CA2579851A1

CA2579851A1 - Use of uridine for improving cognitive and neurological functions

Info

Publication number: CA2579851A1
Application number: CA002579851A
Authority: CA
Inventors: Richard J. Wurtman; Carol Watkins
Original assignee: Individual
Current assignee: Massachusetts Institute of Technology
Priority date: 2004-09-15
Filing date: 2005-09-13
Publication date: 2006-03-23
Anticipated expiration: 2025-09-13
Also published as: AU2005285090A1; WO2006031683A2; JP2013064026A; JP6141643B2; EP1802314A2; WO2006031683A3; JP2008513453A; CA2579851C; CN102600199A; EP1802314A4

Abstract

The present invention is directed to methods of improving cognitive and neurological functions and increasing synthesis and release of neurotransmitters and membrane synthesis by neural cells and brain cells, comprising administering a composition comprising a uridine.

Description

COMPOSITIONS CONTAINING URIDINE, AND METHODS UTILIZING SAME
FIELD OF THE IN'VENTION

[0001] The present invention is directed to methods of improving cognitive and neurological functions and increasing synthesis and release of neurotransmitters and membrane synthesis by neural cells and brain cells, comprising administering a composition comprising a uridine or a source thereof.

BACKGROUND OF THE INVENTION

[0002] Uridine is a pyrimidine nucieoside and is essential in the synthesis of ribonucleic acids and tissue glycogens such as UDP glucose and UTP glucose. Prior medical uses of uridine alone include treatment of genetic disorders related to deficiencies of pyrimidine synthesis such as orotic aciduria. Choline, a dietary component of many foods, is part of several major phospholipids that are critical for= normal membrane structure and function.
Choline is included with lipid emulsions that deliver extra calories and essential fatty acids to patients receiving nutrition parenterally.

SUMMARY OF THE INVENTION

[0003] The present invention is directed to methods of improving cognitive and neurological functions and increasing synthesis and release of neuiotransmitters and membrane synthesis by neural cells and brain cells, comprising administering a composition comprising a uridine or a source thereof:

[0004] In one embodiment, the present invention provides a method of improving a co;nitive function in a subject, comprising administering to the subject a uridine, a source thereof, or a composition comprising a uridine and a choline.

[0005) In another embodiment, the present invention provides a method of improving a neurological function in a subject, comprising administering to the subject a uridine, a source thereof, or a composition comprising a uridine and a choline.

[0006] In another embodiment, the present invention provides a method of treating or ameliorating a decline in a cognitive function in a subject, comprising administering a uridine, a source thereof; or a composition comprising- a uridine and a choline to the subject.

SUBSTITUTE SHEET (RULE 26)

[0007] In another embodiment, the present invention provides a method of increasing or enhancing an ability of a brain cell or a neural cell of a subject to synthesize a neurotransmitter, comprising adniinisfiering to the subject or the brain cell or neural cell a uridine, a source thereof, or a composition comprising a uridine and a choline.

[0008] In another embodiment, the present invention provides a method of increasing a level of a neurotransrrritter in a synapse, comprising contacting a neural cell adjacent to the synapse witli a uridine, a source thereof, or a composition comprising a uridine and a choline, wliereby the composition enhances synthesis of a phospholipid or a precursor thereof, thereby increasing a level of a neurotransmitter in a synapse.

[0009] In another embodiment, the present invention provides a method of increasing a level of' a cytidine in a tissue, plasma, or cell of a subject, coniprising administering a uridine, a source thereof, to the subject.

[0010] In another embodiment, the present invention provides a method of increasing a level of a cytidine in a tissue, plasma, or cell of a subject, comprising administering a composition comprising a uridine or a source thereof and a choline to the subject.

[0011 ] In another embodiment, the present invention provides a method of stimulating or enl]ancing a production of a membrane of a brain cell or a neurai cell of a subject, comprising contacting the subject with a uridine, a source thereof, or a composition comprising a uridine and a choline, whereby the composition enhances synthesis of a phospholipid or a precursor thereof, thereby stimulating or enhancing a production of a membrane of a brain cell or a neural cell of a subject.

[0012] In another embodiment, the present invention provides a method of stimulating or=
enhancing an outgrowth of a neurite of a neural cell, comprising contacting the neural cell with a uridine, a source thereof, or a composition comprising a uridine and a choline, whereby the .. 25 composition enhances syntliesis of a phospholipid or a precursor thereof, thereby stimulating or enhancing ae outgrowth of a neurite of a neural cell.

BRIEF DESCRIPTION OF THE DRA.WINGS

[0013] Figure l illustrates the coincidence of'cytidine and tyrosine pealcs (6.59) when tested by a standard HPLC method.

[0014] Figure 2 illustrates distinct cytidine (3.25) and tyrosine (2.92) peaks when tested by a modified HPLC method, which utilizes elution buffer with low methanol.

[0015] Figure 3., Oral LJMP administration raises blood uridine levels in liumans. Depicted is the ratio of uridine (set as 100% value) to cytidine in plasma after oral administration of 250 milligram per kg of body weight (mg/kg) of uridine.

[0016] Figure 4. Oral uridine administration raises blood uridine levels in gerbils. Depicted are plasma uridine levels 60 minutes following mock administration or administration of cytidine or uridine. **: p < 0.01 vs. mock-fed control; ##: p < 0.01 vs, cytidine.

[0017] Figure 5. Oral UMP administration raises blood uridine levels in gerbils. Depicted are plasma uridine levels at various time points following administration or administration of water or UMP.

[0018] Figure 6. A UMP-supplemented diet raises blood uridine levels in gerbils. Depicted are plasma uridine levels in gerbils fed a diet containing the indicated percentages of UMP.

[0019] Figure 7. Oral uridine administration raises brain uridine levels.
Depicted are brain uridine levels 60 minutes following mock administration or administration of' cytidine or uridine. **: p < 0.01 vs. mock-fed control; ###: p< 0.01 vs., cytidine.

[0020] Figure 8. Oral UMP administration raises brain uridine levels. Depicted are brain uridine...levelsat various time points following administration or administration of water or UMP.

[0021] Figure 9.. Uridine is readily converted to cytidine in the brain.
Depicted is the ratio of uridine (100%) to cytidine in plasma (A) and in the brain (B) after oral administration of' 250 milligram per kg of' body weight (mg/kg) of uridine.

[0022] Figure 10. Oral UMP administration raises brain CDP-clioline levels.
Depicted are brain CDP-choline levels at various time points following administration or administration of water . _ ... ....
_----_..... ... ........ ..........
,25 or UMP.

[0023] Figure 11. Uridine increases intracellular levels of CDP-choline'in a neural cell Iine.
Cells were incubated for 6 h with the indicated concentrations of uridine.
Depicted are the means +/- S..E.M. of six dishes, expressed as picomole (pmol) CDP-choline/rng protein. The experiment was repeated 3 times. *: p < 0.05.

[0024] Figure 12. UMP dietary supplementation significantly increases potassium-evoked dopamine (DA) release in striatal dialysate. (A) Effect of dietary UMP
supplementations on K}-evoked striatal DA release. Data were calculated from six to nine measurements at each point (means standard error of measurement [S.E.M.])., The 100% value represented the mean of the four measurements before potassium stimulation was set at 100%. (B) Data were pooled according to UMP treatment groups. "*" denotes p< 0..05 compared to corresponding controls.
[0025] Figure 13. Effect of potassium on DOPAC and HVA 'levels in striatal dialysate with LIMP dietary supplementation. (A): DOPAC (B): HVA. *: p< 0.05 compared to corresponding controls..

[0026] Figure 14. Increased acetylcholine basal concentration witli UMP
treatment. Depicted are means +/- SEM. "'"" denotes p value of >0.05.

[0027] Figure 15. Effect of UMP dietary supplemention on neurofilament protein levels in contralateral striatum. (A): NF-70. (B): NF-M *: p < 0.05, **: p < 0..01 compared to corr-esponding coritrols.

[0028] Figure 16. Uridine treatment enhanced neurite outgrowth in PC 12 cells.
A. PC 12 cells treated for 4 days with NGF (50 ng/ml) in the presence or absence of uridine (5.0 M).. B.
Number of neurites per cell after 2 or 4 days of treatment. C. Number of neurites per cell after 2 or 4 days of NGF plus different concentrations of uridine (50, 100 and 200 M). D.
Quantification of the number of branch points for each cell. E. Levels of the structural proteins NF-70 and NF-M, as determined using Western blotting. N NGF, U
Ur'idine.
Values represent means + SEM. p < 0.01, p < 0.001 vs. NGF treatment.

[0029] Figure 17. Uridine treatment increased intracellular levels of UTP and CTP in PC 12 cells exposed to NGF for 2 days. Uridine treatment (50 M) significantly increased intracellular-UTP levels (A) and intracellular CTP levels (B): N= NGF, U = Uridine, C=
Cytidine. Values represent means + SEM.. *: p < 0.05 vs. NGF treatment..
........ ..... ....... ....... ............ ....-._........ .......
.....__............................ ..... ..__..-..---.............
..._....__.........._.__.._............. ._..._-_........ -......_...
_....._.__.._._............. ..... _._.......... .... ---............
........... ..__...... -- --....-...... _.._.......... ..__..... ....
[0030] Figure 18. UTP treatment increased neurite outgrowth. Treatment of PC
12 cells for 4 days with NGF and UTP significantly enhanced the nuniber of neurites produced per cell, compared to treatment with NGF alone. Values represent means + SEM. **p <
0.01.

[0031] Figure 19. NGF-differentiated PC 12 cells express pyrimidine-sensitive P2Y receptors.
A. Levels of P2Y2, P2Y4 and P2Y6 receptor expression afl:er incubation of'cells with NGF for varying lengths of time. B. Following 4 days of NGF treatment, cells were fixed and NF-70 (red) and P2Y receptor (green) proteins were visualized using immunofluorescence. Left panel:
P2Y2. Middle panel: P2Y4. Right panel: P2Y6. Values represent means + SEM. ***
p <
0.001..

[0032] Figure 20. P2Y2 receptor co-localizes with the neuronal marker= MAP-2.
Left panel:
P2Y2 receptor. Middle panel: MAP-2. Right Panel: Merge.

[0033] Figure 21. P2Y receptor antagonists inhibited the effect of uridine on neurite outgrowth.
Cells were treated for 4 days with NGF and with or without uridine (100 M) and the P2Y
receptor antagonists PPADS, surarnin, or RB-2. Values represent means + SEM.
1;**p < 0.001 vs. NGF treatment; # p< 0.05, ### p< 0..001 vs., NGF plus uridine treatment.

[0034] Figure 22. Phosphatidylinositol (PI) turnover is stimulated by UTP and uridine. Cells were metabolically labeled with [3H]inositol overrright, stimulated with UTP, uridine, or UTP
plus PPADS in the. presence of lithium at the indicated concentrations, and radio-labeled inositol phosphates derived from PI breakdown were measured by scintillation counting.
Values represent means + SEM. * p< 0.05, *"~.7 < 0,01 vs. control; # p< 0.05 vs.. 100 M UTP
treatment.

[0035] Figure 23.. Oral UMP improves learning and spatial memory in rats. 18-month old rats in restricted environinents consumed a control diet or a UMP diet for 6 weeks, and then were tested, using a Morris Water Maze, 4 trials/day for 4 days. Mean time to locate the platfonn is given in seconds.

[0036] Figure 24. Oral UMP irnproves learning and spatial memory in gerbils..
Learning and spatial memory of gerbils fed a control diet or diets containing the indicated amount of'UMP
were tested in a radial arm maze. Results are depicted as the amount of'time remaining before the 3-minute deadline..

[0037] Figure 25. Oral iJMP improves working memory and reference memory. The memory ....._._,....._ .........._..... _........... _.....-----....... ..... .........._..__......-_...__._..._~. __._..... .__... -....... -_.................
of gerbils fed a control or a 0.1 % UMP diet for four weeks was tested using modification of the test depicted in Figure 24, which measured both working memory errors (A) and reference memory errors (B).. Diamonds represent data points from control gerbils;
triangles represent data points from gerbils fed 0.1 % UMP diet..

[0038] Figure 26. Uridine and choline incr-ease neurotransmitter release in striatal slices (top panel), hippocampal slices (middle panel), and cortical slices (top panel).
Data are expressed as nanomoles per milligram protein per two hour, and depicted as means SEM.
'"I" = P < 0.001 relative to values obtained in the absence of choline. The first series in each panel was performed in the absence of choline; the second series was performed in the presence of choline. The bars in each series represent, from left to right, no additional compound added;
cytidine added; and uridine added (eacli in addition to the choline, where appropriate).

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention is directed to methods of improving cognitive and neurological functions and increasing synthesis and release of neurotransmitters and membrane synthesis by neural cells and brain cells, comprising administering a composition comprising a uridine or a source thereof.

[0040] In one embodiment, the present invention provides a method of improving a cognitive function in a subject, comprising.administering to the subject a uridine or a source thereof;
thereby improving a cognitive function in a subject.

[0041] In one embodiment, the present invention provides a method of improving a cognitive function in a subject, compr-ising administering to the subject a composition comprising a uridine or a sour-ce thereof and a choline, thereby improving a cognitive function in a subject..
[0042] Tlie phrase "uridine or a, source thereof and a choline" refers to 2 embodirnents of the present invention: a) a combination of'uridine and choline; b) a combination of a uridine source and clioline. The terms "uridine," "choline," and "uridine source" refer to any of their respective meanings that are mentioned herein. Each possibility represents a separate embodiment of the present invention..

[0043] In one embodiment, the cognitive function is memory. The memory is, in other embodiments, spatial memory, working memory, reference memory, short-term memory, long-.................. ........ - - ...... __-.........._.. _-...- ..... =
ry, or medium-term memory. In another embodiment, the men~ory isany othe....
ype- --term memo of memory known in the art. Each type of memory represents a separate embodiment of the present invention.

[0044] As provided herein, the data in Figures 21-23 show directly that uridine improves several types of memory. The consistency of the effect across difl:erent species in different types of assessments of'memory verifies the findings of the present invention.. The data in Example 15 further show that the effects of uridine are enhanced by inclusion of a choline.
Thus, administration of compositions comprising uridine and choline are effective at improving memory- more effective, in one embodiment, than administration of'either uridine or choline alone.

[0045] In another embodiment, the cognitive function is learning,. The learning is, in other embodiments, cognitive learning, affective leaming, or psychomotor learning.
In another embodiment, the learning is any other type of learning known in the art. Each type of learning represents a separate embodiment of the present invention.

[0046] In another embodiment, the cognitive function is intelligence. In other embodiments, the intelligence is linguistic intelligence, musical intelligence, spatial intelligence, bodily intelligence, interpersonal intelligence, intrapersonal intelligence, interpersonal intelligence, or logico-mathematical intelligence. In another e-nbodiment, the intelligence is any other type of intelligence known in the art. Each type of intelligence represents a separate embodiment of'the present invention,.

[0047] In anotlier embodiment, the cognitive function is mental fitness. In another embodiment, the cognitive function is any other type of cognitive function lcnown in the art. Each type of cognitive function represents a separate embodiment of the present invention.

[0048] In one embodiment, "improving" a cognitive function, or "improvement"
of a cognitive function refer to increasing the capacity of the subject to perforni the cognitive function. In another embodiment, the terms refer to an increased or improved baseline level of'the cognitive furlction in the subject. ln anotller embodiment, the terms refer to an increased or improved level of the cognitive function in response to a challenge or test.

[0049] In another embodiment, improving a cognitive function refers to effecting a 10%
improvement tliereof. In another embodiment, a 20% improvement is attained..
In other embodiments, a 30% improvement, a 40% improvement, a 50% improvement, a 60%
improvement, a 70% improvement, an 80% improvement, or a 90% improvement is attained. In another embodiment, improving a cognitive function refers to effecting a 100%
improvement thereof. L-ach possibility represents a separate embodiment of the present invention.

[0050] In another embodiment, improvement of a cognitive function is assessed relative to the cognitive function before beginning treatment. In another embodiment, improvement of a cognitive function is assessed relative to an untreated subject. In anotlier embodiment, improvement of a cognitive function is assessed according to a standardized criterion such as, for example, a test or the like. Lach type of' improvement of cognitive activity represents a separate embodiment of the present invention.

[0051 ] In another embodiment, improvement of a cognitive function is assessed by the number of connections between neurons in the subject's brain. In another embodiment, the improvement is assessed by the number of capillaries in the subject's brain, or in a specific region of the subject's brain. In another embodiment, the improvement is assessed by neural activity. In other embodiments, the improvement is assessed by neural function, linguistic function, or ability to convnunicate. In another embodiment, the improvement is assessed by measurement of levels of' acetylcholine or other neurotransmitters or brain chemicals correlated with cognitive function. In other embodiments, the improvement is assessed by Positron Emission Tomography (PET) scanning of the subject's brain, magnetic resonance imaging (MRI) scanning of the subject's brain. In another embodiment, the improvement is assessed by Cognitive Abilities Screening Instrument (CASI) (Peila R et al, Stroke. 32:
2882-9, 2001). In another embodiment, the improvement is assessed by a test such as, for example, the tests disclosed herein (Example 13). Additional methods for assessing improvement of cognitive function are well known in the art, and are described, for example in Antonova E et al (S.chizophr .Res...2004... nct 1 .;7.0(2-3 ):.1.1.7.-45) and....i.n,..Cogniti..v...e., Funciion.._Analysis..,(G.reenwood ...................... .
Pub Group, 1998). Each method represents a separate embodiment of'the present invention,.

[0052] In one embodiment of methods of the present invention, a composition of the present invention increases a level of cytidine, in the subject, thereby mediating one of the effects described herein (e.g. improving cognitive or neurological function, stimulating neural function, membrane synthesis, neurotransmitter release, etc). In another embodiment, the effect is mediated by incr-easing a level of cytidine tr7phosphate (CTP) in the subject.
In another ....... ..... ............. ............ embodiment; the-effect.-is-mediated-.by increasing.-.a-..level....of..-CDP-choline...in..the.-subjeet..-.In......-.- ...................-another embodiment, the effect is mediated by increasing a level of' a derivative of cytidine, CTP, CDP-choline in the subject. In another embodiment, the effect is mediated by increasing a level of a metabolite of cytidine, CTP, CDP-choline in the subject. In another embodiment, the effect is mediated witliout increasing a level of cytidine, CTP, CDP-choline, or a derivative or metabolite thereof Facli possibility represents a separate embodiment of the present invention.
Each possibility represents a separate embodiment of the present invention.

[0053] As described herein, Figures 9-11 show that orally administered uridine acts rapidly and effectively to raise levels of cytidine in the brain. In combination with Figures 3-8, which show that uridine is effectively and rapidly absorbed into the bloodstream, in several species, including humans, these findings demonstrate that administration of uridine raises levels of cytidine, CTP, and CDP-choline: The data in Example 15 further show that the effects of uridine are enhanced by inclusion of a choline.

[0054] In one embodiment, the cytidine level is a systemic level. In another embodiment, the cytidine level is a brain level., In another embodiment, the cytidine level is a nervous system level. Each possibility represents a separate embodiment of the present invention.

[0055] In another embodiment, the potential benefit of uridine administration is greater than the benefit of cytidine administration. This is due to the fact that cytidine, as opposed to uridine, either cannot cross or is much less efficient than uridine in crossing the blood-brain 6arrier (Cornford et al., Independent blood-brain barrier transport systems for nucleic acid precursors.
Biochim. Biophys. Acta 349:211-219, 1975).

[0056] In another embodiment, the increase in cytidine, CTP, or CDP-choline or a derivative or metabolite thereof enables the cell to increase levels of a phospholipid, thereby mediating one of the efI'ects described herein. In one embodiment, the phospholipid is phosphatidylcholine ...............................................................................
................. ........... ........... ........ ................ . . . .
.... ..... . ........ . .......................................... ......... .
. . ..... . .. .. ......... . . . . ........ ..... ...........................
...................
(PC).. In another embodiment, the phospholipid is phosphatidylethanolamine (PE). In another embodiment, the phospholipid is phosphatidylserine (PS). In another embodiment, the phospholipid is or a derivative or metabolite of PC, PE, or PS. Each possibility represents a separate embodiment of the present invention.

[0057] In another embodiment, the present invention provides a method of improving a neurological function in a subject, comprising administering to the subject a uridine or a source tliereof, thereby improving a neurological function in a subject.

[0058] In another embodiment, the present invention provides a method of improving a neurological function in a subject, comprising administering to the subject a composition comprising a uridine or a source tiiereof' and a choline, thereby improving a neurological function in a subject.

[0059] In another embodiment, the neurological function that is improved by a method of the present invention is a synaptic transmission. In one embodiment, the synaptic transmission is adjacent to a motor neuron. In another embodiment, the synaptic transmission is adjacent to an interneuron. In one embodiment, the synaptic transmission is adjacent to a sensory neuron.
Each type of synaptic transmission represents a separate embodiment of the present invention.
[0060] In another embodiment, the synaptic transmission is improved or enhanced by means of stimulating or enhancing an outgrowth of a neurite of a neural cell. In another embodiment, stimulating or enliancing an outgrowth of a neurite of a neural cell is partially responsible for improving or enhancing the synaptic transmission. In another embodiment, a composition of the present invention improves or enhances synaptic transmission without stimulating an outgrowth of a neurite. Each possibility represents a separate embodiment of the present invention.

[0061 ]"Neurite" refers, in one embodiment, to a process. growing out of a neuron. In one embodiment, the process is a dendrite. In another embodiment, the process is an axon. Each type of neurite represents a separate embodiment of'the present invention.

[0062] In another embodiment, the synaptic transmission is improved or enhanced by increasing the nuniber of neurites of the neural cell. In another embodiment, improvement or enhancement of the synaptic transmission occurs without increasing the number of neurites of the neural cell. Each possibility represents a separate embodiment of the present invention..

[0063] In another embodiment, the synaptic transmission " is improved or er-dianced by ............... . . .... ............. .......
................................ .. ... .
stimulating or er-flhancing branching of a neurite of a neural cell.. In another embodiment, improvement or enhancement of the synaptic transrnission occurs without stimulating or enliancing branching of a neurite of a neural cell.. E-ach possibility represents a separate enibodiment of the present invention.

[0064] The data of' Example 9 shows that when levels of inembrane precursors are increased, neurons produce morre neurites, with more branches. By increasing its surface area and size, a cell is able, in one embodiment, to form more connections with neighboring cells. Moreover, an increase in the arnount or composition of plasma tnembrane alters, in one embodiment, neurotransmitter synthesis and release, which also, in one embodiment, affects memory formation. Thus, compounds that promote neurite outgrowth, such as uridine, are useful for treatment of neuro-degenerative disorders like Alzheimer's disease, wliich involves loss of neuronal connections and memory impairment.

[0065] In another embodiment, improving the synaptic transmission in the subject is achieved by increasing an amount of a membrane of a neural cell as a result of administration of the uridine and/or choline. In anotlier embodiment, the improvement is achieved by stimulating a synthesis of a membrane of a neural cell. In another embodiment, the improvement is achieved by enhancing a synthesis of'a membrane of a neural cell. In another embodiment, stimulating or erillancing an amount of or a synthesis of a membrane of a neural cell is partially responsible for mediating improving the synaptic transmission in the subject. In another embodiment, the uridine and/or choline improves the synaptic transmission without stimulating or enliancing an amount of or a synthesis of a membrane of a neural cell. Each possibility represents a separate embodiment of the present invention..

[0066] Ln another embodiment, the neurological function that is improved or enhanced is a function of a neurotransmitter. In one embodiment, the improvement occurs by means of increasing a level of the neurotransmitter in a synapse. In another embodiment, the improvement occurs by means of increasing the release of the neurotransmitter into a synapse.
In another embodiment, the improvement occurs without clianging the level or release of the neurotransmitter in a synapse. Each possibility represents a separate embodiment of the present invention.

[0067] As provided herein, the data in Figures 12-13 show that uridine significantly improves neurotransmitter function, highlighting the ability of uridine to improve neurological function.
The data in Figures 14-17 show a beneficial effect of uridine on the morphology of neurites, ..... .... .... ............... .
...............................................................................
................................................... .. ....
.....................................
.................................................... .......
............................... .................. ..........................
. . .
further demonstrating the ability of uridine to improve neurological function,. The data in Example 15 further show that the effects of uridine are enhanced by inclusion of a choline..
Thus, administration of cornpositions comprising ur-idine and choline are effective at improving neurological function - more effective, in one embodiment, than administration of eitller uridine or choline alone.

[0068] In another embodiment, the present invention provides a method of treating or anieliorating a decline in a cognitive function in a subject, comprising administering a uridine ...... ................ ... .............. ... .. ..... . ..
................... ........... ._........... -- _....... ............... -....... ._.... .......... ...... ............ . _....... .... ....... ..___ ....._ .....
or a source tliereof to the subject, tliereby treating or ameliorating a decline in a cognitive function in a subject.

[0069] In another embodiment, the present invention provides a method of, treating or ameliorating a decline in a cognitive function in a subject, comprising administering a

11 composition comprising a uridine or a source thereof and a clioline to the subject, thereby inhibiting or preventing a decline in a cognitive function in a subject..

[0070] "Treating or ameliorating a decline in a cognitive function" refers, in one embodiment, to mitigating the decline.. In another embodiment, the phrase refers to preventing the decline. In another embodiment, the phrase refers to reversing the decline. In another embodiment, the plrrase refers to slowing the decline. In anotlier embodiment, the phrase refers to lialting the decline.. Each possibility represents a separate embodiment of the present invention.

[0071] In another embodiment, the decline in a cognitive function results from a neurological disorder. In one embodiment, the neurological disorder is a rnemory disorder.
The memory disorder comprises, in one. embodiment, a memory decline. In another embodiment, the niemory decline is associated with brain aging. In other embodiments, the memory disorder is selected from Pick's disease, Lewy Body disease, or a dementia. In other embodiments, the dementia is associated with Huntington's disease or AIDS dementia. Each possibility represents a separate embodiment of the present invention.

[0072] In another embodiment, the decline in a cognitive function results from a neurodegenerative disease. In one embodiment, the neurodegenerative disease is Alzheimer's disease. In other embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis, multiple system atrophy, Parkinson's disease, progressive supranuciear palsy, frontotemporal dementia, Huntington's disease, or a prion disease. In another embodiment, the neurodegenerative -disease is any other neurodegenerative disease .known in the art. Eacli possibility represents a separate embodiment of the present invention,.

[0073] In another= embodiment, the decline in a cognitive function results from a cardiovascular disease.. In one embodiment, the cardiovascular disease is a strokeõ In another embodiment, the cardiovascular disease is a multi-infarct dementia. In another embodiment, the cardiovascular disease is any other cardiovascular disease known in the art. Each possibility represents a separate embodiment of the present invention.

[0074] In one embodiment, the neurological disorder is associated with a dopaminergic pathway.. In another embodiment, the neurological disorder is not associated with a dopaniinergic pathway. Each possibility represents a separate embodiment of the present invention.

12 [0075] In another embodiment, the neurological disorder is a cognitive dysfunction. In one embodiment, the cognitive dysfunction is dyslexia. In other embodiments, the cognitive dysfunction coniprises a lack of attention, a lack of alertness, a lack of concentration, or a lack of focus. In other embodiments, the cognitive dysfunction comprises minimal cognitive inlpairment or age-related memory impairment. Each possibility represents a separate embodiment of the present invention.

[0076] In another embodiment, the neurological disorder is an emotional disorder. In other embodiments, the emotional disorder comprises mania, depression, stress, panic, anxiety, dysthymia, or psychosis: In another embodiment, the emotional disorder comprises a seasonal effective disorder. In another embodiinent, the emotional disorder comprises a bipolar disorder.
[0077] In another, embodiment, the neurological disorder is a psychiatric disease. In another embodiment, the neurological disorder is a depression. In one embodiment, the depression is an endogenous depression. I:n another embodiment, the depression is a major depressive disorder.
In another embodiment, the depression is depr=ession with anxiety. In another embodiment, the depression is bipolar depression. Each type of depression represents a separate embodiment of the present invention.

[0078] In another embodiment, the neurological disorder is selected from the group consisting of ataxia and Friedreich's ataxia..

[0079] In another embodiment, the neurological disorder is a movement disorder. The .. .................... .............................................
................. ................... ......... ........ .....................
......... .... .......................
movement disorder comprises, in other embodiments, a tardive dyskinesia, a dystonia, or a Tourette's syndrome. In another embodiment, the movement disorder is any other movement disorder Icnown in the art.

[0080] In another embodiment, the neurological disorder is a cerebro-vascular disease. The cerebro-vascular disease results, in one embodiment, from hypoxia. In another embodiment, the disease results from any other cause capable of causing a cerebro-vascular=
disease. In another embodiment, the disease is cerebral thrombosis. In another embodiment, the cerebro-vascular disease is iscliemia.

[0081] In another embodiment, the neurological disorder is a beliavioral syndrome. In another embodiment, the neurological disorder is a neurological syndrome. In other embodiments, the

13 behavioral syndrome or neurological syndrome follows brain trauma, spinal cord injury, or anoxia.

[0082] In another embodiment, the neurological disorder is a peripheral nervous system disorder. In other embodiments, the peripheral neivous system disorder is a neuromuscular disorder, myasthenia gravis, or post-polio syndrome. In another embodiment, the peripheral nervous system disorder is any other peripheral nervous system disorder Icnown in the art.. In another embodiment, the neuromuscular disorder is a muscular dystrophy..

[0083] Each type of neurological disorder mentioned herein represents a separate embodiment of the present invention.

[0084] In another embodiment, the present invention provides a method of increasing or enhancing an ability of a brain cell or a neur=al cell of a subject to synthesize a neurotransmitter, comprising administering to the subject or.the brain cell or neural cell a uridine or a source thereof, thereby increasing or enhancing an ability of a brain cell of a subject to synthesize a neurotransmitter, [0085] In another embodiment, the present invention provides a method of increasing or enhancing an ability of a brain cell or a neural cell of'a subject to synthesize a neurotransmitter, comprising administering to the subject or the brain cell or neural cell a composition comprising a uridine or a source thereof and a choline, thereby increasing or enhancing an ability of a brain cell of a subject to synthesize a neurotransmitter.

[0086] In another embodiment, the present invention provides a method of increasing or enhancing an ability of a brain cell or a neural cell of a subject to repeatedly release an effective quantity of a neurotransrrritter into a synapse, comprising administering to the subject or the brain cell or neural cell with a uridine or a source thereof, thereby increasing or enhancing an ability of a brain cell or a neural cell of a subject to repeatedly release an effective quantity of' a neurotransmitter into a synapse.

[0087] In anothei- embodiment; theTpresent -iriveiition provides a method- o incieasing or_-'. - ..
enhancing an ability of a brain cell or a neural cell of a subject to repeatedly release an effective quantity of a neurotransmitter= into a synapse, comprising adniinistering to the subject or the brain cell or neural cell with a composition comprising a uridine or a source thereof' and a choline, thereby increasing or enhancing an ability of a brain cell or a neural cell of a subject to

14 repeatedly release an effective quantity of a neurotransmitter into a synapse.
As described herein, findings of the present invention show that uridine enliances the ability of neurons to synthesize neurotransmitters and repeatedly release them (Exaniple 7). The data in Example 15 further show that this effect of uridine is enhanced by inclusion of choline.

[0088] In one embodiment, the release which is enhanced by a method of the present invention occurs following a stimulation of the.neuron. In one embodiment, the release which is erilianeed occurs following a depolarization of the neuron. In one embodiment, the release which is enhanced is a basal neurotransmitter release. In one embodiment, the stimulation of the neuron comprises exposure of the neuron to a potassium ion. In another= embodiment, the stimulation of the neuron comprises any other means of neural stimulation Icnown in the art.
Methods for assessing neural stintulation and release of'neurotransmitters are well known in the art, and are described, for example, in Bewick GS, J Neurocytol.. 32: 473-87, 2003. Each possibility represents a separate embodiment of the present invention..

[0089] In another embodiment, the present invention provides a method of increasing a level of a neurotransmitter in a synapse, comprising contacting a neural cell adjacent to the synapse with a uridine or a source thereof, whereby the composition enhances synthesis of'a phospholipid or a precursor tliereof, thereby increasing a level of a neurotransmitter in a synapse.

[0090] In another embodiment, the present invention provides a method of increasing a level of a neurotransmitter in a synapse, comprising contacting a neural cell adjacent to the synapse with - a eomposition comprising a uridine or a source thereof and a cholinei-whereby the composition enhances synthesis of a phospholipid or a precursor thereof, thereby increasing a level of a neurotransmitter in a synapse.

[0091] In another embodiment, the present invention provides a method of increasing a sensitivity of a neuron to a stimulus, comprising contacting the neuron witll a ur-idine or a source thereof; whereby the composition er-Alances synthesis of a pliospholipid or a precursor thereof, thereby increasing a sensitivity of'a neuron to a stirnulus, [0092] In another embodiment, the present invention provides a method of' incr-easing a sensitivity of a neuron to a stimulus, comprising contacting the neuron with a composition comprising a uridine or a source thereof and a choline, whereby the composition enliances synthesis of a phospholipid or= a pr-ecursor tliereof, thereby increasing a sensitivity of a neuron to a stimulus..

[0093] In one embodiment, the neurotransmitter whose levels or activity, or release is affected by methods of the present invention is acetylcholine. In another embodiment, the neurotransmitter is dopamine.. In another embodiment, the neurotransmitter is serotonin. In another embodiment, the neurotransmitter= is 5-hydroxytryptaniine (5-I-iT). In another embodiment, the neurotransmitter is GABA. In another embodiment, the neurotransmitter is any other neurotransmitter Icnown in the art. Each type of netrrotransmitter represents a separate embodiment of the present invention..

[0094] In another embodiment, the present invention provides a method of stimulating a pr=oduction of a phosphatidylcholine (PC) by a brain cell or neural cell of a subject, comprising administering to the subject or brain cell or neural cell a uridine or a source thereof, thereby stimulating a production of a PC by a brain cell or neural cell.

[0095] In another embodiment, the present invention provides a metliod of stimulating a production of a phosphatidylcholine (PC) by a brain cell or neural cell of a subject, comprising administering to the subject or brain cell or neural cell a composition comprising a uridine or a source thereof and a choline, thereby stimulating a production of a PC by a brain cell or neural cell. As described herein, findings of the present invention show that uridine enhances synthesis of the PC precursor CDP-choline (Example 6). The data in Example 15 further show that this effect of uridine is enhanced by inclusion of clioline.

[0096] In another enibodiment, the present invention provides a method of stimulating or .......... enhancing an amount of- or a synthesis of a--component- of a cell membrane, comprising-contacting the cell with a uridine or a source thereof, thereby stimulating or enhancing an amount of or a synthesis of a cell membrane.

[0097] In another enibodiment, the present invention provides a method of stimulating or enliancing an amount of or a synthesis of a component of a cell menibrane, con-iprising 7 5 contacting the cell with a composition comprising a uridine or a source thereof and a clloline, fihereby stimulating or enhancing an amount of or a syntliesis of a cell membrane.

[0098] In anotller embodiment, the component whose synthesis is enhanced by a method of the present invention is a PC. In another embodiment, the component is a glycerophospholipid. In another embodiment, the coinponent is a phosphatidic acid. In another embodiment, the component is a PE. In another embodiment, the component is a lecitliin. In another embodiment, the component is a Pl. In another embodiment, the component is a PS, In other embodiments, the component is a 2-lysolecithin, a plasmalogen, a choline plasmalogen, a.
phosphatidylglycerol, a choline diphosphatidylglycerol, a choline sphingolipid, or a choline sphingomyelin. In another embodiment, the component is any other phospholipid known in the art.. Each type of phospholipid represents a separate embodiment of the present invention.

[0099] In another embodiment, the present invention provides a method of stimulating or enhancing an amount of or a synthesis of a phospholipid precursor, comprising contacting the cell with a uridine or a source thereof, thereby stimulating or enhancing an amount of or a synthesis of a phospholipid precursor.

[00100] In another embodiment, the present invention provides a method of stimulating or enhancing an amount of or a synthesis of a phospholipid precursor, comprising contacting the cell with a composition comprising a uridine or a source thereof and a choline, thereby stimulating or enliancing an amount of or a synthesis of a phospholipid precursor. ln another embodiment, the phospholipid precursor is CDP-choline (Example 6). In another embodiment, the phospholipid precursor is CTP.. In another embodiment, the phospholipid precursor is inositol. ln another embodiment, the phospholipid precursor is choline. In another embodiment, the phospliolipid precursor is glycerol.. In another embodiment, the phospholipid precursor is acetate.. In another embodiment, the phospholipid precursor is any other phospholipid precursor known in the art. Each phospholipid precursor represents a separate embodiment of the present invention.

-[00101 ]:_-... ..... In another-embodiment;-the present invention provides a metliod of stimulating or enhancing a production of a membrane of a brain cell or a neural cell of' a subject, compr-ising contacting the subject with a uridine or a source thereof, wher'eby the composition enhances synthesis of a phospholipid or a precursor thereof, thereby stimulating or enhancing a production of a membrane of a brain cell or a neural cell of a subject..

[00102] In another embodiment, the present invention provides a method of stimulating or enhancing a production of' a membrane of a brain cell or a neural cell of a subject, - c - oinprisiii~'contactingthe"subject with'a composition comprising'a uridine ora source thereof and a choline, whereby the composition enliances synthesis of a phospholipid or a precursor thereof, thereby stimulating or enhancing a production of a membrane af'a brain cell or- a neural cell of'a subject,.

[00103] In one embodiment, the membrane is a neurite membrane. In another embodiment, the membrane is a dendritic membrane. In another embodiment, the membrane is an axonal membrane. In another embodiment, the membrane is any otlier type of membrane known in the art. Each type of' membrune represents a separate embodiment of' the present invention.

[00104] In another embodiment, stimulating an aniount of or a synthesis of the cell mernbrane is accomplished by stimulating or enliancing a synthesis of a phospholipid (Example 6). In anotlier embodiment, stimulating or enhancing an amount of or a synthesis of a membrane of a neural cell is accomplished by stimulating or eriliancing a synthesis of a phospholipid precursor (Exarnple 6). In aiiother enibodiment, stimulating or enhancing a synthesis of a phospholipid or= a precursor thereof is partially responsible for stimulating an aniount of or a syntliesis of a membrane of a neural cell. In another embodiment, a composition of the present invention stimulates the amount of or a synthesis of a membrane without stimulating or enhancing a synthesis of a pllospholipid or a precursor ther=eof. Each possibility represents a separate embodiment of the present invention..

[00105] Methods for assessing production of' a brain cell membrane or neural cell membrane are well known in art. In another embodiment, membrane production is assessed by measuring the level of neurite outgrowth or branching (Example 9). In another embodiment, membrane production is assessed by measuring the level of a rrrembrane marker protein (Exanzple 8): In anotlier embodiment, membrane production is assessed by measuring synthesis ..................
...............................................................................
..................................................................
................. .........:.................... ...........................
.............. .... ..................... ................................
............. ................. .
of a membrane precursor. In another embodiment, membrane production is assessed by measuring amounts of membrane prior to and following uridine treatment. In another embodiment, membrane production is assessed by measuring biological indicators of membrane turnover. Indicators or cellular membrane turnover are well known in the art, and are described, for example, in Das KP et al, Neurotoxicol Teratol 26(3): 397-406, 2004,. Each method of assessing membrane production represents a separate embodiment of' the present invention.
..... . . . . .... . ... .. . . ......... . .... _........ .. ........ ... .
.....v. . . ...... .. .. ......... ......... .._ ........ ......... ....., .............
.[00106] I.n another einbodiment, the present invention provides a method of stimulating or enhancing an outgrowth of a neurite of a neural cell, comprising contacting the neural cell with a uridine or a source thereof, whereby the composition enhances synthesis of a phospholipid or a precursor thereof, thereby stimulating or enhancing an outgrowth of a neurite of a neural cellõ

[00107] In another embodiment, the present invention provides a metliod of stimulating or enhancing an outgr=owth of a neurite of a neural cell, comprising contacting the neural cell with a composition comprising a uridine or a source thereof' and a choline, whereby the composition enhances synthesis of a phospliolipid or a precursor thereof, thereby stimulating or enlrancing an outgrowth of a neurite of a neural cell. As described herein, findings of the present invention show that uridine enhances outgrowth and branching of neurites (Lxai7rpie 9).
The data in Example 15 further show that this effect of uridine is enhanced by inclusion of choline.

[00108] In another embodiment, the present invention provides a method of increasing a number of neurites of a neural cell, comprising contacting the neural cell with a uridine or a source thereof, wliereby the composition enhances synthesis of a phospholipid or a pr'ecursor thereof, thereby increasing a number of neurites of a neural cell.

[00109] In another embodinient, the present invention provides a method of increasing a number of neurites of a neural cell, comprising contacting the neural cell with a composition comprising a uridine or a source thereof and a choline, whereby the composition enhances syntliesis of a phospholipid or a precursor thereof, thereby increasing a number- of neurites of a neural cell.

[00110] In another embodiment, the present invention provides a method of stimulatiiig or enhancing a branching of a neurite of a neural cell, comprising contacting the neural cell with 20-~~1-.auridine ora souree thereof: thereby stimulating or enhancing-a branching of a neuriteof a neural cell.

[001111 In another embodiment, the present invention provides a method of stiniulating or enliancing a brancliing of a neurite of a neural cell, comprising contacting the neural cell with a composition comprising a uridine or a source tliereof and/or a choline, thereby stimulating or enhancing a branching of a neurite of a neural cell.

[OOl 1.2] ,,_ ,, . In one embodiment, the cell that is the target of inetliods of the present invention .. ..........
or is contacted in the methods is a neural cell. In anotlter embodiment, the cell is a brain cell,. In another embodiment, the cell is any cell in which synthesis of a membrane or a component thereof'is enhanced by contact with a composition conlprising a uridine and/or a choline. In another embodiment, the cell is any cell in which a neurological function is enhanced by contact with a compositioii comprising a uridine and/or a choline. Each possibility represents a separate embodiment of the present invention.

[00113] - In anotlier embodiment, the neural cell, neurite, or brain .cell of methods of the present invention is newly differentiated. In another embodiment, the cell is not newly differentiated. In one embodiment, "newly differentiated" r-efers to a neuron that has differentiated in the 24 hours prior to commencing administration of the uridine and/or choline.
In another embodinient, the neuron has differentiated in the 4$ hours prior to administration. In another embodiment, the neuron has differentiated in the 72 hours prior to administration. In another= embodiment, the neuron has differentiated in the 1 week prior to adrninistration. In another embodiment, "newly differentiated' refers to a neuron that cornpletes its differentiation following cornmencement of administration of the composition of. the present invention. Each possibility represents a separate embodiment of the present invention.

[00114] Methods of assessing neuronal differentiation are well lcnown in the art, and are described, for example, in Contestabile A et al (Neurochem Int, 45: 903-14, 2004). Each such method represents a separate embodiment of the present invention.

[00115] In another embodiment, the present invention provides a metliod of increasing a level of a cytidine in a tissue, plasma, or cell of a subject, coniprising administering a uridine or a source thereof to the subject, thereby increasing a level of a cytidine in a tissue, plasma, or cell.
....................... ...................................... ..
[00116] In another embodiment, the present invention provides a method of increasing a level of a cytidine in a tissue, plasma, or cell of a subject, comprising administering a composition comprising a uridine or a source thereof and a choline to the siibject, thereby increasing a level of a cytidine in a tissue, plasma, or cell. In another embodiment, the present invention provides a method of increasing a level of' a CTP in a tissue, plasma, or cell of a subject, comprising administering a composition of the present invention to the subject. In another- embodiment, the present invention provides a method of increasing a level of'a CDP-- chaline in a tissue; plasma;--or-cell of a subject;- comprising-administering a-composition of the present invention. ln anotller embodiment, the present invention provides a method of increasing a level of a derivative of a cytidine, a CTP, or a CDP-choline in a tissue, plasma, or 3 0 cell of a subject, comprising administering a composition of the present invention. In another embodiment, the present invention provides a method of increasing a level of a metabolite of a cytidine, a CTP, or a CDP-choline in a tissue, plasma, or cell of a subject, comprising administering a composition of the present invention. Each possibility represents a separate embodiment of the present invention.

[00117] In one embodiment, the tissue is a brain tissue. In one embodiment, the tissue is a neural tissue. In another embodiment, the tissue is a spinal tissue. In another embodiment, the tissue is any other tissue I<nown in the art.

[00118] In one embodiment, the cell is a brain cell. In one embodiment, the cell is a neural cell. In another embodiment, the cell is a spinal cell. In another embodiment, the cell is any other cell known in the art. Each possibility represents a separate embodiment of the present invention.

[00119] In one embodiment, the uridine that is administered in the present invention is a uridine-5'-monophosphate (UMP). In another embodiment, the uridine is a uridine-5'-diphosphate (UDP).. In anotlier embodiment, the uridine is a uridine-5'-triphosphate (UTP). In another embodiment, the uridine is UDP glucose. Each possibility represents a separate embodiment of the present invention..

[00120] In another embodiment, a uridine precursor is adininistered in methods of the present invention.. ln one embodiment, the uridine precursor that is administered is a cytidine-5'-monophosphate. In anotlier embodiment, the uridine precursor that is administered is a cytidine-5'-diphosphate (CDP). In another embodiment, the uridine precursor that is administered is a CDP-glucose. In another embodiment, the uridine precursor that is ................. ................ ............... ......... ........
......... ......................... ............. ................ ...
.................... ......................................
...............................................................................
.................................................. .........
administered is any pharmacologically acceptable uridine precursor, derivative or metabolite k.nown in the art.

[00121] In another embodiment, a uridine derivative is administered in methods of the present invention. The term "derivative" in one embodiment refers to a compound chemically related to uridine in such a way that uridine is converted-to the derivative in a subject's body. In another embodiment, "derivative" refers to a compound chemically related to uridine in such a way that the derivative is converted to uridine in a subject's body~ In one embodiment, the conversion occurs via one or more stable intermediates. In anotlier embodiment, the conversion occurs directly. Each possibility represents a separate embodiment of the present invention.
[00122] In another embodiment, a uridine metabolite is administered in methods of the present invention..

[001.23] In other embodiments, uridine-based compounds otlier than uridine itself serve as uridine sources or uridine precursors. These are, in some embodiments, uridine-rich food or dietary products like algae; salts of uridine like uridine phosphates, acylated uridine or the like.
In another embodiment, therapeutically or pharmacologically effective doses of acyl derivatives of uridine or mixtures thereof, e.g. those disclosed in U.S. Pat. No.
5,470,838, are administered.
[00124] In anotlier embodiment, the uridine sourse is cytidine-diphosphocholine (CDP-choline; citicholine). While citicholine contains choline as well as uridine in a 1:1 molar ratio, it is not, in one embodiment, sufficient to supply all the clioline required by the subject. Thus, in this embodiment, citicholine serves a the source of all the uridine and some of' tlle choline required by the subject.

[00125] In another embodiment, a salt of the uridine precursor, derivative or source is utilized in a method of the present invention. In one embodiment, the salt is UMP disodium (Examples 2-3).. In another embodiment, the salt is any other pharmacologically acceptable salt of a uridine precursor or derivative. In another embodiment, the composition that is administered comprises the salt of the uridine or precursor or derivative thereof as the sole active ingredient. Each uridine salt represents a separate embodiment of the present invention.
[00126] In another- embodiment, a mixture of two or more of the above uridine-related compounds is administered. Each type of uridine precursor, derivative, nietabolite, or source represents a separate embodiment of the present invention.
.. . ....... ....... .. .................. ............
[00127] The tenn "uridine" as used herein refers, in one embodiment, to any uridine phosphate, uridine precursor, uridine metabolite, uridine-based compound, or salt thereof mentioned above. In another embodiment, "uridine" refers to any uridine or related compound that is known in the art. Each possibility represents a separate embodiment of the present invention..

[00128] In one embodiment, the uridine, derivative, source, or precursor thereof is administered in methods of the present invention in a dosage of between about 20 milligrams (mg) and 50 grams (g) per day. In another- embodiment, the uridine or related compound is administered in a dosage of about 50 mg-30 g per day. In other embodiments, the dosage is about 75 mg-20 g; 100 mg-20 g; 100 mg-10 g; 200 mg-8 g; 400 mg-6 g; 600 n1g-4 g; 800 mg-3 g; 1-2.5 g; 1..5-2 g; 5 mg-5 g; or 5 mg-50 g per day. Each dosage or dosage range represents a separate embodiment of the present invention.

[00I29] ln one embodiment, the choline administered in methods of the present invention is a choline salt. In one embodiment, the salt is choline chloride. In another embodiment, the salt is choline bitartrate. In another embodiment, the salt is choline stearate. In other embodiments, the salt is choline alfoscerate, choline dehydrocholate., choline dihydrogen citrate, or choline salicylate. In another embodiment, the salt is any other choline salt known in the art. Each possibility represents a separate embodiment of'the present invention.

[00130] In another embodiment, the choline is a choline-based compound, e.g. a choline ester.

[00131] In another embodiment, the choline is a compound that dissociates to choline. In one embodiment, the compound is sphingomyelin. In another embodiment, the compound is an acylglycerophosphocholine. In another embodiment, the compound is lecitliin.
In another embodiment, the compound is lysolecithin. In another embodiment, the compound is glycerophosphatidylcholine. In another embodiment a mixture of two or more of the above choline-related compounds is administered., [00132] The term "choline" as used herein refers, in one embodiment, to any choline phosphate, choline precursor, choline metabolite, choline-based compound, or salt tlaereof mentioned above.. In another embodiment, "choline" refers to any choline or related compound that is known in the art. Each possibility represents a separate embodiment of the present invention..

[00133] In another embodiment, the choline or choline-related compound is administered in such a nzanner, and dosage that a choline level of at-least 20-30 nanomoles is attained in the subject's blood or brain.. In another embodiment, a choline level of 10-50 nanomoles is attained.
ln another embodiment, a choline level of 5-75 nanomoles is attained. In another embodiment, a choline level of 25-40 nanomoles is attained. In another embodiment, a choline level of 30-35 nanomoles is attained. Each possibility represents a separate embodiment of the present invention.

[00134] In another embodiment, the choline, derivative, source, or precursor thereof is administered in methods of the present invention in a dosage of 20 mg-50 g per day.. In other embodiments, the choline or related compound is administered in a dosage of' about 50 mg-30 g; 75 mg-20 g; 100 mg-20 g; 100 mg-10 g; 200 mg-8 g; 400 mg-6 g; 600 mg-4 g;
800 mg-3 g;

1-2.5 g; 1.5-2 g; 5 mg-5 g; or 5 mg-50 g per day. Each dosage range represents a separate embodiment of the present invention.

[00135] In anotlier= enlbodiment, a composition of the present invention is administer=ed at a dose that produces a desired effect in at least 10% of a population of treated patients. In another embodiment, the dose is that which produces the effect in at least 20%
of treated patients. In other embodiments, the effect is produced in at least 30%, in at least 40%, in at least 50%, in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the treated patients.
In another embodiment, the effect is produced in over 90% of the patients.
Each possibility represents a separate embodiment of the present invention.

[00136] In one embodiment, the subject of' methods of the present invention is a mammal_ In another embodiment, the subject is a human. In otlier- embodiments, lhe subject is a r=odent or a laboratory animal. In another embodiment, the subject is a male.
In another=
embodiment, the subject is a female. In another embodiment, the subject is any other type of subject known in the art. Each possibility represents a separate embodiment of the present invention..

[00137] In one embodiment, the terms "administering" or "administration" refer to bringing a subject in contact with a compound of the present invention. In other embodiments, administration comprises swallowing or imbibing the composition of the present invention. In another embodiment, the step of administration utilizes a pharmaceutical composition, a ?O nutritional supplementi. or the like. Each possibility _represents a separateembodiment. of.the present invention.

[00138] In one embodiment, administration is perfornied by the subject.= In another embodiment, administration is perforn-ied by a care provider=_ In another embodiment, administration is performed by a tlzird party.. Each type of adnlinistration represents a separate embodiment of the present invention.

[00139] In another embodiment, an additional therapeutic compound is administered to the subject as part of the method of the present invention. In another embodiment, the uridine or precursor, -derivative or source thereof is the sole active ingredient in the composition. In another embodiment, the uridine or precursor, derivative or source thereof and choline or precursor, derivative or source thereof are the sole active ingredients in the composition. Each possibility represents a separate embodiment of the present invention.

[00140] In one embodiment, the additional therapeutic compound is a drug that acts as a uridine phosphorylase inhibitor; e.g. benzyl barbiturate or derivatives thereof. In another embodiment, the compound is a drug that increases uridine availability. In another embodiment, the compound is a uridine secretion-inhibiting compound, e.g. dilazep or-hexobendine. In anotlier embodiment, the compound is a uridine renal transport competitors, e.g. L-uridine, L-2',3'-dideoxyuridine, and D-2',3'-dideoxyuridine. In another embodiment, the compound is a dnrg which acts in synergy with uridine in generation of a phospholipid. In another embodiment, the compound is a compound which competes with uridine in kidney clearance, e.g. L-uridine, L; 2',3'-dideoxyuridine, and D-2',3'-dideoxyuridine or mixtures tliereof as disclosed in U.S. Pat. Nos. 5,723,449 and 5,567,689. In another embodiment, the compound is any other compound that is beneficial to a subject..

[00141] In another embodiment, a method of the present invention causes one of the above effects by means of stimulating a P2Y receptor of a neural cell, neuron, or brain cell. In another embodiment, one of the above effects is caused partially as a result of stimulating a P2Y receptor of a neural cell or neuron. In another embodiment, one of the above effects is caused partially or fully by means of stimulating a P2Y receptor of another cell type. In another embodiment, one of the above effects is caused without stimulating a P2Y
receptor. Each possibility represents a separate embodiment of'the present invention.

[00142] In one embodiment, the stimulation of a P2Y receptor is mediated by uridine or a related compound in a composition of the present invention. In another=
embodiment, the .
......................................................
...............................................................................
........................ . .............. ..................... ..........
..................... .................. .................................
......... ........... .............. ...................
uridine is converted to a second compound that stimulates a P2Y receptor in the cell.. In one embodiment, the second compound is uridine-5'-triphosphate., In another embodiment, the second compound is any metabolic product luiown in the art of uridine or derivative or source thereof. Each compound represents a separate embodiment of the present invention. In other embodiments, the uridine or derivative or source thereof is converted into the second compound intracellularly or extracellularly.. In another embodiment, the uridine or der=ivative or source thereof is secr-eted from a cell after being converted into the second compound. In another --- embodiment,--the uridine-orderivative-or---source--thereof-contacts-a-different.cell-afterbeing-.-.--------secreted from the cell in which it was converted to the second compound,and stimulates a P2Y
receptor in the different cell. Each possibility represents a separate embodiment of the present invention.

[00143] P2Y receptors are a farnily of receptors known to be involved in platelet activation and other biological functions. They are reviewed in Mahaut-Smith MP et al, Platelets. 2004 15 :131-44, 2004.

[00144] In one embodiment, the P2Y receptor of the present invention is a P2Y2 receptor. In another= embodiment, the P2Y receptor is a P2Y4 receptor. In another embodiment, the P2Y receptor is a P2Y6 receptor. In another, embodiment, the P2Y receptor is any other P2Y
receptor known in the art. Each possibility represents a separate embodiment of the present invention.

[00145] In another embodiment, the P2Y receptor stimulates a second messenger.
In one embodiment, the second messenger is a G alpha protein. In another embodiment, the second messenger is a G alpha(q) protein. In another embodiment, the second messenger is cAMP. In anotlier embodiment, the second messenger is any other second messenger known in the art.
Second messengers, and their associated signaling pathways, are well known in the art, and are described, for example, in Ferguson S, Pharm Rev 53: 1-24, 2001; Huang E et al, Ann Rev Biochem 72: 609-642, 2003; and Blitterswijlc W et al, Biochem.. .1. 369: 199-211, 2003. Each second messenger represents a separate embodiment of the present invention..

[00146] In other embodiments, the second messenger stimulates a phospholipase C
enzyme, modulates intracellular calcium levels, or increases protein kinase C
activity. In one embodiment, one or more of the above pathways stimulates membrane production.
In another embodiment; - the second messenger modulates _ or..stimulates .
another....cel.lularpathway-that_ stimulates membrane production. Each possibility represents a separate embodiment of the present invention.

[00147] In one embodiment, ur-idine or a related compound in a composition of the present invention stimulates a receptor other than a P2Y receptor'.

[00148] In another embodiinent of the methods of the present invention, the uridine and/or choline is carried in the subjects' bloodstream to the subject's brain cell or neural cell. In another embodiment, the substance is carried by diffusion to the subject's brain cell or neural cell. In another embodiment, the substance is carried by active transport to the subject's brain cell or neural cell. In another embodiment, the substance is administered to the subject in such a way that it directly contacts the subject's brain cell or neural cell. Each possibility represents a separate embodiment of the present invention..

[00149] In one embodiment, "pharrnaceutical composition" refers to a tllerapeutically effective aniount of the active ingredients, i.e., the uridine and/or choline, together with a pharmaceutically acceptable carrier or diluent. "Tlierapeutically effective amount," in another embodiment, refers to that amount which provides a therapeutic effect for a given condition and administration regimen.

[00150] In other embodiments, the pharmaceutical composition containing the uridine and/or choline is administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, iiitradermall,y, subcutaneously, intraperitonealy, intraventricularly, intracranially, intravaginally or intratumorally.

[00151] In another embodiment, the pliarmaceutical compositions are administered orally, and thus is formulated in, a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include, for example, tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention, the composition containing the uridine and clloline is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprises a hard gelating capsule, in addition to the active compounds and the inert carrier or diluent.

[00152] In another embodiment, the pharmaceutical compositions are administered by -20 -intravenous, intraarterial, or intramuscular -injection .-of._ a_..liquid_ prepar.ation, Suitable .liquid ..........
formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus forniulated in a fornl suitable for intravenous administration. In anotlier embodirnent, the pharmaceutical compositions are administered intraarterially, and are thus formulated in a form suitable for intraarterial administration.. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.

[00153] Further, in another embodiment, the pbarmaceutical compositions are administered as a suppository, for= exaniple a rectal suppository or a uretlual suppository. In another embodiment, the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In anotlier embodiment, the pellet provides for controlled release of uridine and/or choline over a period of time.

[00154] Pharmaceutically acceptable carriers or diluents are well l:nown to those skilled in the art. The carrier or diluent is, in one embodiment, a solid carrier or diluent for solid formulations, a liquid carrier= or diluent for liquid formulations, or mixtures thereof.

[00155] Solid carriers/diluents iriclude, in other embodiments, a gum, a starch '(e.g, corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g.
polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[00156] For liquid formulations, pharinaceutically acceptable carriers are, in other embodiments, aqueous or non-aqueous solutions, suspensions, emulsions or oils.
Non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoliolic/aqueous solutions, emulsions or suspensions, including saline and buffered media.. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[00157] In another embodiment, the compositions ftrrther conlprise binders (e.g. acacia, cornstarch, gelatin, carboiner, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl nieth,yl cellulose, povidone), disintegrating s(e..g..
cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodiuni, crospovidone, guar guin, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, ....20....... .additives..such.as .albumin..or..gelatin .toprevent.absorption..to.surfaces,detergents. (e.g.,..Tween ................
20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e..g. sodium lauryl sulfate), permeation enhancers, solubilizers (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.
hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing s(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweetners (e.g.
aspartame, citr=ic acid), preservatives (e..g.., Thimerosal, benzyl alcohol, parabens), lubricants (e.g.
stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e..g,. colloidal .......... _-_....__..........
.._...__._.....__....._..._._......._................. ..._...........
.............................._.._..._...........__......._............. -_._...._.......__._.......... ................ .......__._..... _......
........... ..... _......... .... ............. ......................
..............
silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium laury) sulfate), polymer coatings (e.g., poloxamers or poloxanlines), coating and film forming s (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[00158] In another embodiment, the pharmaceutical compositions provided herein are controlled release compositions, i.e. compositions in which the uridine and/or choline is released over a period of'time after administration. Controlled or sustained release compositions include forniulation in lipophilic depots (e.g. fatty acids, waxes, oils). In anotlier embodiment, the composition is an immediate release composition, i.e. a composition in which all of the uridine and/or choline is released immediately after administration.

[00159] In anotlier embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the composition is administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (see Langer', supra; Sefton, CRC Crit, Ref.
Biomed.. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. .1..
Med. 321:574 (1989). In another embodiment, polymeric materials are used. In another embodiment, a controlled release system is placed in proxirnity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systeniic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol., 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).

[00160] The preparation of pharmaceutical compositions which contain an active component is well understood in the art, for example by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients which are phar7naceutically acceptable and compatible with the -active ingredient. For oral administration, .
...............................................................................
...............................................................................
...............................................................................
...............................................................................
.......................................
the uridine and/or choline or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are nlixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable fomis for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. For parenteral administration, the uridine and/or choline or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, susperision, or= emulsion, if desired with the substances customary and suitable _........for this purpose;..for-example,.solubilizersor-other...._ [00161] An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Phannaceutically acceptable salts include the acid addition salts (formed with the free aniino groups of the polypeptide or antibody molecule), whieh are formed with inorganic acids such as, for example, hydrochloric or phosplioric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed from the free carboxyl groups can also be derived from inorganic bases sucli as, for example, sodium, potassium, annnionium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylaniine, 2-ethylamino ethanol, histidine, procaine, and the like.

[00162] For use in medicine, the salts of the uridine and/or choline are pharmaceutically acceptable salts. nther salts are, in one embodiment, useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.
Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the conipound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, funiaric acid, maleic acid, succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

EXPERIMENTAL DETAILS SECTION

Measurement Of Cytidine Sy HPLC Without Interference From Tvrosine MATERIALS AND METHODS

Santple preparatioir [00163] 1-milliliter (mL) samples of heparinized plasma were spilced with I g fluoro-...................................... ...................
......................
uridine for use as an internal standard, then deproteinized by adding methanol (5 mL,). Samples were centrifuged, lyophilized, reconstituted in 5 mL of 0.25 N anlmonium acetate (pH 8.8), then irnmediately purified over boronate affinity columns.

Boi-oatate affr-tity colrunns [00164] All steps were perfonned at 4 C.. Boronate affinity colurnns (Affigel-601, Bio-Rad) were primed with two 5-mL, ammonium acetate washes, saniples were applied, and columns were washed again with ammonium acetate, after which the nucleosides were eluted ........ .......................... .....
._.._..................._..._........................... ....... --....
..........._.................... ........ ...... .......__... .............
.... _..___............. ................ ........
.._..........._.........._........................----._... ..............
...... ..... .... ............ _.__.... _................ _...... ......
with 0.1 N formic acid (7 mL). Eluates were lyophilized, then reconstituted in 100 L water for IiPLC analysis. Bor=onate affinity colunins bind many biological niolecules, including the nucleotide bases adenosine, cytidine, guanosine, thymidine, and uridine.

HPLC

[00165] I-IPLC analysis was performed using a Beckman System Gold apparatus (Beclcman Instruments) equipped with a Rainin Dynamax Microsorb Cl8 column (3 [rm packing; 4.6 X 100 mm) at room temperature. The standard HPLC method is described in Lopez-Coviella et al, (J. Neurochem 65: 889-894, 1995). For= modified HPLC, an isocratic elution buffer was used containing 0.004 N potassiuni phosphate buffer (pH
5.8) and 0.1%
methariol instead of formic acid, flowing at l mL/min and heated to 35 .

RESULTS
[00166] A standard HPLC method for measuring nucleosides yields separate peaks for I0 uridine and cytidine; however, a coincidence of the cytidine and tyrosine peaks precludes accurate rneasuremeitt of cytidine levels, as shown for human plasma samples (Figure 1).
Tyrosine is present in many biological fluids, e.g., plasma or cerebrospinal fluid (CSF).. In the present Exainple, a modified HPLC method was used which distinguished cytidine and tyrosine peaks, perTnitting accurate measurement of cytidine levels (Figure 2).

Oral administration of UMP increases plasma uridine levels in humans MATERIALS AND EXPERIMENTAL METHODS

Study desigrr -[00167] Eight-healtlry subjects (5 malei 3 female;-27-67-years old) were instructed to fast overnight and given sequentially increasing doses (500, 1000, and 2000 mg) of disodium UMP
(Numico, Wageningen, NL) at 7 - 8 AM on each of three days, sepaiated by at least a three-day washout period. All subjects were given lunch. Blood samples were drawn over an eight-hour period into heparinized tubes.. Plasma was treated with methanol to precipitate protein, extracted with chloroforin, and an aliquot of the aqueous layer lyophilized, dissolved in water, and assayed by HPLC with UV detection.

.....
........... tatisticnl ana yses--................. ....... [00168] Statistical analyses were carried out with SPSS 12-0. Data were represented as mean SEM. Unpaired Student's t test, one-way analysis of variance (ANOVA), ANOVA with repeated measures, two-way ANOVA were used to assess the statistical effects, as described in detail in the context_ Tukey's HSD post hoc analyses were conducted when appropriate.. The significance level was set atp <0.05.

RESULTS
[00169] Subjects were administered 500, 1000, or 2000 mg UMP orally, and blood uridine levels were measured at baseline and 1, 2, 4 and 8 hours (hr) following dosing.. Plasma uridine levels were assayed as described in Example 1. Plasma uridine levels increased in response to oral UMP in a dose-dependent fashion, then returned to baseline levels within 8 hr (Figure 3).

Oral administration of uridine or UMP increases plasma uridine levels in gerbils MATERIALS AND EXPERIMENTAL METHODS

E'xperimental design [00170] Groups of eight to nine male gerbils (60-80 g) were fasted overrright, administered (a) uridine (Sigma, St. Louis, MO; 250 nig/Icg body weight) (Figure 4) or disodium UMP (l mmol/kg body weight, a dose equivalent to 250 mg/lcg uridine by gavage) (Figure 5) and sacrificed by decapitation under Telazol anesthesia one hour later. For Figure 6, gerbils were fed chow (Harlan Teklad, Madison, WI) ad lib containing either 0.
0..1, 0,5 or 2.5%
L1MP by weight for 4 weeks, fasted overnight, then sacrificed one hour after consumption of a . last rneal of the sanie composition:.. Blood-collected. from .the _neck...was. coIlected...into..tubes containing EDTA and was treated as described above for Example 2.

RESULTS
[00171] To asceitain whether oral administration of uridine can raise plasma uridine levels, gerbils were fed by gavage 250 mg/lcg cytidine or uridine. 60 minutes (min) later, plasma uridine levels were assessed by the method described in Exaniple 1.
Both dietary cytidine and uridine increased plasma uridine levels by a statistically significantly margin ._........
..... ...... ........... ......... __-...,_..._..._..-............_..._..........._-........ _.. _......... _...__....-_--......_....-.-..---......__.....-...._.-. ._.....__...... -_._.__.._.....
...... .............
relative to a control group that was fed cliow not containing cytidine or uridine, both dietary uridine resulted in plasma uridine levels approximately 3-fold higher than dietary cytidine (Figure 4)., [00172] In a separate experiment to assess the time course of the increase in plasma uridine levels, gerbils were administered eitlier water or I millimole (mrnol) UMP per kilogram (kg) body weight, were sacrificed at various time points in the following 60 min, and plasma uridine levels were assessed.. Plasma uridine levels increased within 10 min of administration, reaching peak levels by 30 min (Figure 5).

[00173] - In another experiment, gerbils were fed either a control diet or a diet containing 0.1%, 0.5%, or 2.5% UMP. One hour later, plasma uridine levels were assessed.
As depicted in Figuie 6, plasma uridine levels increased in response to dietary UMP in a dose-dependent manner. These results indicate that orally administered uridine is absorbed into the bloodstream..

Oral administration of uridine or UMP increases brain uridine levels in gerbils MATERIALS AND METHODS

Gerbil brain tissue preparation [00174] Brains were quickly removed from the skull after decapitation, frozen on dry ice, homogenized in 80% metlianol, centrifuged, lyophilized and analyzed as described for Faample 3., RLSULTS
[00175] To ascertain whether oral administration of uiidine can raise brain uridine levels, brains of the gerbils from the first experiment in Example 3 were honiogenized, and the uridine levels were assayed_- Oral- administration of cytidine resulted in a two-fold increase in brain uridine levels, and oral administration of uridine resulted in a greater than a three-fold increase in brain uridine levels, relative to the control animals (Figure 7).
All differences between groups were statistically significant., [00176] In order to assess the time course of the increase in plasma uridine levels, brain uridine levels were assessed in the gerbils from the second experiment of Example 3. Brain uridine levels increased within 10 min of uridine administration, reaching peak levels within 30 . 30 min; similar-to-the-results observed with-plasma-uridine-levels (Figure-8): These-results-indicate-that orally administered uridine is efficiently transported into the brain.

Uridine is Readilv Converted to Cvtidine in the Brain [00177] In a separate experiment, gerbils were orally administered 250 mg/kg body weight uridine, and 60 min later plasma and brain levels of cytidine and uridine were assessed.
The fold-increases relative to control animals was calculated and are depicted in Figure 9A
(plasma) and Figure 9B (brain). In each case, the fold-increase of cytidine was normalized to the fold increase of uridine, which was arbitrarily set as 100%. These results indicate that (a) uridine in the bloodstreani is transported into the brain and (b) uridine is metabolically processed differently in the brain than in.plasma; specifically, it is more efficiently converted to cytidine than in plasma.

Uridine Incrcases Levels of the Phospholipid Precursor CDP-Choline in the Brain and in a Neural Cell Line METHODS
Experimeirtal desib n [00178] Data was pooled from three experiments, with group sizes ranging from 5 to 16 animals. Male gerbils (60 - 80 g) were given UMP (1 mmole/kg body weight) by gavage and sacrificed at the indicated times. After brain homogenization, protein precipitation, and lyophilization as described for Example 4, samples were analyzed by HPLC-UV.

...
Assessment of CDP=..choline-levels .............................._................
........................................_..................._..................
.................._.........................................

[00179] Brain tissue or cells was dissolved in methanol/chloroforrn (1:2 vol/vol), centrifuged, and the aqueous phase was dried under vacuum, resuspended in 100-200 L water and separated by HPLC on an ion-exchange column (Alltech Hypersil APS-2, 5 M, 250 x 4.6 mm).. CDP-choline was eluted with a linear gradient of' NaHzPO4 buffers A
(1.75 mM
NaH2PO4, pH 2.9) and B (500 mM, pH 4.5), which allowed resolution of CDP-choline from closely co-eluting substances such as UMP over 40 min. The retention time for CDP-choline ........... ,,was 9.5 min. Individual nucleot.ide_P. eaks_were detected by UV
absorption at 380 nm, and were identified by comparison with the positions of autlientic standards, as well as by the addition of nucleotide standards to selected samples..

PCIl cells [00180] PC12 cells were maintained in Minimal Essential Medium (MEM;
Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37 C.
Experimental incubations were for 2 or 4 days in medium containing 50 ng/ml mouse 2.5 S(2..5 subunit) NGF and 1% FBS, with or without test compounds. NGF and FBS were obtained from Invitrogen.

RESULTS
[00181] In order to assess the effect of orally administered uridine on levels of phospholipid precursors in the brain, brains of the gerbils from the second experiment of Example 3 were assayed for levels of CDP-choline, a key intermediate in phospholipid biosynthesis via the Kennedy pathway. Levels of CDP-choline rose significantly in a linear fashion (regression analysis, r= 0.98, p < 0.02) for 30 min after administration of UMP (Figure 10).

[00182] To directly demonstrate conversion of uridine to CDP-choline in neural cells, PC
12 cells, a cell line capable of differentiation into neural cells, were treated with uridine, and intracellular levels of CDP-choline were measured. Uridine treatment resulted in a statistically significant increase in CDP-choline levels after 50 minutes (Figure 11). These results sliow that, after transport to the brain, uridine is converted to phospholipid precursors such as CDP-choline, perhaps via the intermediate CTP, and therefore augrnents cognitive function by iiicreasing synthesis of phospholipid precursors in brain cells.

Oral Administration of UMP Increases Neurotransmitter Release in Brains of Aaed Rats METHODS
Animals and dietary LIMP supplementation [00183] Male middle aged Fischer 344 rats, 22-24 months old at the time of doing 30" rriicrodialysi"s; "were obtairied froii~ Natioiial Iiistitute oii"Agriig---(Har an' prague=Dawley, Indianapolis, IN). Rats were housed individually under standard husbandry conditions and exposed to 12 hr light/ dark cycle with food and water provided ad libitun7,.
Each rat consumed approximately 500 mg/kg/day of UMP=2Na (LD50 by i,p. of uridine is about 4.3 g/Kg).

[00184] Rats were acclimated to the animal facility for more than 7 days before fed a control laboratory diet (Teklad Cilobal 16% protein rodent diet, TD.00217, Harlan Teldad, Madison, WI), or this diet fortified with i.IMP=2Na+ (2.5%, TD.03398, UMP=2Na+; Nurrrico Research, the Netherlands) for, 6 weeks.

[00185] Rats were not fed with the research diet until at least 7 days later after their arrival. They were weighed at the time of beginning feeding (t=0), as well as 1, 2, 4, 6 weeks later'. At time 0, rats were randomly assigned into two groups. There were no significant differences of body weight between groups (F1,1 1= 3.03, p> 0.05); average weight was 455 5 (N = 13 rats). Repeated measures with weeks as within-subjects factor showed feeding time (0, 1, 2, 4, 6 weeks) significantly clianged body weight (F4,44 = 2.65, p< 0.05), while neitlier UMP-diet (vs. control) nor UMPxtime interaction affected body weight (F1.11 =
0.01, F4,44 = 1.25, respectively; all p > 0..05)..

[00186] The experiment described in this Example was performed twice, each time witli 7 control rats and 9 rats administered the UMP diet..

Ci1L't7tiClYls flI1CI sOl111iOltS
[00187] . Dopaniine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and 3,4-dihydroxybenzoic acid (DHBA; inten--al standard) were purchased from Sigma (St. Louis, MO) and were dissolved in HC1O4 (0.1 M) to make 1 mM stock solutions, and aliquots were kept at -80 C.
--Ketamine hydrochloride (100 mg/ml) was purchased from FortDodge Animal Health (Fort Dorge, IA). Xylazine (20 mg/ml) originated from Phoenix Scientific, Inc. (St.
Joseph, MO).
[00188] Ringer solution consisted ofNaCl 147, KCI 2.7, CaC12 1.2 and MgCl.2 0.85 mM.
For high potassium solution, KCI was increased to 80 mM, with NaCI decr=eased to 69..7 mM to rriaintain osmolarity. All solutions were made from doubly distilled deionized water and filtered by Steriflip'r' (Millipore, Bedford, MA),.

Irl vivo...llricrodirr/ysis .......... ...................
._..._._._........................... ............
.._....._.............................. .... ................
_._.......__............................... ..........................
...............,............... ...............
..........__........_.............. .... ._......
[00189] Rats were anesthetized with a mixture of ketaniine and xylazine (80 and 10 mg/Kg of body weight, respectively, intraperitoneally), and were placed in a Kopf stereotaxic frame.. All surgical instruments were sterilized by a hot bead dry sterilizer or 70% ethanol. A
small hole was drilled into the skull by a 2-mm trephine bone drill. CMA/11 14/04 Cupr probe (O.D. 0.24 mm, 4inm membrane, 6,000 Da, CMA microdialysis, Sweden) was implanted into the right striatum (AP =+0.5, ML =-3_0 from Bregma, DV =-7.3 mm from Dura, as described in Paxinos G et al, The Rat Brain in Stereotaxic Coordinates, 2"d ed., Academic Press, San Diego) with incisor bar set at -5.0 mm. Probes were secured permanently in position using dental cement and three anchor screws to the skull. After= surgery, rats were injected intraperitoneally with saline (5 ml/kg) and kept on a heating pad maintaining body temperature at 37 C until awaking.

[00190] The freely moving rat was perfused in a circular bowl on a rotating platform obviating the need for a liquid swivel (see Wang L et al, Neurochem Int 42:
465-70, 2003), and was habituated to the environment on the first day after surgery. Experiments were performed approximately 48 hr after the surgery, a1id were carried out between 10:00 am to 4:00 pm.
Ringer's solution was perfiised continuously using Fluorinatedethylenepropylene (FEP) Resin tubing and a gas-tight syringe (Exmire type 1, CMA), at a constant rate of 1.5 l/min by a microinfusion pump (CMA/100). Dialysates were collected at 15-min intervals. 5 l of antioxidant mixture, consisting of 0.2 M HCIQ4 and 0.1 mM EDTA, was added to the sampling vial prior to collection to protect dopamine and its metabolites. The sainples within the frrst 60 min were discarded from analysis. Subsequently, 3 consecutive sessions of samples were collected. Except for the last session (1.5 llrs, 6 samples), the others were collected for 1 lir (4 samples), The order was as follows: session 1(aCSF), 2 (High IC+), .3 (aCSF).
All samples were collected on crushed ice, instantly frozen and kept at -80 C until HPLC
analysis.

Braiii rlissectiotr for the proteins aiul ionoarnines ..... .. . .. . ....... ............. . .................... .. . ... ........
... . . .
[00191] After microdialysis experiments, rats were anesthetized with lcetarnine and xylazine (80 and 10 mg/Kg, i.p.). A black ii~k was pushed thr=ough the probe to stain the tissue ar-ound the probe. Rats were decapitated with a guillotine. Brains were quickly dissected on a chilled dissection board. The left striatum was snap-frozen in an Eppendorf tube placed in liquid nitrogen for future protein assays. The right striatum was further dissected, and the position of probe was determined by visual observation. Data were not included if probe was found not within the striatum..

[00192] An additional group of rats (20 months old; n = 6 for both control and UMP) were fed for 6 weeks. No microdialysis was carried out in these rats. Stt-iata (both left and riglit) were collected as above to determine tissue levels of dopamine and its metabolites.

Extraction of tisstte dopainine smnples [00193] The striatum were weighed and homogenized in an Eppendorf tube on ice for 1 min with 1 ml of H20 containing 0..1 M HC1O4 and I M EDTA. After vortexing for 10 seconds, an aliquot was used for Bicinchoninic Acid (Sigma, St.. Louis, MO) protein assay. The liomogenafies were then filtered with Ultrafree-MC centrifugal filter units (Millipore, 14,000 rpm/15 min/4 C). A 1: 10 dilution was made before the aqueous layer was subjected to HPLC.
DHBA was added to the samples prior to homogenization as the internal standard.
Concentrations of dopamine and its metabolites were determined by IIPLC, and values from the tliree repeated measures were averaged and nomialized to the amount of protein per sample.
Analysrs of dopantine and ntetabolite.s [00194] DA and metabolites in dialysates and tissue samples were determined using an ESA Coulochem 11 5100A detector (E.i = -175 mV; E2 = +325 rnV; Egõzrd = 350 mV) with an ESA Microdialysis Cell (model 5014B, ESA, North Chelmsford, MA). The mobile phase (MD-TM, ESA) consisted of 75 mM NaN--)P04, 1.7mM I-octanesulfonic acid, 100 l/L
Triethylarnine, 25 M EDTA, 10% acetonitrile, pH 3Ø The flow rate was 0.4 mL/min. The column (ESA MD 150, 3 x 150 inm, 3 m, 120 A) was kept in a 40 C column oven.. Samples were injected to HPLC by an Alltech 580 autosampler (Alltech, Deerfield, IL) and maintained to 4 C with a cooling tray during analysis.. Data were captured by Alltech A1lChromT" data system, and analyzed with AllChrom plusr" software.. A timeline program, which could change the detection gain during sample separation and detection, was used to make it possible to get low DA and high metabolites concentration data in dialysate through one injection.
...............................................................................
.......................... .. . . ... ...... .....................
..................................... .............. ......................
........... ....................... .............................. .
Data analysis [00195] Data were represented according to sampling time of six to nine measurements each point (means standard error of measurement [S.E.M.]).. Basal values of DA and major metabolites were determined based on the averages of the first four consecutive samples prior to Ki* stimulation (mean value in the dialysate was 10..2 0.4 nM, n = 22), which was assigned a value of 100%. Statistics were performed using two-way ANOVA
(Treatmentxtime) with Turkey's HSD post hoc test.. One-way ANOVA was used to compare the differences among the ........_. ..
_ _pom.._t: A p value of > 0::05 was used to assess statistical signi .icance.
three groups iri ead .fi tirne_ . .
Basal levels of dopamine were homogeneous between the two replicated experiments and were therefore pooled into the corresponding groups (Fi,z0 = 3.99, p> 0.05). Basal DA levels in the dialysates were stable after I hr equilibration, in the four consecutive samples prior to K+

stimulation (F3,57 = 0.15, p > 0.05; one-way ANOVA with repeated measures -using sampling time (0, 15, 30, 45 min) as within-subjects factor).

[00196] Similar to basal DA levels, basal levels of DOPAC and HVA in the dialysates were 612 14 and 369 7 nM (n = 22 rats), and were stable (F3,57 = 1 ~06, F3,57 = 0.84, respectively; in each case, p > 0.05). There were no effects of UMP treatment on the basal DOPAC and HVA levels (Control vs. UMP-1 week vs. UMP-6 weeks; F2,19 = 0.27, F2,19 =
0.03,.respectively; in each case, p> 0.05).

RESULTS
[00197] In order to assess the effect of orally administered uridine metabolites on neurotransmitter release in the brain, aged rats maintained in a restricted environment consumed for 1 or 6 weeks either a control diet or a diet supplemented with 2.5% UMP., UMP
supplernentation did not affect basal DA levels in the dialysate among treatment groups (control vs. LJMP- I week vs. UMP- 6 weeks; F2,19 = 0.98).. DA concentration in the dialysate was 10.2 0..4 nM (n = 22 rats)..

[00198] The effect of dietary UMP supplementation on K.{*-evoked striatal DA
release (following perfusion with the high-K+ solution) is depicted in Figure 12A. A
statistically significant difference (F~1,266 = 3.36) was found in DA levels in the dialysates among the control, UMP- I week, and UMP- 6 weeks treatment groups. Posi hoc multiple comparisons revealed a significant difference between control and UMP-6 weeks' groups.
Data were further divided into tluee sections (before, K.+-evoked and after), which also revealed a significant enhancement of K+-evoked DA release between control and UMP-6 weeks' groups, from 283 9% to 341 f21%(Figure 1213). The UMP-1 week group also exhibited increased DA
release (316 t 15%) relative to the control group; however, this increase was not significant.

[00199] Next, the effect of'dietary UMP supplementation on the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) in striatal dialysate was assessed.. K+-depolarization, significantly deceased DOPAC (Figure 13A) and HVA (Figure 13B) to 65 4% and 51 4% compared to baseline levels in all groups (F~,9$ =
51.90, F2,95 =
92.74, respectively; all p < 0.01). Tliere were no differences in K*-decreased DOPAC and HVA
levels among treatment groups (F~1,266 = 1.01, F-1,1-66 = 1.20, respectively).
Changing the solution from high K+ back to normal Ringer's solution at 105 min increased both DOPAC
and HVA

levels in the dialysate, with maximum levels attained at 30 min after changing (DOPAC, 169 9%; HVA, 149 5%). However, no significant differences were found among the three groups..
[00200] In addition, dietary UMP was shown to increase the basal release of the neurotransmitter acetylcholine from neurons in the corpus striatum (Figure 14).

[00201] These results show that (a) orally administered uridine improves neurotransmitter release in the brain; (b) uridine-mediated augmentation of brain function is a multi-species phenomenon, not limited to gerbils; and (c) augmentation of brain function by uridine occurs biologically relevant animal model for age-impaired cognitive dysfunction.

Oral Administration of UTP Increases Levels of NF-70 and NF-M in Brains of Aged Rats METHODS
Data analysis [00202] Data were represented according to UMP treatment of six to sixteen measurements each group (means S.E,M.). One-way ANOVA with Turkey's HSD post hoc tests were used to compare the difference among the treatments the Newman-K.euls multiple range test was used for the data in Figure 16.

H'estern blotting ...................... ................. ................._............... .
............................ .... ............ ............. .. ..
............. .. . .........................................
................................... ............ ...... ......................
.....
[00203] Striatal tissues were placed in Eppendorf tubes containing 200 gl lysis buffer (60 mM Tris-HCI, 4% SDS, 20% glycerol, 1 mM dithiotlireitol, I mM. AEBSF, 8 gM
aprotinin, 500 M bestatin, 15 M E64, 200 M leupeptin, 10 M pepstatin A)_ The saniples were sonicated, boiled (10 min), and centrifuged (14,000 g for I min at room temperature). The supernatant fluid was transferred to a clean tube, and total protein content was determined using the Bicinchoninic Acid assay (Sigma, St. Louis, MO).

[.OQ204.]...._.._........ .....__E.qual._amounts protein/lane)wereloaded forsodium dodecyl sulfate-polyacrylamide gel electrophor=esis (4-15% SDS PAGE; Bio-Rad, Hercules, CA). Prior to gel electrophoresis, bromphenol blue solution (0.07%) was added to each sample.. Proteins were separated, transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore), and blocked with 5% bovine serum albumin (Tris-buffered saline/0.15% Tween 20) for I h. After 3 10 min rinses in Tris-buffered saline (TBST), blots were incubated in TBST

with various antibodies against the proteins of interest, including NF-70, NF-M (1: 2000, 1:
5000, respectively; Calbiochem, La Jolla, CA) at 4 C overnight on an orbital shaker. Protein-antibody complexes were detected and visualized using the ECL system (Amersham, Piscataway, NJ) and Kodalc X-AR film, respectively, as suggested by the manufacturer. Films were digitized using a Supervista S-12 scanner with a transparency adapter (UMAX
Technologies, Freemont, CA). Analysis was performed using the public domain NIH Image prograni (NII-I V.1..61).

RCSULTS
[00205] In order to assess whether increasing uridine levels can augment the production of new niembrane in the brain, levels of neurofilament-70 (NF-70) and neurofilament-M (NF-M), biomarkers of neurite outgrowth, were assessed in the brains of the rats fi=-om the experiment described in Example 7... As shown in Figure 15, UMP dietary supplementation for 6 weeks significantly increased the levels of NF-70 (Figure 15A) and NF-M
(Figure 15B), to 182 25% (F2,31 = 6..01, p < 0.05) and 221 34% (F2,21 = 8.86, p < 0.01) of control values, respectively. Consurnption of a UMP diet for I week did not incr=ease the levels of these two proteins compared to control gioup in a statistically significant manner.
Levels of NF-70 and NF-M in striatum increased to 204 36% and 221 34% of control values, respectively.

Oral Administration of Uridine or UTP Increases Neurite Outgrowth and Branching and ........ .. ... . ...
...........1... ....
Levels of NF-70 and NF-M .'n PC 2 cells METHODS
Data analysis [00206] Data are presented as mean +/- S.E.M. Analysis of variance (ANOVA) was used to determine differences between groups (significance level, p<0_05).
When differences were detected, means were separated using the Newman-Keuls multiple range test.
... ............................... ................ ... ....... ......
....... ....................................... . ...
........... ........_.............. ............. ._._..,....... ............
......... .
Nerrrite outgroivtli studies [00207) PC12 cells were sparsely plated on collagen-coated 60 mm culture dishes in MEM containing 1% fetal bovine serum. Experimental groups were as follows:
uridine, uridine triphosphate, cytidine, reactive blue 2, suramin and PPADS (Sigma, St. Louis, MO). All treatments were performed 24 h after plating. At the end of'the treatment period, images were obtained with a phase-contrast Zeiss Axioplan 2 microscope, using OpenLab software. Six digital images were captured for each dish, for a total of 18 to 24 images per treatment group.
Approximately 300 cells were quantified for each treatment group for each experiment..
Experiments were performed in triplicate. Quantification of neurites, including neur=ite branching and neurite length, was performed by one more researchers blinded to experimental groups. Neurite length was measured using the public domain NIH software "Image J."
Processes longer than the diameter of the cell body were counted as neurites.
Only process-bearing cells were analyzed.

Detection of irztrncellular UTP amid CTP
[00208] Levels of.' intracellular UTP and CTP were aiialyzed by HPLC as described for Example 6, except that 5 mM NaH2POa, pH 2.65 was used as buffer A..

RiCSULTS
[00209] The effect of uridine treatment (10 - 200 M) on NGF-induced neurite outgrowth was next tested. In the absence of NGF, PC12 cells did not sprout neurites (fewer than I%). Uridine treatment (50 M, 2 or 4 days) in the absence of NGF did not result in the production of neurites. In the presence of NGF, uridine (50 - 200 M) significantly (p < 0.01 or 0.001) enhanced the number of neurites per cell after 4 days of' treatment (Figure 16A-C), whereas 2-day treatment or lower uridine concentrations (10, 25 M) had no effect. Treatment . . . ............ .........
................................................... .............. ..........
........_......... ..................... ............ .... ..... ........ ....
.........
...............................................................................
...................._..........................................................
....................... ..................... .
of the NGF-exposed cells witll cytidine also had no effect on neurite outgrowth.

[00210] Since uridine increased the number of neurites per cell, the effect of uridine on neurite branching and length in the presence of NGF was also assessed. After 4 days of treatment witli uridine (50 M) and NGF, the numbers of neurite branch points per cell were significantly (p < 0.01) increased, compared with those in cells treated with only NGF (Figure 16D). Uridine did not significantly affect average neurite length in NGF-differentiated cells.

- [00211 ] .................... . Neurofilament- proteins "'are--highly --enriched-- within--neurites;--therefore;- -an------increase in neurite number should be associated with increased expression of neurofilament proteins. NF-70 (70 kD) and NF-M (145 IcD) levels following 4-day treatment of PC 12 cells with NGF alone, or NGF plus uridine (50 gM) were thus measured (Figure 16E).
Both NF-70 and NF-M expression significantly (p < 0.01, p < 0.001, respectively) increased following uridine treatment, compared to cells treated only witli NGF. In the absence of' NGF, uridine treatment had no effect on levels of either neurofilament protein. Thus, uridine augments neurite outgrowth in PC 12 cells.

[00212] In the absence of NGF, the addition of exogenous uridine increases intracellular.
UTP and CDP-choline levels in PC12 cells (Example 6). To determine whether uridine affects UTP or CTP levels in the presence of NOF, levels of UTP and CTP were measured in PC 12 cells for 2 days with NGF, treated with no nucleotide, (control), uridine, cytidine or UTP, in the presence of'NGF. Uridine (50 M) significantly (p < 0.05) increased both UTP
and CTP levels (Figure 17 A-B, respectively) compared to cells receiving only NGF treatment..
UTP (100 M) or cytidine (50 M) did not significantly affect the intracellular levels of either nucleotide, [00213] In order to ascertain whether UTP may mediate the effect of uridine on neurite outgrowth, PC12 cells were treated with NGF and various doses of UTP. After 4 days of treatment, UTP (10 and 50 M) significantly (p < 0.01) enhanced neurite outgrowtlr, compared to that in cells treated only witli NGF. Thus, either uridine or UTP augments neurite outgrowth.

[00214] In conclusion, uridine or UTP dietary supplementation increased the levels of two major neurofilament proteins in rat brain, and was directly shown to induce neurite outgrowth in PC 12 cells.

_ ....................... ........ .... .......... _. . . . .................
... ......,..... .... . ...................... ............. .. _......
......................... ................
NGF-differentiated PC 12 cells express pyrimidine-sensitive P2Y2, P2Y4 and receptors METHODS
Detectian of P2Y receptors [00215] Western blots were performed as described for Example 8, using rabbit anti-P2Y2, anti-P2Y4 (both from Calbiochem); or rabbit anti-P2Y6 (Novus Biologicals, Littleton, 5 .... .......... .
_.. ~ ..._..._..... . . .........._...._ ................................_............
Inuturocytochemistri, [00216] PC 12 cells were treated as described above, except they were gr=own on 12mm glass cover slips (A. Daigger & Co.., Vernon Hills, IL) coated with collagen.
Proteins were visualized using irnmunofluorescence. Briefly, the cells were fixed with 4%
paraformaldehyde, permeabilized with 0.25% Triton X-100, blocked in 10% normal goat serum, and incubated ovemight in the appropriate antibodies (mouse anti-NF-70, and either rabbit anti-P2Y2, rabbit anti-P2Y4 or rabbit anti-P2Y6). For P2Y2 and P2Y4 visualization, control cultures wer-e incubated with primary antibody plus a control antigen in order to ensure that the immuno-staining would be specific. Control antigen was not available for the P2Y6 receptor. Cells were then incubated in fluorochrome-conjugated secondary antibodies for..1.. hour.:
(goat, anti.-rabbit ALEXA 488 arld goat anti-mouse ALEXA 568; Molecular Probes, L-ugene, OR) and,mounted on glass slides with mounting media with or without DAPI (Vector Laboratories;
Burlingame, CA). Control antigens provided with the primary antibodies were used to ensure that immuno-staining was specific. Digital images were obtained on a Zeiss (Oberkochen.
Germany) Axioplan microscope witli OpenLab software, using a Zeiss Plan-Neofluor 40x oil-immersion objective.

RESULTS
[00217] UTP is an agonist of the pyrimidine-activated class of P2Y receptors, namely P2Y2, P2Y4 and P2Y6 receptors. To determine whether these receptors participate in the mechanism by which extracellular [JTP affects neurite outgrowth, it was first determined wllether the receptors are expressed in PC12 cells, and whether exposure to NGF alters their expression, PC 12 cells were treated for 0- 7 days with NGF and levels of the receptors measured. After 3 days of NGF treatment, expression of the P2Y2 receptor reached maximal levels, which were significantly (p < 0.001) higlier than those seen at less than 3 days of NGF
..............
treatment (Figure 19A). To visualize the expression and localization of the P2Y2, as well as the P2Y4 and P2Y6, receptors, cells were grown in the presence or absence of NGF for 4 days and then immuno-stained them for the neuritic marker NF-70, and for P2Y2, P2Y4, or P2Y6 (Figure 19B, left to right, respectively). All three receptors were highly expressed in NGF-differentiated PC12 cells.. In addition, P2Y2 co-localized with the neuronal marker M.AP-2 (Figure 20), In the absence of NGF, receptor protein expression was undetectable by irnmuno-staining.. Moreover=, the presence of uridine did not affect the expression of the receptors .. _ conipared-with t ie quantrties preseilt tn ce s &_'-o_'s-ed't6'NGF a ori: ius;
P2Y2,-P2Y4 , _ ii~~n P2Y6 receptor=s are present in neural cells, but not in their precur=sors.

Antagonism of P2Y receptors inhibits the effect of uridine on NGF-induced neurite outgrowth [00218] To ascertain wliether signaling by P2Y receptors mediate induction of neurite outgrowth by uridine, PC 12 cells were incubated for 4 days with NGF, uridine (100 M) aiid the P2Y receptor antagonists suramin (30 M), pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid .(PPADS; 30 M) and reactive blue 2 (RB-2; 10 M). Each of the antagonists significantly' (p < 0..05 or 0.001) blocked uridine enhancement of NGF-stimulated neurite outgrowth (Figure 21)., None of the P2Y receptor antagonists inhibited the uptalce of uridine into the PC12 cells., These results show that signaling via P2Y receptors mediates uridine induction of neurite outgrowtli.

Phosplratidylinositol (IP) signaling is stimulated by UTP and uridine METHODS
Metabolic labeling atirl PI trrrnover analysis [00219] Analysis of PI turnover was perfornied as described by (Nitsch RM et al, J
Neurocliem 69: 704-12, 1997). Briefly, cells were labeled metabolically for 36 h with 1.25 microCurie ( Ci)/dish of myo-[2-3H]inositol (17.0 curie/mmol; Amersham Biosciences) in serum-free MEM, washed twice with Hank's balanced salt solution (HBSS), and treated for 15 ............ ........................... _...........................
............. ........_.................. ....................
..................................... ............. .......
................... ................. ............... .....................
.................. ..........
min with 10 mM lithium chloride in HBSS. Drugs were added in the presence of 10 mM
lithium for 60 min at 37 C. Cells were lysed witli ice-cold methanol, and lipids were removed by extraction with chloroform/methanol/water (2:2:1 by volume). Labeled water-soluble inositol phosphates were separated from free [3H]inositol by ion-exchange clu-omatography, using AG 1-XS colunms (Bio-Rad), and 1M ammonium formate and O.1M fonnic acid as eluent.
Radioactivity was quantified by liquid scintillation spectrometry.

RESULTS
[00220] P2Y2, P2Y4 and P2Y6 receptors activate the phospholipase C/diacylglycer=ol/inositol triphosphate (PLC/DAG/IP3) signaling pathway. To determine whether- concentrations of uridine or UTP that promote neurite outgrowth activate these receptors, NGF-differentiated PC 12 cells were labeled with [3H]-inositol (50 M) or UTP (10, 100 M) for 1 hour, and IP signaling was assessed by measuring turnover of radio-labeled IP

(Figure 22). Formation of' IP was signif cantly increased by addition of 100 M UTP (p < 0,05) and by 50 M uridine (p < 0.01). The P2Y receptor antagonist PPADS (100 lV1) significantly (p < 0.05) blocked the stimulation of' IP signaling by UTP. These findings indicate that UTP
promotes neurite outgrowth via P2Y receptors-mediated stimulation of the IP
signaling pathway.

[00221) The findings of L-xamples 10-12 provide a mechanism by which uridine and its metabolites stimulate neurite outgrowth: namely, by activation of P2Y
receptors.= At least part of the action of the P2Y receptors is mediated by IP signaling.. Taken together, the findings from Lxamples 7-12 provide further evidence that uridine treatment can iniprove cognitive function by enhancing neurotransmission by multiple mechanisms: (1) enhancing neurotransmitter release; (2) acting, through CTP, as a precursor for membrane phosphatides;
(3) activating, through UTP, the P2Y receptor-coupled intracellular signaling pathway.
Mechanisms (2) and (3) may act together to increase neurite formation.

UMP-supplemented diets enhance learning and memory in multiple species MATERIALS AND EXPERIMENTAL METHODS

Morris Water Maze [00222] Aging r=ats (18 months, 500 g) were fed a control diet or a diet containing 2.5%
................ _._.. .... _.._.._..__.._._...
........................,..............
........................................... . ..
..........
UMP diets for six weeks. They were then shown a hidden platform iri a six=foot diarneter pool of water, placed somewhere in each of the four quadrants of the pool in turn, and were allowed 90 seconds in each trial to attempt to relocate tiie platform by swimming, and the swimming time "mean escape latency" recorded. The set of four trials was repeated on each of.four consecutive days. The platform was in the same place each day. This test, known as the Morris water- maze, is an indicator of'spatiat memory.

Footl pellet learniitg assay _...................... .-.-----._........ _------ .......................__ .__........... _...__............. .......... _...... _.......
......_.__._...._................... ......._...._._..._.. __....._.....-......__............ .............. ......---.......
[00223] Male young adult gerbils fed control or UMP-containing chow (0, 0.1, 0.5 or 2.5%) ad lib for three weeks were tested in a radial arm maze, consisting of a central chamber witll four branches primed with a small food pellet at the end of each. Before testing, animals were fasted overnight; each animal was then placed in the central chamber and allowed up to 180 seconds to find all of the pellets.. A shorter time required to find the pellets is indicative of improved learning and spatial memory.

Working mernory aiid reference nrennor,p assay [00224] Groups of ten gerbils fed control or 0.1 % UMP diet for four weeks and trained to successfully find all of the food pellets as described above were then given a modified test, in wliich only two arms of the maze (but always the same two) contained food pellet rewards. In this test, a worlcing memory er7or is orie in which a gerbil revisits an arm from which it has alr-eady taken. the pellet that day. A reference menlory error is one in which the gerbil enters an arm whicll never had food pellets (during the modified tests).

RESULTS
[00225] Previous Examples showed that orally administered uridine improves augments-the ability of neural cells to function in several ways. The present Example directly shows that uridine augments cognitive function. Aging rats (18 months, 500 g) were fed a control diet or a diet containing 2.5% UMP=2Na4' for six weeks, and their memory was tested using the Morris water maze, an indicator, of spatial memory. Rats administered the UMP-2Na+-fortified diet showed a statistically signifcant reduction in the time required to reach the location of the platform (Figure 23), indicating that UMP enhances spatial memory.

[00226] The effect of orally administered uridine upon learning and spatial memory was -- also examined in gerbils: Male young adult gerbils-fed-control or UMP-eontaining" chow-(0, 0:.L:
0.5 or 2.5%) ad lib for three weeks were tested in a radial arm maze, consisting of a central chamber with four branches prirned with a small food pellet at the end of each. Before testing, animals were fasted overnight; each animal was then placed in the central chamber and allowed up to 180 seconds to find all of the pellets. The reduction in time needed to find the pellets requires spatial learning.. UMP-supplemented diets reduced the time required for gerbils to find the pellet in a dose-dependent manner (Figure 24).

[00227] 1ri 'additiori,-'the effect of orally administered- uridine" on--wor ang-memory-and reference meniory was examined. Gerbils fed a control or a 0.1% UMP diet for four weeks and trained to successfully find all of the food pellets as described above were then given a modified test, that measures working memory and reference memory.. Gerbils fed the UMP-supplemented diet exhibited reduced numbers of both working memory errors (Figure 25A) and reference memory errors (B).

[00228] These findings directly show that (a) uridine dietary supplementation improves leaming and various types (spatial, worlcing, and reference) of memory; (b) the effect is not limited to a particular species; and (c) the eff:'ect is manifested in biologically relevant models of age-impaired cognitive function.

[00229] In summary, the findings presented herein demonstrate that orally administered uridine positively affects neurological signaling, neural cell anatomy and cognitive function..
The findings also implicate several mechanisms by which uridine exerts its effects.

URIDINE AND CHOLINE INCREASE NEUROTRANSMITTER RELEASE
MATERIALS AND EXPERIMENTAL METHODS

Brain slice preparation [00230] Male Sprague-Dawley rats, 9-11 montlis old, were anesthetized with lcetamine (85 mg/kg of body weight, intramuscularly) and were decapitated in a cold room at 4 C. Brains were rapidly removed and placed into chilled (4 C) oxygenated Krebs buffer (119.5 mM NaCI, 3.3 mM KCI, 1.3 mM CaC12, 1.2 mM MgSO4, 25 mM NaHCO3, 1.,2 mM, KH2PO4, 11 mM
glucose, and 0.03 mM EDTA, pH 7.4) containing 1 mM ketamine and 15 g/ml eserine. After removal of remaining meninges and chorioid plexus, 30 m slices of striatum, hippocampus, and cortex were immediately prepared with a McIllwain tissue chopper, washed 3 times, and 20.,,.. placed u~to custom-made superfusion chambers (Wamer Instr=ument, Hamden, CT).

Superfir.sion and electrical stimulatioir.
[00231] Slices were equilibrated for 60 min at 37 C by superfusing the chambers with oxygenated Krebs/ketamine/eserine buffer described above at a flow rate of 0.8 ml/min.
Superfusion chambers contained two opposing silver mash electodes that were connected to an electrical stimulator (model S88; Grass lnstruments).. A custom-made polarity reversal device was used to prevent chamber polarization and also to monitor both the current and voltage 50 ................ ....... .._micr.oseconds .after_.ahe....onset of._each_pulse..._to.._ensure_..uniform.,_c..h,.,amber.._resistance. Afier the equilibration period, slices were depolarized by perfusion with a high-K+ (52 mM) version of the Krebs/ketanline/eserine buffer in the presence or absence of 20 M
choline, 25 M
cytidine, and/or 25 M ur'idine. Perfusates were collected during the entire 2-hour period and assayed for acetylcholine.. Values were normalized for protein content of slices..

RESULTS
[00232] To determine the effect of uridine and choline on acetylcholine release, slices of striatum, hippocampus, and cortex (n=8) were incubated in the presence or absence of choline and then depolarized, and acetylcholine release was measured. In some groups, cytidine or uridine was added as well. Choline increased acetylcholine release whetlier or not uridine was also present (Figure 26).

[00233] These findings show that when neurons are repeatedly stimulated to release acetylcholine, choline increases the aniount of neurotransmitter that is released, by replenishing stores of choline in membrane-phospholipids (e.g. PC). The above Examples have shown that ] 0 uridine augments synthesis of CDP-choline, wliich is then used to synthesize new PC. Together with the findings of this Example, these results show that the ability of neurons to synthesize new phospholipids, and thus repeatedly release neurotransmitters, will be increased in an additive or synergistic fashion by addition of uridine together with choline.

URIDINE AND CHOLINF INCRFASE NEUROTRANSMITTER RELEASE
ADDITIVELY FOLLOWING REPLATED DEPOLARIZATION

[00234] Brain slices are repeatedly stimulated as described in the previous Example, in this case for 8 cycles or alternating 20-minute periods of stimulation and rest. In all groups, the amount of neurotransmitter release decreases with each successive stimulation period; however, .. . . .............................................................. ...
........................... ............... .................
........................................ .......................
._..........................._....................................... ....
............................................................. ........ .... .
this decrease is significantly less in the presence of either uridine or choline. This effect is enlianced by the presence of both uridine and choline. Thus, uridine and choline the total amount of neurotransmitter release after repeated stimulation is increased by the presence of uridine or choline, and is fur-ther increased by the presence of uridine and choline.

Claims

1. A method of improving a cognitive function in a subject, comprising administering to said subject a uridine or a source thereof, thereby improving a cognitive function in a subject.

2. A method of improving a cognitive function in a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby improving a cognitive function in a subject.

3. A method of treating or ameliorating a decline in a cognitive function in a subject, comprising administering a uridine or a source thereof to said subject, thereby inhibiting or preventing a decline in a cognitive function in a subject.

4. A method of treating or ameliorating a decline in a cognitive function in a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby inhibiting or preventing a decline in a cognitive function in a subject.

5. The method of claim 3 or 4, wherein said decline is a result of a cardiovascular disease, neurodegenerative disease, or psychiatric disease.

6, The method of claim 1, 2, 3, or 4, wherein said cognitive function is memory, learning, intelligence, or mental fitness.

7. A method of improving a neurological function in a subject, comprising administering to said subject a uridine or a source thereof, thereby improving a neurological function in a subject.

8. A method of improving a neurological function in a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby improving a neurological function in a subject.

9. The method of claim 7 or 8, wherein said neurological function is a synaptic transmission or a function of a neurotransmitter.

10. A method of increasing a level of cytidine, cytidine triphosphate, or CDP-choline in a brain of a subject, comprising administering to said subject a uridine or a source thereof, thereby increasing a level of cytidine, cytidine triphosphate, or CDP-choline in a brain of a subject.

11. A method of increasing a level of cytidine, cytidine triphosphate, or CDP-choline in a brain of a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby increasing a level of cytidine, cytidine triphosphate, or CDP-choline in a brain of a subject.

12. A method of increasing or enhancing an ability of a brain cell or neural cell of a subject to synthesize a neurotransmitter, comprising administering to said subject a uridine or a source thereof, thereby increasing or enhancing an ability of a brain cell or neural cell of a subject to synthesize a neurotransmitter.

13. A method of increasing or enhancing an ability of a brain cell or neural cell of a subject to synthesize a neurotransmitter, comprising administering to said subject a composition, said composition, comprising a uridine or a source thereof and a choline;
thereby increasing or enhancing an ability of a brain cell or neural cell of a subject to synthesize a neurotransmitter.

14. A method of increasing a level of a neurotransmitter in a synapse, comprising contacting a neural cell adjacent to said synapse with a uridine or a source thereof, whereby said uridine enhances synthesis of a phospholipid or a precursor thereof, thereby increasing a level of a neurotransmitter in a synapse.

15. A method of increasing a level of a neurotransmitter in a synapse, comprising contacting a neural cell adjacent to said synapse with a composition, said composition comprising a uridine or a source thereof and a choline, whereby said composition enhances synthesis of a phospholipid or a precursor thereof, thereby increasing a level of a neurotransmitter in a synapse, comprising

16. The method of claim 9, 12, 13, 14, or 15, wherein said neurotransmitter is acetylcholine.

17. A method of stimulating or enhancing a synthesis of a membrane of a brain cell or neural cell of a subject, comprising administering to said subject a uridine or a source thereof, thereby stimulating or enhancing a synthesis of a membrane of a brain cell or a neural cell of a subject.

18. A method of stimulating of enhancing a synthesis of a membrane of a brain cell or neural cell of a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby stimulating or enhancing a synthesis of a membrane of a brain cell or a neural cell of a subject.

19. A method of stimulating or enhancing an outgrowth of a neurite of a neural cell of a subject, comprising administering to said subject a uridine or a source thereof, thereby stimulating or enhancing an outgrowth of a neurite of a neural cell of a subject.

20. A method of stimulating or enhancing an outgrowth of a neurite of a neural cell of a subject, comprising administering to said subject a composition, said composition comprising a uridine or a source thereof and a choline, thereby stimulating or enhancing an outgrowth of a neurite of a neural cell of a subject.

21. The method of claim 17, 18, 19, or 20, whereby said composition enhances a production of a phospholipid, thereby stimulating or enhancing said synthesis of a membrane or outgrowth of a neurite.

22. The method of any of claims 1-21, wherein said uridine is uridine-5'-monophosphate, uridine-5'-diphosphate, or uridine-5'-triphosphate.

23. The -method of any of claims 1-21, wherein said source is citicoline (CDP-choline).

24. The method of any of claims 2, 4, 5, 6, 8, 9, 11, 13, 15, 16, 18, 20, 21, 22, or 23, wherein said choline is a choline salt.

25. The method of any of claims 1-11, 22, or 23, whereby said uridine or a metabolite thereof mediates its effect by stimulating a P2Y receptor in a neural cell or brain cell of said subject.

26 The method of any of claims 12-21 whereby said uridine or a metabolite thereof mediates its effect by stimulating a P2Y receptor in said neural cell or brain cell.

27. The method of either of claims 25 or 26, wherein said metabolite is uridine-5'-triphosphate.