Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02579851 2012-08-20
USE OF URIDINE FOR IMPROVING COGNITIVE AND
NEUROLOGICAL FUNCTIONS
FIELD OF THE INVENTION
[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 nucleoside and is essential in the synthesis of
ribonucleic acids
and tissue glycogens such as U]DP glucose and UT? 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 as broadly disclosed 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.
[0004] In one embodiment, the present invention provides a method of improving
a cognitive
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.
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[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.
[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 administering 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 neurotransmitter in a synapse, comprising contacting a
neural
cell adjacent to the synapse 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 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,
comprising
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 enhancing a production of a membrane of a brain cell or a
neural 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
2
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 composition enhances synthesis
of
a phospholipid or a precursor thereof, thereby stimulating or enhancing an
outgrowth of a neu rite of a neural cell.
[0012-A] The invention as claimed is however more specifically directed to the
use
of (a) uridine, an acyl derivative thereof, or uridine phosphate and (b)
choline, a choline
salt or a choline ester in the preparation of a medicament for improving
cognitive
function in a subject in need of such treatment, wherein the uridine is
present in an
amount of about 400 mg to 6 g and the choline is present in an amount of about
200
mg to 8 g of choline.
[0012-B] The invention as claimed is also directed to the use of of (a)
uridine, an
acyl derivative thereof, or a uridine phosphate and (b) choline, a choline
salt, or a
choline ester in the preparation of a medicament for treating a decline in
cognitive
function in a subject, wherein the uridine is present in an amount of about
400 mg to
0.8 g and the choline is present in an amount of about 200 mg to 8 g of
choline.
[0012-C] In another embodiment the invention relates to a use of uridine or a
uridine
phosphate, and choline, a choline salt or a choline ester in the preparation
of a
medicament for stimulating the synthesis of a neural cell membrane in a
subject in
need of such treatment, wherein the uridine or uridine phosphate is present in
a dose of
400 mg to 6 gr, and the choline, choline salt or choline ester is present in a
dose of
200mg to 8 gr, and wherein both ingredients are present in a synergistically
effective
amount.
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[0012-D] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the choline is a choline phosphate.
[0012-E] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the choline salt is choline chloride, choline bitartrate, or
choline
stearate.
[0012-F] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the uridine phosphate is uridine-5'-monophosphate (UMP),
uridine-5'-diphosphate (UDP), uridine-5'-triphosphate (UTP), or a salt of the
UMP,
UDP, or UTP.
[0012-G] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the salt of uridine-5'-monophosphate is uridine-5'-
monophosphate disodium.
[0012-H] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the medicament further comprises sphingomyelin,
acylglycerophosphocholine, lysolecithin, glycerophosphatidylcholine, a fatty
acid or
a combination thereof.
[0012-1] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the uridine or the uridine phosphate stimulates or enhances
an
outgrowth of a neurite or dendritic spine of a neural cell, thereby improving
or
enhancing the synaptic transmission.
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[0012-J] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the neural cell is newly differentiated.
[0012-K] In another embodiment the invention relates to the use defined
hereinabove
for the preparation of the medicament for stimulating the synthesis of a
neural cell
membrane, wherein the uridine or the uridine phosphate increases a number of
neurites of a neural cell, thereby improving or enhancing the synaptic
transmission.
[0012-L] In another embodiment the invention relates to the use as defined
hereinabove, wherein the neural cell membrane is dendritic membrane.
[0012-M] In another embodiment the invention relates to the use as defined
hereinabove, wherein the neural cell membrane is axonal membrane.
[0013-N] In
another embodiment the invention relates to a use of uridine or a
uridine phosphate, and choline, a choline salt or a choline ester in the
preparation of a
medicament for stimulating the synthesis of neural cell membrane in a subject
in need
of such treatment, wherein said medicament is formulated in a fixed dose of
said
uridine or uridine phosphate of 400mg, 600mg or 800mg, and a fixed dose of
said
choline, choline salt or choline ester of 200mg, 400mg, 600mg or 800mg, and
wherein
both ingredients are present in a synergistically effective amount.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 illustrates the coincidence of cytidine and tyrosine peaks
(6.59)
when tested by a standard HPLC method.
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[0014] Figure 2 illustrates distinct cytidine (3.25) and tyrosine (2.92) peaks
when tested by a
modified HPL,C method, which utilizes elution buffer with low methanol.
[0015] Figure 3, Oral LIMP administration raises blood uridine levels in
humans. 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; 0: 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 bruin 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; #1#: p< 0.01 vs. cytidine.
[0020] Figure 8, Oral UMP administration raises brain uridine levels_ Depicted
are brain
uricline levels at 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-choline 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 line.
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/mg
protein. The
experiment was repeated 3 times, *: p <0.05.
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[0024] Figure 12. UMP dietary supplementation significantly increases
potassium-evoked
dopamine (DA) release in striatal dialysate, (A) Effect of dietary UMP
supplementations on Kt
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 LIMP 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
UMP dietary supplementation. (A): DOPAC (B): HVA. *: p <0.05 compared to
corresponding
controls
[0026] Figure 14. Increased acetylcholine basal concentration with 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
corresponding controls.
[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
(50 p.M)
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
uM). 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 =
Uridine.
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 (504M) 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 number 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 after incubation of cells
with NGF for
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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
immunolluorescence. 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 IrM)
and the P2Y
receptor antagonists PPADS, suramin, or RB-2. Values represent means + SEM.
***p < 0.001
vs. NOF 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 overnight, 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, **p < 0,01 vs. control; #p < 0.05 vs
100 u.M UTP
treatment.
[0035] Figure 21 Oral UMP improves learning and spatial memory in rats. 18-
month old rats
in restricted environments 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 platform is
given in seconds.
[0036] Figure 24. Oral UMP improves 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 UMP 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.
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[0038] Figure 26. Uridine and choline increase 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. ""
= P <0001
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 (each 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, comprising administering to the subject a composition
comprising a
uridine or a source thereof and a choline, thereby improving a cognitive
function in a subject.
[0042] The phrase""uridine. or a source thereof 'and a- choline" refers' to. 2
embodiments-of' the
present invention: a) a combination of uridine and choline; b) a combination
of a uridine source
and choline. 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-
term memory, or medium-term memory, In another eibdithnç the memory is any
other type
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 different
species in different
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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 learning, 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 embodiment, 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 another embodiment, the cognitive function is mental fitness. In
another embodiment,
the cognitive function is any other type of cognitive function known 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 perform the
cognitive function. In
another embodiment, the terms refer to an increased or improved baseline level
of the cognitive
function in the subject. In another embodiment, the terms refer to an
increased or improved
lei/el 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 thereof. 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. Each possibility represents a separate embodiment of the present
invention.
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[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 another
embodiment,
improvement of a cognitive- function is assessed according to a standardized
criterion such as,
for example, a test or the like. Each 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 communicate 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
(Schizophr Res. 2004 Oct 1;70(273):117-45) and in Cognitive Function Analysis
(Greenwood
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 increasing a level of cytidine triphosphate (CTP) in the subject.
In another
embodiment, the effect -is mediated-by. increasing-a level of CDP-choline in-
the-subject. .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 without increasing a level of cytidine, CTP, CDP-choline,
or a derivative or
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metabolite thereof. Each 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 Farrier
(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 effects 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
thereof, 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 thereof and a choline, thereby improving a
neurological
function in a subject,
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[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
intemeuron. 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 enhancing 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 number 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
enhanced by
stimulating or enhancing branching of a neurite of a neural cell. In another
embodiment,
improvement or enhancement of the synaptic transmission occurs without
stimulating or
enhancing branching of a neurite of a neural cell. Each possibility represents
a separate
embodiment of the present invention.
[0064] The data of' Example 9 shows that when levels of membrane precursors
are increased,
neurons produce more 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 amount or composition of plasma membrane 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, which
involves loss of
- neuronal connections and memory impairment.
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[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 another 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
enhancing 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 enhancing an
amount of or a synthesis of a membrane of a neural cell. Each possibility
represents a separate
embodiment of the present invention..
[0066] In 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 changing 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
neur=otr=ansmitter 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 compositions comprising uridine and choline are
effective at improving
neurological function - more effective, in one embodiment, than administration
of either uridine
or choline alone.
[0068] 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
or a source thereof to the subject, thereby 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
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composition comprising a uridine or a source thereof and a choline 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
phrase refers to slowing the decline. In another embodiment, the phrase refers
to halting 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 memory disorder.
The memory
disorder comprises, in one embodiment, a memory decline. In another
embodiment, the
memory 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 supranuclear palsy,
frontotemporal
dementia, Huntington's disease, or a prion disease. In another embodiment, the
.. netuodegenerative disease is any other neurodegenerative disease known in
the art. Each
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 stroke. 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
dopaminergic pathway. Each possibility represents a separate embodiment of the
present
invention.
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[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 comprises 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
impairment 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. hi 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 embodiment, 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. In another embodiment, the depression is a major
depressive disorder.
In another embodiment, the depression is depression 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
burette's syndrome_ In another embodiment, the movement disorder is any other
movement
disorder known 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 ischemia.
[0081] In another embodiment, the neurological disorder is a behavioral
syndrome. In another
embodiment, the neurological disorder is a neurological syndrome. In other
.embodiments, the
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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 nervous 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 known
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 neural 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 neurotransmitter 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 another enibbiliment, die present invention provides a meth-od of
increging 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 administering 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
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repeatedly release an effective quantity of a neurotransmitter into a synapse.
As described
herein, findings of the present invention show that uridine enhances the
ability of neurons to
synthesize neurotransmitters and repeatedly release them (Example 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 enhanced
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 known in the art.
Methods for
assessing neural stimulation 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 thereof, 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
= 20 = = a composition comprising. a-uridine or a.source thereof and
=a.choline, whereby-the.eornposition-
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 with a
uridine or a
source thereof; whereby the composition enhances synthesis of a phospholipid
or a precursor
thereof, thereby increasing a sensitivity of a neuron to a stimulus,
[0092] In another embodiment, the present invention provides a method of
increasing 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 enhances
synthesis of a phospholipid or a precursor thereof, thereby increasing a
sensitivity of a neuron
to a stimulus.
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[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-hydroxytryptamine (5-HT). In
another
embodiment, the neurotransmitter is GABA. In another embodiment, the
neurotransmitter is
any other neurotransmitter known in the art. Each type of neurotransmitter
represents a separate
embodiment of the present invention.
[0094] In another embodiment, the present invention provides a method 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 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 method 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 choline.
[0096] In another embodiment, the present invention provides a method of
stimulating or
enhancing an .arnount...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 embodiment, 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 composition comprising a uridine or a source
thereof and a choline,
thereby stimulating or enhancing an amount of or a synthesis of a cell
membrane.
[0098] In another 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 component is a phosphatidic acid. In another
embodiment, the
component is a PE. In another embodiment, the component is a lecithin. In
another
embodiment, the component is a Pl. In another embodiment, the component is a
PS. In other
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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 enhancing an amount of or a synthesis of a phospholipid
precursor. In 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. In another embodiment, the phospholipid precursor is choline. In
another embodiment,
the phospholipid 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.
20.........[00101] 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,
comprising contacting the subject with a uridine or a source thereof, 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.
[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,
comprising contacting"the subject"with-a-composition-tomprising auridine ora =
source-thereof
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,.
17
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[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 other type of
membrane
known in the art. Each type of membrane represents a separate embodiment of'
the present
invention.
[00104] In another embodiment, stimulating an amount of or a synthesis
of the cell
membrane is accomplished by stimulating or enhancing a synthesis of a
phospholipid (Example
6). In another embodiment, stimulating or enhancing an amount of or a
synthesis of a
membrane of a neural cell is accomplished by stimulating or enhancing a
synthesis of a
phospholipid precursor (Example 6). In another embodiment, stimulating or
enhancing a
synthesis of a phospholipid or a precursor thereof is partially responsible
for stimulating an
amount of or a synthesis 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 phospholipid or a precursor
thereof.. 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 membrane marker
protein
(Example 8). In another 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 ICP et al, Neurotoxicol Teratol 26(3): 397-406,
2004. Each
method of assessing membrane production represents a separate embodiment of'
the present
invention.
[00106] 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 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 cell,
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[00107] 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 composition comprising a uridine or a source thereof and a choline,
whereby the
composition enhances synthesis of a phospholipid or a precursor thereof,
thereby Stimulating or
enhancing 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 (Example 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, whereby the composition enhances synthesis of a phospholipid
or a precursor
thereof, thereby increasing a number of neurites of a neural cell.
[00109] 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 composition
comprising a uridine or a source thereof and a choline, whereby the
composition enhances
synthesis 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 stimulating
or enhancing a branching of a neurite of a neural cell, comprising contacting
the neural cell with
....................................................................... a
uridine .or. a source thereof,- .thereby stimulating or enhancing a -branching
of a-neurite .of .a
neural cell.
[00111] In another embodiment, the present invention provides a method
of stimulating
or enhancing a branching of a neurite of a neural cell, comprising contacting
the neural cell with
a composition comprising a uridine or a source thereof and/or a choline,
thereby stimulating or
enhancing a branching of a neurite of a neural cell.
[00112] ..... In one embodiment, the cell that is the target of methods of
the present invention
or is contacted in the methods is a neural cell. In another 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 comprising a uridine and/or
a choline. In
another embodiment, the cell is any cell in which a neurological function is
enhanced by
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contact with a composition comprising a uridine and/or a choline. Each
possibility represents a
separate embodiment of the present invention.
[00113] ¨ In
another 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" refers to a neuron
that has
differentiated in the 24 hours prior to commencing administration of the
uridine and/or choline.
In another embodiment, the neuron has differentiated in the 48 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
administration. In
another embodiment, "newly differentiated" refers to a neuron that completes
its differentiation
following commencement 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 known 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 method of increasing a
level of a cytidine in a tissue, plasma, or cell of a subject, comprising
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
subject, 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-
tholine-in a-tissue; plasma;--orcell .of a-subject;-comprising-administering a
composition-ofthe
present invention. In another 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
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
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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 known 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 another 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 administered in
methods of the
present invention. In one embodiment, the uridine precursor that is
administered is a cytidine-
5'-monophosphate. In another 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
known 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 another 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..
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[00123] In
other embodiments, uridine-based compounds other 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
another 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 choline required by
the subject. Thus, in
this embodiment, citicholine serves a the source of all the uridine and some
of the 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,
metabolite, or source
represents a separate embodiment of the present invention.
[00127] _ The term "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 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 or dosage
range represents a
separate embodiment of the present invention_
. . .
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=
[00129] In 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 lecithin. 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 thereof
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 manner 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.
In 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-I 0 g; 200 mg-8 g; 400 mg-6 g; 600 mg-4 g;
800 mg-3 g;
23
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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 another embodiment, a composition of the present invention
is administered 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 other embodiments,
the subject is a
rodent 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
= = = =20====== nutritional -supplementi-or-the ==like.-- Each- possibility
--represents -a-separate .embodiment =of the
present invention.
[00138] In one embodiment, administration is performed by the
subject.. In another
embodiment, administration is performed by a care provider_ In another
embodiment,
administration is performed by a third party. Each type of administration
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.
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[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
another 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
drug 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, I.,-2',3'-dideoxyuridine, and D-2',3'-dideoxyuridine or
mixtures thereof 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 known 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
derivative or source
thereof is secreted from a cell after being converted into the second
compound. In another
embodiment; =the uri dine- or=-= d erivati ve - or source. thereof. contacts-
a -different - cell after = being-
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.
.)5
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[00143] P2Y receptors are a family 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
another 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 Blitterswijk W et al, Bioehem .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 cellular
pathway that
stimulates membrane production. Each possibility represents a separate
embodiment of the
present invention.
[00147] In one embodiment, uridine or a related compound in a
composition of the
present invention stimulates a receptor other than a P2Y receptor
[00148] In another embodiment 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
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[00149] In one embodiment, "pharmaceutical composition" refers to a
therapeutically
effective amount of the active ingredients, i.e. the uridine and/or choline,
together with a
pharmaceutically acceptable carrier or diluent. "Therapeutically 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, intradermally, subcutaneously, intraperitonealy,
intraventricularly, intracranially,
intravaginally or intratumorally.
[00151] In another embodiment, the pharmaceutical compositions are
administered
orally, and thus is formulated inn 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 choline 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
intravenous,=intraarterial, or intramuscular injection of a liquid
preparation. Suitable liquid
formulations include solutions, suspensions, dispersions, emulsions, oils and
the like. In one
embodiment, the pharmaceutical compositions are administered intravenously,
and are thus
formulated in a form suitable for intravenous administration. In another
embodiment, 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 pharmaceutical compositions
are
administered as a suppository, for example a rectal suppository or a urethral
suppository. In
another embodiment, the pharmaceutical compositions are administered by
subcutaneous
implantation of a pellet. In another embodiment, the pellet provides for
controlled release of
uridine and/or choline over a period of time.
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[00154] Pharmaceutically acceptable carriers or diluents are well known
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 include, in other embodiments, a gum, a
starcl¶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, pharmaceutically acceptable carriers
are, in other
embodiments, aqueons 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, alcoholic/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 further comprise binders
(e.g. acacia,
cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl
cellulose,
hydroxypropyl methyl cellulose, povidone), disintegrating s (e.g..cornstarch,
potato starch,
alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum,
sodium starch
glycolate), buffers (e.g., Tris-FICI., acetate, phosphate) of various pH and
ionic strength,
......................................................................
additives such as alburnin or gelatin to prevent 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, citric 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 lauryl sulfate), polymer coatings (e.g.,
poloxamers or
poloxamines), coating and film forming s (e.g. ethyl cellulose, acrylates,
polymethacrylates)
and/or adjuvants.
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[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 formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In
another 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 another 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 proximity to the
therapeutic target, i.e., the
brain, thus requiring only a fraction of the systemic 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
pharmaceutically 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 mixed with additives customary for this purpose, such
as vehicles,
stabilizers, or inert diluents, and converted by customary methods into
suitable forms 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, suspension, or emulsion, if desired with the substances
customary and suitable
for this purpose, for example, solubilizers or other.
[00161] An active component can be formulated into the composition as
neutralized
pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts
include the acid
addition salts (formed with the free amino groups of the polypeptide or
antibody molecule),
which are formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids,
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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 such as, for
example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 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. Other 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 compound
according to the
invention with a solution of a pharmaceutically acceptable acid such as
hydrochloric acid,
sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic
acid, acetic acid,
benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid.
EXPERIMENTAL DETAILS SECTION
EXAMPLE 1
Measurement Of Cytidine By HPLC Without Interference From Tyrosine
MATERIALS AND METHODS
Sample preparation
[00163] 1-milliliter (mL) samples of heparinized plasma were spiked
with 1 lig 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 ammonium
acetate (pH 8.8),
then immediately purified over boronate affinity columns.
Boronate affinity columns
[00164] All steps were performed at 4 C. Boronate affinity columns
(Affige1-601, Bio-
. Rad) were primed with two 5-mL ammonium acetate washes, samples 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 )..IL water for
HPLC analysis, Boronate affinity columns bind many biological molecules,
including the
nucleotide bases adenosine, cytidine, guanosine, thymidine, and uridine.
HPLC
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[00165] HPLC analysis was performed using a Beckman System Gold
apparatus
(Beckman Instruments) equipped with a Rainin Dynamax Microsorb C18 column (3
i_trn
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 potassium phosphate buffer (pH 5.8)
and 0.1%
methanol instead of formic acid, flowing at 1 mL/min and heated to 35 .
RESULTS
[00166] A standard HPLC method for measuring nucleosides yields
separate peaks for
uridine and cytidine; however, a coincidence of the cytidine and tyrosine
peaks precludes
accurate measurement 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 Example, a modified HPLC method was used which distinguished cytidine
and tyrosine
peaks, permitting accurate measurement of cytidine levels (Figure 2).
EXAMPLE 2
Oral administration of UMP increases plasma uridine levels in humans
MATERIALS AND EXPERIMENTAL METHODS
Study design
[00167] Eight healthy subjects (5 male,=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, separated 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 chloroform, and an aliquot of the aqueous layer lyophilized,
dissolved in water,
and assayed by HPLC with UV detection.
Statistical analyses
[00168] Statistical analyses were carried out with SPSS 12Ø 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
31
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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).
EXAMPLE 3
Oral administration of uridine or UMP increases_plasma uridine levels in
gerbils
MATERIALS AND EXPERIMENTAL METHODS
Experimental design
[00170] Groups of eight to nine male gerbils (60-80 g) were fasted
overnight,
administered (a) uridine (Sigma, St, Louis, MO; 250 mg/kg body weight) (Figure
4) or
disodium UMP (1 mmol/kg body weight, a dose equivalent to 250 mg/kg 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%
UMP by weight for 4 weeks, fasted overnight, then sacrificed one hour after
consumption of a
-- last meal=of the same composition. Blood collected from the neck was
collected into tubes
containing EDTA and was treated as described above for Example 2.
RESULTS
")5
[00171] To ascertain whether oral administration of uridine can raise
plasma uridine
levels, gerbils were =fed by gavage 250 mg/kg cytidine or uridine. 60 minutes
(min) later,
plasma uridine levels were assessed by the method described in Example 1. Both
dietary
cytidine and widine increased plasma uridine levels by a statistically
significantly margin
relative to a control group that was fed chow 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 either water or 1 millimole (mmol)
UMP per kilogram
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(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
Figure 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
EXAMPLE 4
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% methanol, centrifuged, lyophilized and analyzed as
described for Example
RESULTS
.)0
[00175] = To ascertain whether oral administration of uridine can raise
brain uridine
levels, brains of the gerbils from the first experiment in Example 3 were
homogenized, 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;-similarto-the results' observed with-plasma-uridine levels
(Figure-8): These results-indicate
that orally administered uridine is efficiently transported into the brain.
EXAMPLE 5
Uridine is Readily Converted to Cytidine in the Brain
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[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 bloodstream 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,
EXAMPLE 6
Uridine Increases Levels of the Phospholipid Precursor CDP-Choline in the
Brain and in
a Neural Cell Line
METHODS
Experimental design
[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/chloroform (1:2
vol/vol),
centrifuged, and the aqueous phase was dried under vacuum, resuspended in 100-
200 pl. water
and separated by HPLC on an ion-exchange column (Alltech Hypersil APS-2, 5 uM,
250 x 4.6
mm) CDP-choline was eluted with a linear gradient of NaH2PO4 buffers A (1.75
mM
NaH7PO4, 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 nucleotide peaks were detected by UV absorption at 380
nm, and were
identified by comparison with the positions of authentic standards, as well as
by the addition of
nucleotide standards to selected samples
PC12 cells
34
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[00180] P012 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)
NOF 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 show that,
after transport to the brain, uridine is converted to phospholipid precursors
such as CDP-
choline, perhaps via the intermediate CTP, and therefore augments cognitive
function by
increasing synthesis of phospholipid precursors in brain cells.
EXAMPLE 7
Oral Administration of UMP Increases Neurotransmitter Release in Brains of
Aged Rats
METHODS
Animals and dietary UMP supplementation
[00183] Male middle aged Fischer 344 rats, 22-24 months old at the time
of doing
fhicrOdialysis, were Obtained- from NatiOnal InstitUte on 'Aging (Harlan -
Sprague4-)awley, = ---
Indianapolis, TN). Rats were housed individually under standard husbandry
conditions and
exposed to 12 hr light/ dark cycle with food and water provided ad libitum.
Each rat Consumed
approximately 500 mg/kg/day of UMP.2Na (LD50 by i p of uridine is about 4,3
g/Kg).
=
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[00184] Rats were acclimated to the animal facility for more than 7
days before fed a
control laboratory diet (Teklad Global 16% protein rodent diet, TD.0021 7,
Harlan Telclad,
Madison, WI), or this diet fortified with UMP.2Na+ (2.5%, TD.03398, UMP-2Na+;
Numico
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 (F1,11 = 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 changed body weight (F4,44 = 2.65,p <0.05),
while neither UMP-
diet (vs, control) nor UMPxtime interaction affected body weight (F1 j1 =
0.01, F4,44 = 1.25,
respectively; all p > 0.05).
[00186] The experiment described in this Example was performed twice,
each time with
7 control rats and 9 rats administered the UMP diet.
Chemicals and solutions
[00187] Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic
acid
(HVA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and 3,4-
dihydroxybenzoic
acid (DHBA; internal standard) were purchased from Sigma (St, Louis, MO) and
were
dissolved in HC104 (0.1 M) to make 1 mM stock solutions, and aliquots were
kept at -80 C.
20. Ketamine hydrochloride (100 mg/ml) was purchased from Fort Dodge Animal
Health (Fort
Dorge, IA). Xylazine (20 mg/m1) originated from Phoenix Scientific, Inc, (St.
Joseph, MO).
[00188] Ringer solution consisted of NaC1 147, KCI 2.7, CaCI21.2 and
MgC11 0.85 mM.
For high potassium solution, KCI was increased to 80 mM, with NaC1 decreased
to 69,7 mM to
Maintain osmolarity. All solutions were made from doubly distilled deionized
water and filtered
by Steriflip (Millipore, Bedford, MA)..
In vivo nticrodialysis
[00189] Rats were anesthetized with a mixture of ketamine 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
(0.D. 0.24 mm, 4 mm membrane, 6,000 Da, CMA microdialysis, Sweden) was
implanted into
= 36
=
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the right striatum (AP = +0.5, ML = -3.0 from Bregnia, DV = -7.3 mm from Dura,
as described
in Paxinos G et al, The Rat Brain in Stereotaxic Coordinates, 2nd 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 lot 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, and were carried out between 10:00 am
to 4:00 pm.
Ringer's solution was perfused continuously using Fluorinatedethylenepropylene
(FEP) Resin
tubing and a gas-tight syringe (Exmire type 1, CMA), at a constant rate of 1.5
pa/min by a
microinfusion pump (CMA/100). Dialysates were collected at 15-min intervals. 5
l.tl of
antioxidant mixture, consisting of 0.2 M HC104 and 0.1 mM EDTA, was added to
the sampling
vial prior to collection to protect dopamine and its metabolites. The samples
within the first 60
min were discarded from analysis. Subsequently, 3 consecutive sessions of
samples were
collected. Except for the last session (1.5 lus, 6 samples), the others were
collected for 1 hr (4
samples) The order was as follows: session 1 (aCSF), 2 (High K ), 3 (aCSF).
All samples were
collected on crushed ice, instantly frozen and kept at -80 C until HPLC
analysis.
Brain dissection for the proteins and monoamines
[00191] After microdialysis experiments, rats were anesthetized with
lcetamine and
xylazine (80 and 10 mg/Kg, fp.). A black ink was pushed through the probe to
stain the tissue
around 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. Striata
(both left and right)
were collected as above to determine tissue levels of dopamine and its
metabolites.
Extraction of tissue dopamine samples
, 37
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[00193] The
striatum were weighed and homogenized in an Eppendorf tube on ice for 1
mm with 1 ml of H20 containing 0.1 M HC104 and 1 I.LM EDTA_ After vortexing
for 10
seconds, an aliquot was used for Bicinchoninic Acid (Sigma, St. Louis, MO)
protein assay. The
homogenates 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 HPLC, and
values from the
three repeated measures were averaged and normalized to the amount of protein
per sample.
Analysis of dopamine and metabolites
[00194] DA and metabolites in dialysates and tissue samples were determined
using an
ESA Coulochem II 5100A detector (E1 = ¨175 mV; E2 = +325 mV; Eguard = 350 mV)
with an
ESA Microdialysis Cell (model 5014B, ESA, North Chelmsford, MA). The mobile
phase (MD-
TM, ESA) consisted of 75 mM Na1-17PO4, 1.7mM 1-octanesulfonic acid, 100 p.1/L
Triethylamine, 25 p.M EDTA, 10% acetonitrile, pH 3Ø The flow rate was 0.4
mL/min. The
column (ESA MD 150, 3x150 mm, 3 1.tm, 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
AllChromTm data
system, and analyzed with AllChrom plus 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 K4 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
three groups in each time point. A p value cif 05-was
used-ti5 assess -sTatiai-e-al signifkance.
Basal levels of dopamine were homogeneous between the two replicated
experiments and were
therefore pooled into the corresponding groups (F1,20 = 3.99, p> 0.05). Basal
DA levels in the
dialysates were stable after 1 hr equilibration, in the four consecutive
samples prior to K+
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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
supplementation did not affect basal DA levels in the dialysate among
treatment groups (control
vs. UMP- 1 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 K1-evoked striatal
DA release _
(following perfusion with the high-K+ solution) is depicted in Figure 12A. A
statistically
significant difference (F1,266 = 3.36) was found in DA levels in the
dialysates among the
control, UMP- 1 week, and UMP- 6 weeks treatment groups. Post hoc multiple
comparisons
revealed a significant difference between control and UMP-6 weeks' groups.
Data were further
divided into three sections (before, K.-evoked and after), which also revealed
a significant
enhancement of Ktevoked DA release between control and UMP-6 weeks' groups,
from 283
9% to 341 21% (Figure 1213), The UMP-1 week group also exhibited increased
DA release
(316* 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. Ktdepolarization, significantly deceased DOPAC (Figure 1 3A) and HVA
(Figure
I3B) to 65 4% and 51 4% compared to baseline levels in all groups (F1,95 =
51.90, F2,95 =-
92.74, respectively; all p < 0.01). There were no differences in Ktdecreased
DOPAC and HVA
levels among treatment groups (F,,266= 1..01, F1.166 = L20, respectively).
Changing the solution
from high IC back to normal Ringer's solution at 105 min increased both DOPAC
and HVA
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levels in the dialysate, with maximum levels attained at 30 min after changing
(DOPAC, 169
9%; FIVA, 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.
EXAMPLE 8
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-
Keuls multiple
range test was used for the data in Figure 16.
Western blotting
[00203] Striatal
tissues were placed in Eppendorf tubes containing 200 1.d lysis buffer (60
mM Tris¨HC1, 4% SDS, 20% glycerol, 1 mM dithiothreitol, 1 mM AEBSF, 8 j_tM
aprotinin,
500 tiM bestatin, 15
NI E64, 200 p.1v1 leupeptin, 10 1.1M pepstatin A). The samples were
sonicated, boiled (10 min), and centrifuged (14,000 g for 1 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).
... _ [00204] Equal amounts
of protein (40 g protein/lane) were loaded for sodium dodecyl_
sulfate-polyacrylamide gel electrophoresis (4-15% SOS 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 1 h. After 3 10 mM rinses in Tris-buffered saline (TBST), blots were
incubated in TBST
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with various antibodies against the proteins of interest, including NF-70, NF-
M (1: 2000, I:
' 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 Kodak 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
program (NI1-1 V.1.61).
RESULTS
[00205] In order to assess whether increasing uridine levels can augment
the production
of new membrane 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
from 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 <O,01) of control
values,
respectively. Consumption of a UMP diet for 1 week did not increase the levels
of these two
proteins compared to control group 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.
EXAMPLE 9
Oral Administration of Uridine or UTP Increases Neurite Outgrowth and
Branching and
Levels of NF-70 and NF-M in PC 12 cells
METHODS
25 Data analysis
[00206] Data
are presented as mean +1- 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.
. ....
Neurite outgrowth studies
30 [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
41
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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 neurite
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 intracellular UTP and CTP
[00208] Levels of intracellular UTP and CTP were analyzed by HPL,C as
described for
Example 6, except that 5 mM NaH2PO4, pH 2.65 was used as buffer A..
RESULTS
[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 1%). Uridine treatment (50 1.tM, 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 JIM)
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 1.1M) had no
effect. Treatment
of the NGF-exposed cells with 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 with uridine (50 i.tM) 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 -=-=hi ghl y=-enri ched---
within-- = n euri tes;-- th erefore.,-- ==
increase in neurite number should be associated with increased expression of
neurofilament
proteins, NF-70 (70 IcD) and NF-M (145 IcD) levels following 4-day treatment
of PC 12 cells
=
with NGF alone, or NGF plus uridine (50 i..t.M) were thus measured (Figure
16E). Both NF-70
and NF-M expression significantly (p < 0.01, p < 0.001, respectively)
increased following
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uridine treatment, compared to cells treated only with 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 NGF, levels of UTP and CTP were measured
in PC12
cells for 2 days with NGF, treated with no nucleotide, (control), uridine,
cytidine or UTP, in the
presence of NGF. Uridine (50 p.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 uM)
or cytidine (50 i..tM) 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
outgrowth, compared
to that in cells treated only with 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.
EXAMPLE 10
NGF-differentiated PC 12 cells express pvrimidine-sensitive P2Y2, P2Y4 and
P2Y6
receptors
METHODS
Detection of P2 1' 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,
CO). .
Inununocytochenzistry
[00216] PC12 cells were treated as described above, except they were
grown on 12mm
glass cover slips (A. Daigger & Co., Vernon Hills, IL) coated with collagen.
Proteins were
visualized using immunofluorescence. Briefly, the cells were fixed with 4%
paraformaldehyde,
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permeabilized with 0.25% Triton X-100, blocked in 10% normal goat serum, and
incubated
overnight 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 were
incubated with primary antibody plus a control antigen in order to ensure that
the irruntmo-
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 and goat anti-mouse ALEXA 568; Molecular Probes, Eagene, OR) and
mounted
on glass slides with mounting media with or without DAN (Vector Laborntories,
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 with 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 UTP affects neurite outgrowth, it was first
determined
whether 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) higher 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
NC& 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 MAP-2
(Figure 20). In the absence of NGF, receptor protein expression was
undetectable by immuno-
staining Moreover, the presence of uridine did not affect the expression of
the receptors
compared with the quantities present in calls exposed to NGF alone. Thus, the
P2Y2, P2Y4 and
P2Y6 receptors are present in neural cells, but not in their precursors.
EXAMPLE 11
44
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Antagonism of PIS( receptors inhibits the effect of uridine on NGF-induced
neurite
outgrowth
[00218] To ascertain whether signaling by P2Y receptors mediate
induction of neurite
outgrowth by uridine, PC 12 cells were incubated for 4 days with NGF, uridine
(100 M) and
the P2Y receptor antagonists suramin (30 M), pyridoxal-phosphate-6-azopheny1-
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-
stimblated neurite
outgrowth (Figure 21). None of the P2Y receptor antagonists inhibited the
uptake of uridine into
the PC12 cells. These results show that signaling via P2Y receptors mediates
uridine induction
of neurite outgrowth.
EXAMPLE 12
Phosphatidylinositol (IP) signaling is stimulated by UTP and uridine
METHODS
Metabolic labeling and PI turnover analysis
[00219] Analysis of PI turnover was performed as described by (Nitsch RM et
al, J
Neurochem 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 with 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
chromatography,
using AG 1-X8 columns (Bio-Rad), and 1M ammonium formate and 0.1M formic acid
as eluent.
Radioactivity was quantified by liquid scintillation spectrometry.
RESULTS
[00220] P2Y2, P2 Y4 and P2Y6 receptors activate the phospholipase
C/diacylglyeerol/inositol triphosphate (PLC/DAG/1P3) 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 1.1M) for 1 hour, and IP signaling was assessed by measuring turnover of
radio-labeled IP
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(Figure 22). Formation of IF was significantly increased by addition of 100
p.M UTP (p < 0.05)
and by 50 p.M uridine (p <0.01). The P2Y receptor antagonist PPADS (100 .tN4)
significantly
(p < 0.05) blocked the stimulation of' IF signaling by UTP. These findings
indicate that UTP
promotes neurite outgrowth via P2Y receptors-mediated stimulation of the IF
signaling
pathway.
[00221] The findings of Examples 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 IF signaling. Taken
together, the findings
from Examples 7-12 provide further evidence that uridine treatment can improve
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.
EXAMPLE 13
UMP-supplemented diets enhance learning and memory in multiple species
MATERIALS AND EXPERIMENTAL METHODS
Morris Water Maze
[00222] Aging rats (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 in a six-foot
diameter 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 the 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 spatial memory,
Food pellet learning 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
with 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
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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 memory and reference memory 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
=
which only two arms of the maze (but always the same two) contained food
pellet .rewards. In
this test, a working memory error is one in which a gerbil revisits an arm
from which it has
already taken. the pellet that day. A reference memory error is one in which
the gerbil enters an
arm which 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% UMP2Na+ for six weeks, and their memory was tested using
the Morris
water maze, an indicator of spatial memory. Rats administered the UMP-
2Natfortified diet
showed a statistically significant 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== UMP-
containing- chow (0; OA ,
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 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. 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] In addition, the effect- of orally administered uridine- on
working-memory- and - ---
reference memory 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)
47
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[00228]
These findings directly show that (a) uridine dietary supplementation improves
learning and various types (spatial, working, and reference) of memory; (b)
the effect is not
limited to a particular species; and (c) the effect 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.
=
= EXAMPLE -14
=
URIDINE AND CHOLINE INCREASE NEUROTRANSMITTER RELEASE
MATERIALS AND EXPERIMENTAL METHODS
Brain slice preparation
[00230]
Male Sprague-Dawley rats, 9-11 months old, were anesthetized with ketamine
(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 NaC1,
3.3 mM KC1, 1.3 mM CaC12, L2 mM .MgSO4, 25 mM NaHCO3, 1.2 mM, KII2PO4, 11 mM
glucose, and 0.03 mM EDTA,
7.4) containing 1 mM ketamine and 15 g/m1 eserine. After
removal of remaining meninges and chorioid plexus, 30 ptm slices of striatum,
hippocampus,
and cortex were immediately prepared with a McIllwain tissue chopper, washed 3
times, and
=
. .2Ø.. .placed into custom-made superfusion chambers (Warner Instrument,
Hamden, CT).
Superfusion and electrical stimulation.
[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 Instruments).. A custom-made polarity
reversal device
was used to prevent chamber polarization and also to monitor both the current
and voltage 50
..............microseconds_ after. the _onset of each õprise
toesrØnIfnn....çh.ambe.e.the.....
equilibration period, slices were depolarized by perfusion with a high-K (52
mM) version of
the Krebs/ketamine/eserine buffer in the presence or absence of 20 p.M
choline, 25 JAM
cytidine, and/or 25 p.M uridine. Perfusates were collected during the entire 2-
hour period and
assayed for acetylcholine.. Values were normalized for protein content of
slices.
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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 whether or
not uridine was
also present (Figure 26).
[00233] These findings show that when neurons are repeatedly stimulated
to release
acetylcholine, choline increases the amount of neurotransmitter that is
released, by replenishing
stores of choline in membrane phospholipids (e.g, PC). The above Examples have
shown that
uridine augments synthesis of CDP-choline, which 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.
EXAMPLE 15
URIDINE AND CHOLINE INCREASE NEUROTRANSMITTER RELEASE
ADDITIVELY FOLLOWING REPEATED 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
enhanced 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 further increased by the presence of uridine and
choline.
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