspect 51 , wherein the subject is a male with normal estrogen levels.Aspect 70. The method of Aspect 69, wherein the male has an estradiol level of <50 pg / mL.Aspect 71 . The method of Aspect 51 , wherein the subject is an estrogen-deficient subject.Aspect 72. The method of Aspect 51 , wherein the subject has low estrogen levels as measured in serum.Aspect 73. A method for improving working memory in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose.Aspect 74. The method of Aspect 73, wherein improvement in working memory efficiency is determined by measuring activity of the subject’s frontal lobe, and more specifically right frontal lobe during a task challenge.Aspect 75. The method of Aspect 74, wherein the task challenge comprises an N-back test, letter number sequencing or a combination thereof.Aspect 76. The method of Aspect 74, wherein the activity is measured by a functional MRI scan after administration of the pharmaceutical composition.Aspect 77. The method of Aspect 76, wherein the activity is measured about 3 hours after the administration of the pharmaceutical composition.Aspect 78. The method of Aspect 73, wherein the administering comprises oral administration.Aspect 79. The method of Aspect 73, wherein the pharmaceutical composition is a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.Aspect 80. The method of Aspect 73, wherein the choline is provided in the form of a choline salt, phospholipid-bound choline, a nucleotide-bound choline, a choline precursor, a choline metabolite, or a combination thereof.Aspect 81 . The method of Aspect 80, wherein the choline salt comprises choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, cholineorotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.Aspect 82. The method of Aspect 80, wherein the phospholipid-bound choline comprises phosphatidylcholine, soy lecithin, sphingomyelin, platelet-activating factor, synthetic phospholipid bound choline compounds, or derivatives thereof.Aspect 83. The method of Aspect 80, wherein the choline metabolite comprises cytidine diphosphate-choline.Aspect 84. The method of Aspect 73, wherein the pharmaceutical composition comprises greater than 800 mg of choline.Aspect 85. The method of Aspect 73, wherein the pharmaceutical composition comprises from 600 to 3500 mg of choline.Aspect 86. The method of Aspect 73, wherein the pharmaceutical composition comprises 1650 mg of choline.Aspect 87. The method of Aspect 73, wherein the pharmaceutical composition is administered in a single unit dose.Aspect 88. The method of Aspect 73, wherein the pharmaceutical composition is administered in multiple unit doses, wherein each of the unit dose may be the same or different.Aspect 89. The method of Aspect 73, wherein the subject is female with low estrogen levels.Aspect 90. The method of Aspect 89, wherein the subject is female with an estradiol level <50 pg / mL.Aspect 91. The method of Aspect 89, wherein the subject is a postmenopausal female.Aspect 92. The method of Aspect 73, wherein the subject is male with low estrogen levels.Aspect 93. The method of Aspect 92, wherein the male has an estradiol level of <10 pg / mL.Aspect 94. The method of Aspect 73, wherein the subject is an estrogen-deficient subject.Aspect 95. The method of Aspect 73, wherein the subject has low estrogen levels as measured in serum.Aspect 96. A pharmaceutical composition for use in improving brain function in a subject in need thereof, the pharmaceutical composition comprising greater than 600 mg of choline in a single unit dose suitable for oral administration.Aspect 97. The pharmaceutical composition of Aspect 96Aspect 96, wherein the pharmaceutical composition is a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.Aspect 98. The pharmaceutical composition of Aspect 96, wherein the choline is provided in the form of a choline salt, phospholipid-bound choline, a choline precursor, a choline metabolite, or a combination thereof.Aspect 99. The pharmaceutical composition of Aspect 98, wherein the choline salt comprises choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.Aspect 100. The pharmaceutical composition of Aspect 98, wherein the phospholipid-bound choline comprises phosphatidylcholine, soy lecithin, and combinations thereof.Aspect 101. The pharmaceutical composition of Aspect 98, wherein the choline metabolite comprises cytidine diphosphate-choline.Aspect 102. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises greater than 800 mg of choline.Aspect 103. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises greater than 1000 mg of choline.Aspect 104. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises greater than 1200 mg of choline.Aspect 105. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises greater than 1500 mg of choline.Aspect 106. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises greater than 2000 mg of choline.Aspect 107. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises from 600 to 3500 mg of choline.Aspect 108. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises from 1000 to 2500 mg of choline.Aspect 109. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises from 1500 to 2200 mg of choline.Aspect 110. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises from 1200 to 1800 mg of choline.Aspect 111. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises from 1400 to 1700 mg of choline.Aspect 112. The pharmaceutical composition of Aspect 96, wherein the pharmaceutical composition comprises 1650 mg of choline.Aspect 113. A combination therapy comprising a combination of greater than 600 mg of choline in a single unit dose and a therapeutically effective amount of an estrogen replacement agent.Aspect 114. The combination therapy of Aspect 113, wherein the estrogen replacement agent comprises medroxyprogesterone acetate, megestrol acetate, clonidine, norethindrone acetate, ethinyl estradiol, conjugated estrogen, natural estrogen, synthetic estrogen, estradiol, progesterone, clomiphene, clomiphene citrate, zuclomiphene, zuclomiphene citrate, enclomiphene, enclomiphene citrate, aspirin, calcitonin, alendronate, etidronate, pamidronate, clodronate, tiludronate, residronate, ibandronate, or any combination thereof.Aspect 115. The combination therapy of Aspect 113, wherein the estrogen replacement agent comprises a phytoestrogen.Aspect 116. The combination therapy of Aspect 115, wherein the phytoestrogen comprises an isoflavone, coumestan, lignan, stibene, a prenylflavonoid, or a combination thereof.Aspect 117. The combination therapy of Aspect 116, wherein the isoflavone comprises daidzein, genistein, glycitein, formononetin, biochanin A, or a combination thereof.Aspect 118. The combination therapy of Aspect 113, wherein the choline and the estrogen replacement agent are formulated in separate compositions.Aspect 119. The combination therapy of Aspect 113, wherein the choline and the estrogen replacement agent are formulated in a single composition.Aspect 120. The combination of Aspect 113, wherein the pharmaceutical composition is a tablet, capsule, pill, sachet, stick pack, powder, granule, lozenge, soluble film, syrup, solution, suspension emulsion, gel, gummy or a combination thereof.Aspect 121. The combination of Aspect 113, wherein the choline is provided in the form of a choline salt, phospholipid-bound choline, a choline precursor, a choline metabolite, or a combination thereof.Aspect 122. The combination of Aspect 121, wherein the choline salt comprises choline bitartrate, choline bitartrate DL, choline chloride, choline dihydrogen citrate, choline salicylate, choline phosphate, choline bicarbonate, choline magnesium trisalicylate, choline orotate, choline ascorbate, choline succinate, choline hydroxide, choline monoxide, or a combination thereof.Aspect 123. The combination of Aspect 122, wherein the phospholipid-bound choline comprises phosphatidylcholine, soy lecithin, and combinations thereof.Aspect 124. The combination of Aspect 122, wherein the choline metabolite comprises cytidine diphosphate-choline.Aspect 125. The combination of Aspect 113, wherein the combination comprises greater than 800 mg of choline.Aspect 126. The combination of Aspect 113, wherein the combination comprises greater than 1000 mg of choline.Aspect 127. The combination of Aspect 113, wherein the combination comprises greater than 1200 mg of choline.Aspect 128. The combination of Aspect 113, wherein the combination comprises greater than 1500 mg of choline.Aspect 129. The combination of Aspect 113, wherein the combination comprises greater than 2000 mg of choline.Aspect 130. The combination of Aspect 113, wherein the combination comprises from 600 to 3500 mg of choline.Aspect 131. The combination of Aspect 113, wherein the combination comprises from 1000 to 2500 mg of choline.Aspect 132. The combination of Aspect 113, wherein the combination comprises from 1500 to 2250 mg of choline.Aspect 133. The combination of Aspect 113, wherein the combination comprises from 1200 to 1800 mg of choline.Aspect 134. The combination of Aspect 113, wherein the combination comprises from 1400 to 1700 mg of choline.Aspect 135. The combination of Aspect 113, wherein the combination comprises 1650 mg of cholineEXAMPLES

[0101] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 : MethodsStudy design

[0102] This human trial was designed to study the impact of an acute dose of choline on brain functions in post-menopausal women. A randomized placebo-controlled trial was conducted.To ensure the scientific rigor of the design, the order of the choline or placebo was randomized across participants.Participant selection

[0103] Participants in the trial were selected based on the inclusion and exclusion criteria presented in Table 1. Potential participants were subjected to a telephone screening to obtain medical history and consent and video conferencing to complete cognitive and neuropsychological screenings. In-person screening visits were conducted for the participants for medical screening and in-person neuropsychological tasks performance.Medical Screening

[0104] A medical history review and physical examination to establish general physical health with the CRC Physician’s Assistant was conducted on all potential participants. Women were examined for physical health. Nonsmokers with a BMI < 35 kg / m2, and no cardiovascular disease other than mild hypertension were selected. Participants were assessed by history, physical exam, and laboratory tests assessing cardiac, hematopoietic, renal, hepatic, and hormonal function. For these assessments, complete blood count (CBC), comprehensive metabolic panel (CMP), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), estradiol (E2), and electrocardiogram (ECG) analysis were conducted. Follicle stimulating hormone (FSH) and estradiol (E2) levels were used to confirm menopausal status.

[0105] Aside from dietary intake, the only source of choline in the body is de novo synthesis of phosphatidylcholine, catalyzed by phosphatidylethanolamine-N-methyltransferase (PEMT). Blood was therefore obtained for a genetic analysis of phosphatidylethanolamine-N- methyltransferase (PEMT) SNPs and other genes related to choline production and metabolism to be completed after the study was finished.

[0106] Subjects were carefully questioned about the history of their perimenopausal and postmenopausal autonomic and vasomotor symptoms utilizing symptom review and structured menopausal symptom checklists. A Reproductive History Questionnaire was used to gather these data. In addition, subjects were asked about any family history of severe perimenopausal symptoms. Such information was recorded for use in data analysis.Cognitive / Behavioral Screening

[0107] All women were cognitively and behaviorally assessed using standard tests designed to exclude subjects with significant cognitive or behavioral impairment. These tests are listed below.Montreal Cognitive Assessment (MoCA): The Montreal Cognitive Assessment test (MoCA) is a 30-point cognitive assessment designed to detect participants at the MCI stage of cognitive dysfunction. This instrument has been shown to have adequate sensitivity and specificity in clinical settings to detect suspected mild cognitive impairment (MCI). Cognitive Change Index (CCI): The Cognitive Change Index is a 20 item self-report and study partner (informant)- report instrument. Items assess primarily memory with some coverage of executive and language changes. A CCI-Memory Total Score obtained by combining both the self and informant forms can yield a total score for memory or overall cognition. Brief Cognitive Rating Scale (BCRS): A scale used to assess functional and cognitive abilities in both normal aging and dementing disorders. Global Deterioration Scale (GDS): A scale used to assess the level of deficits in cognition and function in cognitively impaired patients. Beck Depression Inventory Short Form (BDI-II): The Beck Depression Inventory Short form is a 13-item self-reporting questionnaire that is used to evaluate the severity of depression in normal and psychiatric populations. Beck Anxiety Inventory (BAI): The BAI is a self-report inventory that assesses anxiety symptoms and symptom severity. It contains 21 questions, with answers scored on a scale value of 0 (not at all) to 3 (severely). Pittsburgh Sleep Quality Index (PSQI): The PSQI is a self-report rating form used to assess sleep quality of older adults during the past month. Structured Clinical Interview for DSM-IV (SCID): This is a brief clinical interview designed to establish the presence / absence of Axis I psychiatric disorders. Menopause Symptom Checklist: This is a checklist to assess physical symptoms associated with menopause. Brief Trauma Questionnaire (BTQ): The BTQ is a brief self-report questionnaire that is derived from the Brief Trauma Interview. The BTQ was originally designed to assess traumatic exposure according to DSM-IV but specifically asked only about Criterion A.1 (life threat / serious injury) because of the difficulty of accurately assessing A.2 (subjective response) in a brief self-report format. Criterion A.2 has been eliminated from the PTSD diagnostic criteria in DSM-5, so the BTQ provides a complete assessment of Criterion A.11. Connor-Davidson Resilience Scale (CD-RISC): This is a brief subject-completed form assessing resilience.

[0108] Subjects were required to have a GDS score of 2, a DRS2 score of greater than 131 , and a MoCA score of greater than or equal to 26. All subjects performed the Test of Premorbid Functioning (TOPF) to estimate IQ and were required to score above 80. All women underwent a behavioral screening that consisted of a Structured Clinical Interview for DSM-IV (SCID) to establish the presence / absence of psychiatric disorders, and the Beck Depression Inventory- II (score < 10). Eight women scored below the cutoff of 26 on the MoCA. As this was the first procedure of the screening, these individuals were excluded from the study. Participants were excluded if they had a current psychiatric disorder. Participants completed the Pittsburgh Sleep Quality Index (PSQI) that assessed sleep quality during the past month and a menopause symptom checklist to assess physical symptoms associated with menopause.Neuropsychological Screening

[0109] To assess general neuropsychological functioning, subjects were screened with the tests listed below. All participants were required to score within one standard deviation of the mean for their age.1. Mattis Dementia Rating Scale-2 (DRS): This includes battery-style assessment of overall level of cognitive functioning. The tasks are grouped into five subscales, each one evaluating different cognitive areas, including: Attention, Initiation / Perseveration (l / P), Construction, Conceptualization and Memory.2. Test of Premorbid Functioning (TOPF): TOPF is an assessment tool used to provide a measure of premorbid intelligence, the degree of intellectual function prior to the onset of illness or disease.3. Letter Number Sequencing (LNS): LNS is a measure of working memory. The examinee is read a sequence of numbers and letters and asked to recall the numbers in ascending order and the letters in alphabetical order. The task involves attention, concentration, mental manipulation, sequential processing, memory span, and shortterm auditory memory.4. Repeatable Battery for the Assessment of Neuropsychological Status (RBANS): RBANS is a brief test designed to evaluate neuropsychological status of adults, ages 12-89. The test assesses five cognitive domains including immediate memory, language, visuospatial / constructional ability, attention, and delayed memory and provides a global total measure.5. Delis-Kaplan Executive Function System (D-KEFS): This is used to measure a variety of verbal and non-verbal executive functions. The Trail Making and Verbal Fluency subtests will be used in this study. The Trail Making Test is a visual-motor sequencing task that measures cognitive flexibility. The Verbal Fluency Test measures letter and category fluency.Subjective Cognition

[0110] The Cognitive Change Index - Self and - Informant Ratings were used to asses’ participants for subjective cognition. Self and informant versions of the Everyday Cognition scale (ECog) were also used. Prior studies have shown good discrimination between cognitive normal older adults and those with mild dementia using this measure.Diet History Questionnaire III (DHQIII)

[0111] Current dietary intake was assessed using the Diet History Questionnaire III (DHQIII). This is a food frequency questionnaire and women were asked to complete the version asking for food recall over the past one month. This is a freely available tool for research available on the National Cancer Institute Division of Cancer Control and Population Health Sciences website. These data were used to estimate choline obtained through the diet for each subject.Table 1 : Criteria for selection of participants for the trial

[0112] Based on these criteria, 38 non-smoking (quit >2 year ago) healthy women aged SO- 65 years were selected. Women were postmenopausal and did not have a period in the last 12 months, had FSH>30 I U / L, and estradiol (E2) <50 pg / mL. They were not taking any kind of postmenopausal hormone treatment currently or within the past 12 months. An additional 15 women were screened for the study, and they were not enrolled because they failed thescreening or withdrew consent. Eight women did not pass the cognitive screening, one woman used estrogen cream in the past three months, and six women withdrew consent after screening and were no longer interested in participating.

[0113] Twenty women completed the two study days with choline and placebo and Magnetic Resonance Spectroscopy (MRS) and Magnetic Resonance Imaging (MRI) assessments. Three additional women started the study and completed one study day. They withdrew from the study for the following reasons: claustrophobia in the MRI, headache after the study day, and did not tolerate the procedures. When the blind was broken, each of these subjects had received the placebo on their study day. Their data were replaced with new subjects.Study Davs

[0114] Once participants completed and passed all screening procedures, they were scheduled for the drug challenge / MRI sessions. These procedures are described below.Pharmacological Challenge Procedures

[0115] A randomization schedule was created by an experienced statistician not involved with the trial. For each subject on each study day the pharmacist dispensed the medication according to the randomization schedule. Since there were two medications, the order was divided evenly between day 1 and 2 and then the whole list was be randomized. This was a double-blind study so the subject, Principal Investigator (PI), research assistants, nurses, and MRI technologists were not aware of the medication dispensed on any day. After all data were cleaned and entered after all study procedures were completed, the pharmacist broke the blind and provided the information to the PI.

[0116] Choline was administered at the pre-determined dose as provided in the results. Choline Bitartrate from Nutrabio was used in these trials. These capsules had 275 mg choline cation and were administered to match the pre-determined study drug single dose. Placebos consisted of similar capsules filled with microcrystalline cellulose.

[0117] The two drug challenge days for each participant were spread over no more than one month. No study day followed a previous one by less than 48 hours. This time-line was acceptable to participants for their time commitment to the study and did not negatively impact recruitment. The drug sequence was determined by a random order procedure described above. A schematic of the timeline for a study day is provided in FIG. 1. Following an overnight fast, participants reported to the CRC between 0700 and 0900 depending on the subject’s preference and the availability of the facilities. The study day began with baseline testing and evaluation. Choline or matching placebo was administered 3 hours prior to the MRI session. Vital signs and pupil diameter were recorded at 60 min intervals. Cognitive testing duringfunctional neuroimaging was conducted at 180-120 minutes after Choline / PLC dose. Participants were transported by the experimenter to the MRI suite for the fMRI session that began with the MRS and then functional scans were conducted to maximize the drug dose timing. After scanning, participants returned to the CRC, completed cognition, mood, and physical symptom assessment questionnaires and then received lunch. Each participant was followed until the cognitive testing was completed which was approximately 5 hours after the Choline / PLC dosing. There were no unexpected effects of this single dose of acute choline on vital signs, which were found to be within limits and the subjects were discharged.Assessments used on Study Davs

[0118] A series of assessments were conducted on the participants on study days as listed below:1. Physical Symptom Checklist: this form is a self-report inventory that assesses 21 physical symptoms and the severity of the symptom.2. Stanford Sleepiness Scale: this is a one-item self-report questionnaire measuring levels of sleepiness.3. Subjective Visual Analog Scale (SVAS): participants completed visual analog scale assessing physical symptoms.4. Objective Visual Analog Inventory (OVAI): experimenter completed visual analog scale assessing subjects during study day.5. Brief Psychiatric Rating Scale (BPRS): BPRS is a rating scale used to measure psychiatric symptoms such as anxiety, depression, hallucinations and unusual behavior. Each of the 24 symptoms is rated on a scale of 1 (not present) to 7 (extremely severe).6. Profile of Mood States (POMS): POMS is a psychological rating scale contains 65 selfreport items using the 5-point Likert scale. Participants can choose from 0 (not at all) to 4 (extremely).7. Neuropsychological Evaluations were conducted as described in the participant selection section above.8. Buschke Selective Reminding Test: is a test designed to measure verbal learning and memory through the use of a list-learning procedure over multiple trials. The examinee is read a list of 16 unrelated words and then asked to immediately recall as many of the 16 words as possible. Every trial after the first involved selectively presenting only the words which the examinee did not recall on the immediately preceding trial. Thetrials proceed in this manner until 8 trials have been completed or until the examinee can correctly recall all 16 words on three consecutive trials.9. Symbol Digit Modalities Test (SDMT): The Symbol Digit Modalities Test (SDMT) examines cerebral dysfunction, and specifically processing speed, in adults. Using a reference key, the participant has 90 seconds to pair specific numbers with meaningless figures. They are then scored based on the correct number of substitutions that were made.Procedures used on Study Davs

[0119] In addition to the various tests listed above, participants also underwent various procedures to directly visualize presence and impact of choline administration. These procedures are listed below:1. Magnetic Resonance Spectroscopy (MRS) Procedures: this consisted of two runs using standard procedures targeting one voxel in the right lateral frontal lobe and a second voxel in the right posterior parietal cortex. Each scan took approximately 10 minutes to acquire the data to evaluate choline and other standard metabolite levels in the brain including / V-acetyl-aspartate (NAA) and creatinine.2. fMRI Working Memory Task: The N-Back Test was used as the measure of verbal working memory. In this task, the participant saw a string of consonant letters (except L, W, and Y), one every three seconds during 27-second blocks. Three conditions were presented: 0-back, 1-back, 2-back, and 3-back. In each condition, the task was to decide whether the letter currently on the screen matched the target in the 0-back or the letter that had been presented 1 , 2, or 3 back in the sequence. The N-back task reliably activates a bilateral frontal, parietal, and cerebellar working memory network and this has been previously used it in a number of studies with postmenopausal women. The total task time was 8.15 minutes. Two validated comparable versions were used for each of the N-back task, and were counterbalanced across study days.3. Resting State Scan Participants: were instructed to close their eyes but try to remain awake during the resting state scan. Participants completed two five-minute resting state scans for a total of 10 minutes of rest data.4. MRI Acquisition: A 3T Philips Achieva dStream scanner and 32-channel head coil was used for this procedure. The imaging protocol is based on that developed for the multicenter NIH-funded Adolescent Brain Cognitive Development (ABCD) study, which itself is derived from large studies such as the Human Connectome Project (HCP) and the Lifespan Connectome Project. The protocols make extensive use of simultaneousmultislice imaging (multiband SENSE) to accelerate functional and diffusion MRI acquisitions. MRI Acquisition consisted of T1- and T2-weighted images at 0.8mm isotropic resolution, along with T2-FLAIR at 1.0mm isotropic resolution. The high resolution T 1 - and T2-weighted images are well suited to the HCP processing streams, including the generation of cortical myelin maps, while the T2-FLAIR provides excellent sensitivity to intra-cranial pathology. Task and Resting state fMRI parameters are TR 800ms, TE 35ms, flip angle 52°, 2.4mm isotropic imaging resolution with a 216x216* 144mm3field of view using a multiband acceleration factor of 6 (60 slices, no gap).Magnetic Resonance Imaging (MRI) Analysis

[0120] Structural and BOLD task functional MRI scans of 19 cognitively healthy postmenopausal women were collected on each choline and placebo day.

[0121] Structural and fMRI Preprocessing Task-based and resting-state data were preprocessed and analyzed using CONN toolbox version 20. b, an SPM-based software. Functional and structural images were preprocessed using the preprocessing pipeline for volume-based analyses (direct normalization to MNI-space) with FieldMaps available. Preprocessing steps included the creation of voxel-displacement map for susceptibility distortion correction, realignment, unwrapping, and susceptibility distortion correction (motion correction), slice-timing correction, outlier detection, direct segmentation and normalization, segmentation and normalization, and smoothing. Slice order was interleaved bottom-up and smoothing kernel was 8 mm. Functional connectivity measures were computed for predefined seed regions for an ROI-to-ROI analysis, results that survived a false discovery rate (FDR)- corrected threshold of p < 0.05 were reported.

[0122] Structural scans were used to rule out incidental pathology. FLAIR images were also used to assess white matter damage associated with aging. White matter hyperintensity (WMH) volumes was measured using the Lesion Segmentation Toolbox. Briefly, each voxel on the T 1 image is assigned as gray matter, white matter, or CSF. After bias-correction the FLAIR is co-registered to the T 1 image. The toolbox creates a conservative binary WMH map based on outlier values across the T1 and FLAIR images. Next, a lesion-growth algorithm using Markov Random Fields modeling extends this conservative map to define the extent of the WMH. This lesion map is then used to calculate total cerebral WMH volume which can be used as a covariate in the analyses described below.

[0123] Basal Forebrain Cholinergic System (BFCS) Volume Measures were also determined. Brain scans were manually reoriented, and the stereotaxic origin of each image was set to the anterior commissure at the level of the interhemispheric plane. MRI scans were segmentedinto gray matter (GM), white matter, and cerebrospinal fluid partitions of 1.5 mm isotropic voxel size using the segmentation routine of the VBM8 toolbox. The resulting GM and white matter partitions of each subject in native space was registered to a reference template using the DARTEL algorithm. Individual flow fields obtained from the DARTEL registration to the reference template were used to warp the GM segments. Individual GM volumes of BFCS regions of interest (ROIs) were extracted automatically from the warped GM segments by summing up the modulated GM voxel values within the respective ROI masks.

[0124] The Structural MRI Variable for the outcome analyses was mean left and right hippocampal volume and gray matter density (GMD) adjusted for total intracranial volume and age. A comprehensive set of brain regions of interest for volume, cortical thickness, and gray matter density were used for additional exploratory analyses.

[0125] Additionally, individual participants’ structural T1 scans were parcellated using FreeSurfer version?.4.0 and used in combination with functional data in connectivity analyses preformed using CONN toolbox version 20. b, an SPM-based software. A ROI-to-ROI correlation (bivariate) analysis comparing connectivity on the choline vs placebo days during an Nback task was run among the 68 ROIs (2278 connections) included in the default FreeSurfer atlas. Standard settings for cluster-based inferences with Threshold Free Cluster Enhancement (nonparametric statistics connection threshold: p< 05 p-FWE corrected (TFCE)) were performed.Example 2: Acute Choline Administration Trials

[0126] This study was a cross-over, placebo-controlled trial of the effects of high- concentration acute administration of choline on brain functioning in 19 healthy postmenopausal women. Choline was administered at a dose of 1650 mg choline in the form of choline bitartrate. Each woman participated in two study days with choline or placebo administered on each day three hours before a functional and structural MRI and MRS scans. Choline Bitartrate from Nutrabio was used in these trials. These capsules were 275 mg choline cation equaling 6 capsules per subject.Magnetic Resonance Spectroscopy (MRS)

[0127] Initially, the ability of a single choline dose to influence brain choline metabolites was measured by MRS. The ratios of choline to creatinine and choline to / V-acetyl-aspartate (NAA) in the frontal voxel and parietal voxel were compared separately on both study days (see FIG. 2A and 2B). Using paired t-tests no difference was found between the choline study day and the placebo study day in either voxel for either ratio (ps >30, see Table 2 and Table 3).Table 2: MRS means, standard deviations, standard errors, t-values and p-values for the anterior voxel for the choline and placebo study days. Measures obtained during the MRI were NAA, choline (Cho), and creatinine (Cr). Ratios of Cho / Cr and Cho / NAA were calculated.Table 3: MRS means, standard deviations, standard errors, t-values and p-values for the posterior voxel for the choline and placebo study days, Measures obtained during the MRI were NAA, choline (Cho), and creatinine (Cr). Ratios of Cho / Cr and Cho / NAA were calculated.Cognitive Performance Test

[0128] Working memory was examined during the MRI using the Nbacktest, episodic memory after the MRI using the Buschke Selective Reminding Task, and processing speed using the Symbol Digit Modalities Test (SDMT). For the Nback the outcome measure was the signal detection measure of d’ for each of the 0-, 1-, 2-, and 3-back conditions. No differences could be found in working memory performance for the choline and placebo study days (ps > .20, See Table 4).Table 4: Means, standard deviations, standard errors of the mean, t-values, and p-values for the signal detection measures of accuracy, d’, on the 0-back, 1-back, 2-back, and 3-back conditions of the Nback task on the choline and placebo study days.

[0129] For the Buschke analysis, measures of total number of words recalled, consistency of words recalled, and recall failure were used. There was no episodic memory difference between the choline and placebo study days (ps > .47; Table 4). Finally, for the SDMT, the measures of completed items and number of correctly completed items in the 90 second period were examined (ps > .87, see Table 5).Table 5: Means, standard deviation, standard errors of the mean, t-values, and p-values for the performance measures from the Buschke and SDMT on the choline and placebo study days.Working Memory Task

[0130] fMRI data was collected during working memory task data. Decreased activation was found for the choline day compared to the placebo day during the working memory task in the right frontal pole (p<.05 cluster corrected). Increased activation was observed for the choline compared to placebo day during the working memory task in the central opercular cortex right, insular cortex left, and left postcentral gyrus (p<.05 cluster corrected) - see FIG. 3. This is similar to what has been found with younger adults showing increased posterior activation compared to older adults. Instead, older adults have increased frontal activation when directly compared during working memory tests.

[0131] An increased posterior activation was also observed on the choline day compared to the placebo day in the central opercular cortex right, insular cortex left, and left postcentral gyrus (red areas in glass brains in FIG. 3). Choline reduced frontal working memory-related activation and increased posterior activation compared to placebo.

[0132] These results show that one dose of choline made the brain “look younger” in healthy cognitively normal postmenopausal women.Task Efficiency Measure Across All Tasks

[0133] fMRI image across all task conditions in 19 postmenopausal were collated. Though a direct demonstration of presence of choline in the brain was not sought, functional increase in postcentral cortex (red) and functional decrease in the occipital lobe (blue) for said tasks (see FIG. 5) provides support that choline gets into the brain and impacts functionality in these tasks.Connectivity Analysis

[0134] Connectivity data between different parts of the brain demonstrating the ‘functional’ presence of choline in the brain was analyzed. Decreased connectivity was observed for the choline compared to the placebo study during the working memory task in four clusters. The most significant cluster (p-FWE=0.034) showed a pattern of decreased functional connectivity between inferior parietal (R), and parahippocampus (R), the inferior parietal (R) and parahippocapus (L), and the rostral middle frontal (R) and rostral anterior cingulate left (see FIG. 6). It is known that frontal and parietal brain segments are involved in ‘working memory’ and para-hippocampal areas involved in ‘episodic memory’. Results shown here provide robust and conclusive evidence that choline gets into the brain and impacts working memory.Example 3: Low to Moderate Dose Choline Administration Trials

[0135] The results so far illustrate the effect of high acute dose of choline on working memory related brain activation and functional connectivity. These measures indicate that choline modulates activation and connectivity as compared to placebo with comparable cognitive function indicating improved brain efficiency through reduced functional brain recruitment.

[0136] Further studies were conducted to examine the impact of acute, but lower doses of choline on postmenopausal women. For these trials, two different doses of 550 mg and 1100 mg were tested.. Study design, selection of participants and the test conducted were as provided above in Example 1 .Example 4: Extended Treatment Trials

[0137] Impact of multiple doses of choline over a duration will be tested to study the impact of length of choline administration on behavior and mental acuity in postmenopausal women. Initial trials will be conducted such that an optimized dose of choline determined from the doseranging study will be administered for at least 6 weeks with similar endpoints as described in the previously described protocol. Selection of participants and the test conducted will be as provided above in Example 1 .Example 5

[0138] Objectives

[0139] Aim 1. Examine whether a single dose of 550 mg oral choline influences brain functioning in postmenopausal women during an fMRI working memory task.

[0140] Aim 2. Examine whether a single dose of 1100 mg oral choline influences brain functioning in postmenopausal women during an fMRI working memory task.

[0141] Aim 3. Examine whether choline influences working memory performance during fMRI.

[0142] STUDY DESIGN AND SAMPLE SIZE

[0143] This study is a within-subjects, placebo-controlled trial of two different choline doses on brain functioning in healthy postmenopausal women. To ensure the scientific rigor of the design, the order of the two choline doses and placebo will be counterbalanced across subjects.

[0144] Participants The participants were 20 non-smoking (quit >2 year ago) healthy postmenopausal women (16 completers) aged 50-65 years. Women who are postmenopausal will not have had a period in the last 12 months, have FSH>30 I U / L, and estradiol (E2) <50 pg / ml. They will not be taking any kind of postmenopausal hormone treatment currently or within the past 12 months.

[0145] STUDY POPULATION

[0146] The study enrolled 20 women aged 50-65 years who are medically healthy, willing and healthy as specified in the entry criteria below.

[0147] Inclusion Criteria

[0148] All participants must meet the following criteria:• Women aged 50-65 years• Postmenopausal: Women who are postmenopausal will have not had a period in the last 12 months, have FSH>30 I U / L, and estradiol (E2) <50 pg / ml.• Nonsmokers• Not taking hormone therapy, SSRIs, phytoestrogens, SERMS, or antiestrogen medications and will be at least one year without such treatment• Physically healthyNo cardiovascular disease other than mild hypertension. Subjects will also not have current untreated or unremitted Axis I or II psychiatric or cognitive disorders (see screening below).IQ in the normal range >80• Normal neuropsychological test performance

[0149] Exclusion Criteria

[0150] All participants must not meet the following criteria:• MCI or dementia - Montreal Cognitive Assessment <26, Mattis Dementia Rating Scale <130, and Global Deterioration Scale >2• History of cancer treatment with cytotoxic and / or ongoing (current) maintenance targeted chemotherapy• Blood pressure > 160 / 100 (untreated)• Untreated thyroid disease• Significant cardiovascular disease• Asthma or COPD• Active peptic ulcer• Hyperthyroidism• Epilepsy• Current untreated or unremitted Axis I psychiatric disorders• Use of prohibited medicationsSubjects with major concomitant illnesses will be excluded on the basis of history, physical exam, and laboratory tests assessing hematopoietic, renal, hepatic and hormonal function. (CBC, CMP, TSH, ECG). A minimum of 14 days will elapse after discontinuing prohibited medications and the commencement of this study.

[0151] Exclusion Criteria related to the MRI• Claustrophobia• Having any of the following movable metal objects in the body: cardiac pacemakers or implantable defibrillators, aneurysm clips, neural stimulators, artificial heart valves, ear implants, implanted devices such as an insulin pump or drug infusion device, I UDs, magnetic dental appliances, metal fragments or foreign objects in the eyes, skin or body, metal plates, screws and prosthetics, non-removable metal piercings, tattoos on the head and neck, other certain older tattoos with metal containing inks, and permanent makeup (eyeliner).Metal jewelry, watches, and any other removable metal will need to be removed before entering the MRI room. Medication patches will also be removed before entering the MRI room.

[0152] DESCRIPTION OF STUDY VISITS

[0153] Telephone Pre-Screen

[0154] Potential participants will complete a telephone screen to determine eligibility. Verbal consent to take part in a screening procedure over the telephone will be requested. Questions about medical history and medication use will be asked to ensure subject are not taking any medications on the prohibited medications list will help determine eligibility. The participant will be told that all of the information obtained on the telephone screen will be kept confidential, stored in a locked office, and on a secure computer server. Identifiers obtained during the telephone screening will be kept until the study is completed and closed at the IRB. If it is determined that the person is ineligible, this phone screen with be kept with other ineligible phone screens in study specific folders in locked offices. This information will be maintained to track how many women have a certain medical history that would deem them ineligible to participate. The person will be told on the phone that they have the option to ask for their information to be destroyed or discontinue this study at any point. If the subject passes the telephone screening she will be scheduled for a Screening Visit.

[0155] The subject has the option to complete the screening in 2 parts with one part over a password protected videoconference system and the other part in person at the Clinical Research Center (CRC). The subject can also choose to complete the screening at one time in person at the CRC. This will be arranged with the subject based on her preference and availability. The subject will meet with a study staff member who will explain the study verbally and the subject will be allowed to ask questions. Using REDCap the subject will be able to view the consent form and can print it out if she prefers. If the subject is interested in enrolling in the study and continuing participation and Electronic Informed Consent (elC) will be obtained. The subject will be able to view or print out the signed consent form as well.

[0156] Study Visits1a. Screening Visit - elC, cognitive and neuropsychological screenings that can be completed by video conferencing (all tests except the Trail Making section of the DKEFS, symbol digit matching from the RBANS).1.b Screening Visit - medical screening and in person neuropsychological tasks that require responses from the subject that cannot be converted to an electronic system (like the trail making section of the DKEFS, digit symbol from RBANS).2. Study day 1 - Choline / placebo challenge and MRI3. Study day 2 - Choline / placebo challenge and MRI4. Study day 3 - Choline / placebo challenge and MRISubjects will be encouraged to undergo all procedures.

[0157] Screening Visit

[0158] Use of Electronic Informed Consent and Screening After elC is obtained, the screening visit will continue on the video conference platform. The majority of the assessments can be completed by video conference tools with some exceptions. Those exceptions are the trail making portions of the DKEFS and the symbol digit matching on the RBANS.

[0159] If the subject prefers, she can complete the screening visit in one in person visit at the CRC.

[0160] Medical Screening Women will be physically healthy nonsmokers with a BMI < 35 kg / m2, and no cardiovascular disease other than mild hypertension. Participants will be assessed by history, physical exam, and laboratory tests from a blood draw assessing cardiac, hematopoietic, renal, hepatic, and hormonal function (CBC, CMP, TSH, FSH, E2, ECG) and will have a medical history review and physical examination to establish general physical health. Blood will be obtained for a genetic analysis of PEM and other genes related to choline production and metabolism to be completed after the study is finished. Total blood drawn for these analyses will be approximately 17 ml.

[0161] Eight (8) single nucleotide polymorphisms (SNPs) related to choline production and metabolism will be analyzed by the UVM Vermont Integrated Genetics Resource (VIGR). These analyses will be completed after the study is over. The samples will be coded so that subjects cannot be identified by those doing the SNP analysis before sharing the samples.

[0162] The following hormone levels will be examined: follicle stimulating hormone (FSH) and estradiol (E2) which will be used to confirm menopausal status.

[0163] Subjects will be questioned about the history of their perimenopausal and postmenopausal autonomic and vasomotor symptoms utilizing symptom review and structured menopausal symptom checklists. Reproductive History Questionnaire will be used to gather these data. In addition, subjects will be asked about any family history of severe perimenopausal symptoms. Such information will be recorded for use in data analysis.

[0164] Cognitive / Behavioral Screening All women will be cognitively and behaviorally assessed using standard tests designed to exclude subjects with significant cognitive or behavioral impairment including the Montreal Cognitive Assessment (MoCA (Nasreddine,2005, The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment. J. Am. Geriatr. Soc., 53(4), 695-699), Dementia Rating Scale 2 (DRS2 (Jurica and Mattis, 2001 , Dementia Rating Scale-2, Lutz, FL: Psychological Assessment Resources Inc.).) Brief Cognitive Rating Scale (Reisberg, 1988, Brief Cognitive Rating Scale (BCRS). Psychopharmacology Bulletin, 24, 629-636), and the Global Deterioration Scale (GDS (Reisberg, 1988, Global Deterioration Scale (GDS). Psychopharmacology Bulletin, 24(4), 661- 663)). Subjects will be required to have a GDS score of 2, a DRS2 score of greater than 131 , and a MoCA score of greater than or equal to 26. All subjects will perform the Test of Premorbid Functioning (TOPF) to estimate IQ and will be required to score above 80.

[0165] All women will undergo a behavioral screening consisting of a Structured Clinical Interview for DSM-IV (SCID) to establish the presence / absence of psychiatric disorders, and the Beck Depression Inventory-ll (score < 10) (Beck, Steer, and Brown, 1996, Manual for the Beck Depression Inventory-ll. San Antonio, TX: Psychological Corporation). Participants will be excluded if they have a current psychiatric disorder. Participants will complete the Pittsburgh Sleep Quality Index (PSQI; (Buysse, 1989, The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research. Psychiatry Res 28(2), 193-213)) to assess sleep quality during the past month and a menopause symptom checklist to assess physical symptoms associated with menopause used in prior studies (Dumas, 2013, Increased working memory-related brain activity in middle-aged women with cognitive complaints. Neurobiology of Aging, 34(4), 1145-1147; Newhouse, 2013, Tamoxifen improves cholinergically modulated cognitive performance in postmenopausal women. Neuropsychopharmacology, 38(13), 2632-2643).

[0166] Neuropsychological Screening To assess general neuropsychological functioning, all subjects will be screened with the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS; (Randolph, 1998, The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS): preliminary clinical validity. J. Clin. Exp. Neuropsychol., 20(3), 310-319)). The RBANS assesses five cognitive domains including immediate memory, language, visuospatial / constructional ability, attention, and delayed memory and provides a global total measure. Selected measures from the Delis-Kaplan Executive Function System (D-KEFS; (Delis, Kaplan, and Kramer, 2001 , D-KEFS Executive Function System: Examiner's Manual. San Antonio, TX: Psychological Corporation)) will be included to compliment the RBANS. The Trail Making Test and Verbal Fluency subtests will be included. All participants will be required to score within one standard deviation of the mean for their age.

[0167] Subjective Cognition The Saykin (2013) Cognitive Change Index - Self Rating will be used. The self-version of the Everyday Cognition scale (ECog) will be used. Each questionnaire ask whether the subject notices any changes in their cognitive functioning now compared to around five years ago.

[0168] Diet History Questionnaire III (DHQIII) Current dietary intake will be assessed using the DHQIII. This is a food frequency questionnaire and will ask women to complete the version asking for food recall over the past one month. This is a freely available tool for research available on the National Cancer Institute Division of Cancer Control and Population Health Sciences website. These data will be used to estimate choline obtained through the diet for each subject.

[0169] Study Days

[0170] Once participants have completed and passed all screening procedures, they will be scheduled for the choline challenge / MRI sessions. These procedures are described below.

[0171] Pharmacological Challenge Procedures

[0172] Choline will be administered in doses of 550 mg and 1100 mg choline bitartrate on two different study days. Choline Bitartrate is a commercially available supplement. DA commercially available choline bitartrate like this product from Nutrabio https: / / www.nutrabio.com / product / 45016 / will be used. These capsules are 275 mg choline cation and two doses of 550 mg and 1100 mg choline cation are proposed. The higher dose of 1100 mg requires 4 capsules. To match that for the lower choline dose and the placebo, subjects will take 4 capsules each day. For the lower choline dose, two of the capsules will be choline and the other 2 will be matching placebo.

[0173] Placebos will consist of similar capsules filled with microcrystalline cellulose.

[0174] The randomization schedule will be created. For each subject on each study day the pharmacist will dispense the medication according to the randomization schedule. Since there are three medications, the order will be counterbalanced across days 1 , 2, and 3 and then the whole list will be randomized. This will be a double-blind study.

[0175] The three drug challenge days for each participant will be spread over no more than one month. No study day will follow a previous one by less than 48 hours. The drug sequence will be determined by a counterbalancing procedure described above. Following an overnight fast, participants will report to the CRC between 0700 and 0900 depending on the subject’s preference and the availability of the facilities. The study day will begin with baseline testing and evaluation. Choline or matching placebo will be administered 3 hours prior to the MRI session. Vital signs and pupil diameter will be recorded at 60 min intervals. Cognitive testingduring functional neuroimaging will be conducted at 180 minutes after choline / placebo dose. This time reflects previous data prior studies on when central nervous system effects appear to be maximal following administration of choline (Babb, 2004, Oral choline increases choline metabolites in human brain. Psychiatry Res., 130(1), 1-9). Participants will be transported by the experimenter to the MRI suite for the MRI session that will begin with the MRS scans followed by the functional scans to match the MRI protocol of a prior choline study. After scanning, participants will return to the CRC, complete cognition, mood, and physical symptom assessment questionnaires and will receive lunch. Each participant will be followed until the cognitive testing is completed which is approximately 5 hours after the choline / placebo dosing..

[0176] Cognitive / Behavioral Evaluations

[0177] Assessments used at Screening

[0178] Montreal Cognitive Assessment (MoCA): The Montreal Cognitive Assessment test (MoCA) is, similar to the MMSE, a brief, 30-point cognitive assessment designed to detect participants at the MCI stage of cognitive dysfunction26. This instrument has been shown to have adequate sensitivity and specificity in clinical settings to detect suspected MCI.

[0179] Cognitive Change Index (CCI): The Cognitive Change Index is a 20 item self-report instrument. Items assess primarily memory with some coverage of executive and language changes.

[0180] Brief Cognitive Rating Scale (BCRS): A scale used to assess functional and cognitive abilities in both normal aging and dementing disorders.

[0181] Global Deterioration Scale (GDS): A scale used to assess the level of deficits in cognition and function in cognitively impaired patients.

[0182] Beck Depression Inventory Short Form (BDI-II): The Beck Depression Inventory Short form is a 13-item self-reporting questionnaire that is used to evaluate the severity of depression in normal and psychiatric populations.

[0183] Beck Anxiety Inventory (BAI): The BAI is a self-report inventory that assesses anxiety symptoms and symptom severity. It contains 21 questions, with answers scored on a scale value of 0 (not at all) to 3 (severely).

[0184] Pittsburgh Sleep Quality Index (PSQI): The PSQI is a self-report rating form used to assess sleep quality of older adults during the past month.

[0185] Structured Clinical Interview for DSM-IV (SCID): This is a brief clinical interview designed to establish the presence / absence of Axis I psychiatric disorders.

[0186] Menopause Symptom Checklist: This is a checklist to assess physical symptoms associated with menopause used in prior studies (Dumas 2013 cited above; Newhouse 2013 cited above).

[0187] Reproductive History Questionnaire: This is a self-report questionnaire that asks reproductive history questions like age of menarche and menopause, number of pregnancies, use of hormonal contraceptives, use of postmenopausal hormone therapy, and any hypertensive disorders of pregnancy.

[0188] Assessments used on Study Days

[0189] Physical Symptom Checklist: The form is a self-report inventory that assesses 21 physical symptoms and the severity of the symptom.

[0190] Stanford Sleepiness Scale: This is a one-item self-report questionnaire measuring levels of sleepiness.

[0191] Subjective Visual Analog Scale (SVAS): Subject completed visual analog scale assessing physical symptoms.

[0192] Objective Visual Analog Inventory (OVAI): Experimenter completed visual analog scale assessing subjects during study day.

[0193] Brief Psychiatric Rating Scale (BPRS): The BPRS is a rating scale used to measure psychiatric symptoms such as anxiety, depression, hallucinations and unusual behavior. Each of the 24 symptoms is rated on a scale of 1 (not present) to 7 (extremely severe).

[0194] Profile of Mood States (POMS): The POMS is a psychological rating scale that contains 65 self-report items using the 5-point Likert scale. Participants can choose from 0 (not at all) to 4 (extremely).

[0195] Neuropsychological Evaluations

[0196] (i) Assessments used at Screening

[0197] Mattis Dementia Rating Scale-2 (MDRS): A battery-style assessment of overall level of cognitive functioning. The tasks are grouped into five subscales, each one evaluating different cognitive areas, including: Attention, Initiation / Perseveration (l / P), Construction, Conceptualization and Memory.

[0198] Test of Premorbid Functioning (TOPF): The TOPF is an assessment tool used to provide a measure of premorbid intelligence, the degree of intellectual function prior to the onset of illness or disease.

[0199] Letter Number Sequencing (LNS): The LNS is a measure of working memory. The examinee is read a sequence of numbers and letters and asked to recall the numbers in ascending order and the letters in alphabetical order. The task involves attention, concentration, mental manipulation, sequential processing, memory span, and short-term auditory memory.

[0200] Repeatable Battery for the Assessment of Neuropsychological Status (RBANS): The RBANS is a brief test designed to evaluate neuropsychological status of adults, ages 12-89. The test assesses five cognitive domains including immediate memory, language, visuospatial / constructional ability, attention, and delayed memory and provides a global total measure.

[0201] Delis-Kaplan Executive Function System (D-KEFS): The D-KEFS is used to measure a variety of verbal and non-verbal executive functions. The Trail Making and Verbal Fluency subtests will be used in this study. The Trail Making Test is a visual-motor sequencing task that measures cognitive flexibility. The Verbal Fluency Test measures letter and category fluency.

[0202] The Measurement of Everyday Cognition (ECog): The ECog assesses everyday cognition. Prior studies have shown good discrimination between cognitive normal older adults and those with mild dementia using this measure.

[0203] (ii) Assessments used on Study days

[0204] Buschke Selective Reminding Test: The Buschke is a test designed to measure verbal learning and memory through the use of a list-learning procedure over multiple trials. The examinee is read a list of 16 unrelated words and then asked to immediately recall as many of the 16 words as possible. Every trial after the first involved selectively presenting only the words which the examinee did not recall on the immediately preceding trial. The trials proceed in this manner until 8 trials have been completed or until the examinee can correctly recall all 16 words on three consecutive trials.

[0205] Symbol Digit Modalities Test (SDMT): The Symbol Digit Modalities Test (SDMT) examines cerebral dysfunction, and specifically processing speed, in adults. Using a reference key, the participant has 90 seconds to pair specific numbers with meaningless figures. They are then scored based on the correct number of substitutions that were made.

[0206] PROCEDURES Magnetic Resonance Imaging (MRI)

[0207] fMRI Working Memory Task: The N-Back Test will be used as the measure of verbal working memory. In this task, the participant sees a string of consonant letters (except L, W, and Y), one every three seconds during 27-second blocks. Three conditions are presented: 0-back, 1-back, 2-back, and 3-back. In each condition, the task is to decide whether the letter currently on the screen matches the target in the 0-back or the letter that had been presented 1 , 2, or 3 back in the sequence. The N-back task is ideal for use during fMRI with populations who are expected to perform differently on the task because of the parametric manipulation of working memory load to allow for an examination of activation at different and similar levels of accuracy. The N-back task reliably activates a bilateral frontal, parietal, and cerebellar working memory network (Cohen, 1997, Temporal dynamics of brain activation during a working memory task. Nature 6625, 604-608) and it has been used in a number of studies with postmenopausal women (Dumas, 2008b, Nicotinic versus muscarinic blockade alters verbal working memory-related brain activity in older women. American Journal of Geriatric Psychiatry, 16, 272-282; Dumas, 2010, Cholinergic modulation of hippocampal activity during episodic memory encoding in postmenopausal women: a pilot study. Menopause, 17(4), 852- 859; Dumas, 2012, Estradiol treatment altered anticholinergic-related brain activity in postmenopausal women. Neuroimage, 60, 1394-1403; Dumas, 2018, Dopamine-dependent cognitive processes after menopause: the relationship between COMT genotype, estradiol, and working memory. Neurobiol. Aging, 72, 53-61). The total task time is 8.15 minutes.

[0208] The Face-Name Encoding Task (Sperling, 2001 , Encoding novel face-name associations: a functional MRI study. Hum. Brain Mapp., 14(3), 129-139) will be used as fMRI measure of episodic memory encoding. Subjects see novel faces and names appear on the computer screen one at a time during a 40 second block. There is a rest break followed by 40 seconds of two repeating face name pairs. The Novel and Familiar blocks alternate four times each during one run of the task. Each run of this task takes eight minutes to complete and volunteers will complete two runs on each study day.

[0209] Two validated comparable versions each of the N-back and Face-Name Tasks and will be counterbalanced across study days.

[0210] Resting State Scan: Participants will be instructed to close their eyes but try to remain awake during the resting state scan. Participants will undergo two five-minute resting state scans for a total of 10 minutes of rest data.

[0211] MRI Acquisition: D3T Philips Achieva dStream scanner and 32-channel head coil. The imaging protocol is based on that developed for the multicenter NIH-funded Adolescent Brain Cognitive Development (ABCD) study, which itself is derived from large studies such as the Human Connectome Project (HCP) and the Lifespan Connectome Project. The protocols make extensive use of simultaneous multislice imaging (multiband SENSE) to accelerate functional and diffusion MRI acquisitions.

[0212] Structural MRI Acquisition will consist of T1- and T2-weighted images at 0.8mm isotropic resolution, along with T2-FLAIR at 1.0mm isotropic resolution. The high resolution T1- and T2-weighted images are well suited to the HCP processing streams, including the generation of cortical myelin maps (Glasser, 2014, Trends and properties of human cerebral cortex: correlations with cortical myelin content. Neuroimage, 93 Pt 2, 165-175; Glasser, 2013, The minimal preprocessing pipelines for the Human Connectome Project. Neuroimage, 80, 105-124), while the T2-FLAIR provides excellent sensitivity to intra-cranial pathology. Task and Resting state fMRI parameters are TR 800ms, TE 35ms, flip angle 52°, 2.4mm isotropic imaging resolution with a 216x216x144mm3field of view using a multiband acceleration factor of 6 (60 slices, no gap).

[0213] MRS Procedures will consist of standard procedures targeting a voxel in the lateral frontal lobes and a second voxel in the posterior parietal cortex. Each scan takes 10 minutes to acquire the data to evaluate choline levels in the brain.

[0214] Magnetic Resonance Imaging (MRI) Analysis

[0215] Structural and fMRI Preprocessing will use the pipelines developed as part of the HCP (Glasser 2013 cited above) ,. The HCP structural pipelines are based on FreeSurfer and include subcortical segmentation, reconstruction of white- and pial-cortical surfaces, and folding-based surface registration to an atlas. As these techniques are further developed, the use of multimodal registration will be investigated to further improve cross-subject alignment (Glasser, 2016, A multi-modal parcellation of human cerebral cortex. Nature, 536(7615), 171- 178) using cortical myelin content (Glasser 2011 cited above; Glasser 2014 cited above) and functional imaging features. The HCP functional pipeline corrects for EPI spatial distortions using magnetic field maps, realigns volumes to account for subject motion and registers to the structural data. The functional data are then resampled into CIFTI grayordinate standard space, consisting of 91 ,282 data points, including -30,000 surface vertices per hemisphere and a further -30,000 subcortical gray matter voxels (with vertex and voxel spacing of 2mm). The HCP task fMRI pipelines will be used for first- and second-level analysis of task fMRI data. These pipelines are based on FSL FEAT (FMRIB’s Expert Analysis Tool) and FLAME (FMRIB’s Local Analysis of Mixed Effects), and incorporate high-pass filtering and application of general linear models (GLMs) (Smith, 2004, Advances in functional and structural MR image analysis and implementation as FSL. Neuroimage, 23 Suppl 1 , S208-219; Jenkinson, 2012, Fsl. Neuroimage, 62(2), 782-790).

[0216] Structural scans will be used to rule out incidental pathology. FLAIR images will also be used to assess white matter damage associated with aging. White matter hyperintensity (WMH) volumes will be measured using the Lesion Segmentation Toolbox (Schmidt, 2012, Anautomated tool for detection of FLAIR-hyperintense white-matter lesions in Multiple Sclerosis. Neuroimage, 59(4), 3774-3783). Briefly, each voxel on the T1 image is assigned as gray matter, white matter, or CSF. After bias-correction the FLAIR is co-registered to the T 1 image. The toolbox creates a conservative binary WMH map based on outlier values across the T1 and FLAIR images. Next, a lesion-growth algorithm using Markov Random Fields modeling extends this conservative map to define the extent of the WMH. This lesion map is then used to calculate total cerebral WMH volume which can be used as a covariate in the analyses described below.

[0217] Basal Forebrain Cholinergic System (BFCS) Volume Measures (Grothe, 2010, Reduction of basal forebrain cholinergic system parallels cognitive impairment in patients at high risk of developing Alzheimer's disease. Cereb. Cortex, 20(7), 1685-1695; Grothe, 2016, Cognitive Correlates of Basal Forebrain Atrophy and Associated Cortical Hypometabolism in Mild Cognitive Impairment. Cereb. Cortex, 26(6), 2411-2426): Brain scans will be manually reoriented, and the stereotaxic origin of each image will be set to the anterior commissure at the level of the interhemispheric plane. MRI scans will be segmented into gray matter (GM), white matter, and cerebrospinal fluid partitions of 1.5 mm isotropic voxel size using the segmentation routine of the VBM8 toolbox. The resulting GM and white matter partitions of each subject in native space will be registered to a reference template using the DARTEL algorithm (Ashburner, 2007, A fast diffeomorphic image registration algorithm. Neuroimage, 38(1), 95-113). Individual flow fields obtained from the DARTEL registration to the reference template will be used to warp the GM segments. Individual GM volumes of BFCS regions of interest (ROIs) will be extracted automatically from the warped GM segments by summing up the modulated GM voxel values within the respective ROI masks.

[0218] Given that the BFCS nuclei lack clear anatomical borders that could serve manual delineation on MRI scans, definition of the BFCS mask will be based on recently published cytoarchitectonic maps of BFCS nuclei in the MNI space (Kilimann, 2014, Subregional basal forebrain atrophy in Alzheimer's disease: a multicenter study. J. Alzheimers Dis., 40(3), 687- 700). The cytoarchitectonic BFCS map includes different cholinergic subdivisions within the BFCS. We will consider both the entire volume of the BFCS, as well as specific sub-regions that have showed reduced volume in clinically normal elderly adults with subjective cognitive decline (Scheef, 2018, Subregional volume reduction of the cholinergic forebrain in subjective cognitive decline (SCD). Neuroimage Clin, 101612) . These include the medial septal nucleus (Ch1), the vertical nucleus of the diagonal band of Broca (Ch2), the horizontal limb of the diagonal band of Broca (Ch3), and the nucleus basalis of basal Meynert (NBM, Ch4 sector according to Mesulam’s nomenclature ) (Mesulam and Geula, 1988, Nucleus basalis (Ch4)and cortical cholinergic innervation in the human brain: observations based on the distribution of acetylcholinesterase and choline acetyltransferase. J. Comp. Neurol., 275(2), 216-240).

[0219] Functional Connectivity Analysis: Regions of the brain found to be differentially affected by choline will be used to examine functional connectivity in the sample. Data will be analyzed using a seed-based method similar to that of Fox et al. (Fox, 2005, The human brain is intrinsically organized into dynamic, anti correlated functional networks. Proc. Natl. Acad. Sci. USA, 102(27), 9673-9678) to examine how functional connectivity is modulated by different choline doses. We will utilize the 1000 Functional Connectomes Project scripts. Specifically, after standard preprocessing steps including motion correction, temporal and spatial smoothing, and slice timing correction, the following will be included as nuisance regressors: global signal, white matter, cerebrospinal fluid and motion parameters. Correlations will be produced by extracting the BOLD time course from a priori seed regions and computing the correlation coefficients between the time course for each seed region and the time courses from all the other brain voxels. The resulting correlational maps will then be submitted to a group-level statistical analysis using the ANOVA models described above.

[0220] Statistical Analysis Plan

[0221] Data Analysis for Specific Aims 1 and 2: Aims 1 and 2 will examine the ability of 550 and 1100 doses of choline compared to placebo to influence brain activation on a task that is cholinerically mediated. For the task-based fMRI, the critical significance level for group-level analyses will be based on clusters of activated vertices with the probability threshold set at Pcorr< 0.001. To begin with, whole brain analyses will be used since the working memory network is widely distributed across frontal to parietal lobes as well as lateral and medial regions. In addition to examining the effect of working memory performance or functional brain activation when the independent variables are recorded as continuous measures. The genetic information will be used as a post hoc analysis to examine any effects of PEMT genotype on brain activation and performance. Choline intake will be used as a covariate if variation is observed in dietary choline from the diet questionnaire.

[0222] The effects of choline on brain functioning will improve cognition and result in brain activation that is more efficient and represents a “younger” pattern of activation with increased posterior activation and similar to what was observed in the first study with a choline dose of 1650 mg and in prior work when brain activation patterns in postmenopausal women was studied (Davis, 2008, Que PASA? The Posterior Anterior Shift in Aging. Cerebral Cortex, 18(5), 1201-1209; Dumas 2011 cited above).

[0223] Data Analysis for Exploratory Aim 3: The Aim 3 is to examine whether choline leads to improved performance on tasks that are sensitive to cholinergic manipulations and relatedbrain activation. For performance measures on the N-back task, accuracy and reaction time on hit trials will be examined at each level of N on the N-back task and on the recognition memory task.

[0224] Sample Size and Power Estimate: Sample size estimates are based on ability to detect differences choline / placebo differences in the first study using 1650 mg choline dose. In addition, a prior study by Babb et al. 2004 cited above found effects of acute choline dosing in 11 younger men. Thirty women will be enrolled and screened so that 20 women complete this study similar to the prior study of choline effects on brain functioning in postmenopausal women.

[0225] Example 6

[0226] The study of Example 5 was completed and the results were analyzed. In summary, the 1650 mg dose results were characterized by increased posterior activation together with decreased frontal activation (temporal), as compared to placebo. That pattern was observed with the 1100 mg dose with very strong posterior and occipital activation being observed, along with a maintenance of frontal activation. See Figures 6A and 6B. These patterns are consistent with a “younger” brain pattern. That is, younger adults have increased posterior activation, as compared to older adults. Older adults have increased frontal activation as compared to younger adults. In contrast, the 550 mg dose demonstrated frontal activation, only, with functional presence being demonstrated in the brain after 3.5 hours. See Figure 6C.

[0227] This data confirms the transport of choline across the blood-brain barrier, at the identified doses. This compilation of study data suggests a choline dose response in the brain. Frontal lobe activation was induced with 550 mg of choline. The threshold dose for inducing a posterior and occipital activation was observed with 1100 mg of choline. Posterior activation and frontal deactivation required 1650 mg.

Claims

What is claimed is:1 . A method of increasing brain efficiency in a human subject in need thereof, comprising identifying a human with an estradiol level of <50 pg / mL; orally administering to the human a pharmaceutical composition comprising 1100 mg or greater of choline; wherein the increase in brain efficiency is measured about 3.5 hours after the choline administration, by functional Magnetic Resonance Imaging (MRI), as compared to placebo, when the subject is performing an N-back task.

2. The method of claim 1 , wherein the pharmaceutical composition comprises 1650 mg or greater of choline.

3. The method of any one of the preceding claims, wherein the pharmaceutical composition comprises a single unit dosage form.

4. The method of any one of the preceding claims, wherein the pharmaceutical composition comprises a multiple unit dosage form.

5. The method of any one of the preceding claims, wherein the choline is present in the pharmaceutical composition as a choline salt.

6. The method of claim 5, wherein the choline salt is choline bitartrate.

7. The method of any one of the preceding claims, wherein the subject is a postmenopausal female.

8. The method of any one of claims 1-7, wherein the subject is male.

9. The method of any one of the preceding claims, wherein the increased brain efficiency is an increase in posterior activation including but not limited to parietal and occipital activation as measured by the functional MRI scan.

10. The method of any one of the preceding claims, wherein the increased brain efficiency is an increase in activity in the postcentral cortex as measured by the functional MRI scan.

11. The method of any one of the preceding claims, wherein the increased brain efficiency is an increase in activity in the occipital lobe as measured by the functional MRI scan.

12. The method of any one of the preceding claims, wherein the increased brain efficiency is an increase in activity in the parietal lobe as measured by the functional MRI scan.

13. The method of any one of the preceding claims, wherein the subject has decreased connectivity between regions of the brain associated with working memory as measured by a functional MRI scan during a task challenge.

14. The method of any one of the preceding claims, wherein the administration of at least 1100 mg choline salt results in cerebral activity consistent with that of a human that is of a younger chronological age than the human in need of treatment, as measured by functional MRI at 3.5 hours.

15. The method of any one of the preceding claims, wherein the administration further results in an improvement in the subject’s working memory, as measured by the N- back task.

16. The method of any one of the preceding claims, further comprising administering to the subject an estrogen replacement agent.

17. A method of increasing activation in the frontal lobe of a human subject in need thereof, comprising identifying a human with an estradiol level of <50 pg / mL; orally administering to the human a pharmaceutical composition comprising at least 550 mg of choline; wherein the increase in frontal lobe activation is measured about 3.5 hours after the choline administration, by functional Magnetic Resonance Imaging (MRI), as compared to placebo, when the subject is performing an N-back task.

18. The method of claim 17, wherein the pharmaceutical composition comprises about 550 mg of choline19. The method of claim 17, wherein the pharmaceutical composition comprises about 1100 mg of choline.

20. The method of any one of claims 17-19, wherein the administration further results in an improvement in the subject’s working memory, as measured by the N-back task.