RU2436571C2 - Compositions containing polyunsaturated fatty acids (pufa), and methods of application thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to neurology, and can be used for increasing a level of synaptic proteins in a nerve cell or a brain cell in a subject. That is ensured by introduction a combination of agents to the specified subject, namely omega-3 fatty acid and/or omega-6 fatty acid in a combination with uridine or choline salts.

EFFECT: versions of the method provide the increased level of synaptic proteins in the nerve cell, including the brain cell that can be used for correction of age-related memory disorders.

15 cl, 11 dwg, 7 tbl, 11 ex

Description

FIELD OF THE INVENTION

The present invention provides methods for increasing or enhancing the synthesis and levels of phospholipids, synapses, synaptic proteins and synaptic membranes by a nerve cell or brain cell; methods for treating a subject with a memory disorder, memory impairment, a neurological disease or a brain disease or disorder, comprising administering to the subject a composition comprising an omega-3 fatty acid, omega-6 fatty acid, uridine, a metabolic precursor thereof, or a combination thereof.

State of the art

Dementia (dementia) is a brain disorder that seriously affects a person's ability to exercise his daily activity. The most common form of dementia among older people is Alzheimer's disease (AD), which first affects the parts of the brain that control mental activity, memory, and speech. The deterioration of memory and cognitive (mental) function is considered by most experts as a natural consequence of the aging process. People with age-related diseases of the central nervous system (CNS) demonstrate impaired memory and learning ability, decreased attention and concentration, impaired mental ability, the use of speech and other mental abilities. Methods of treating these diseases and disorders are urgently needed in the art.

Disclosure of invention

The present invention provides methods for increasing or enhancing the synthesis and levels of phospholipids, synapses, synaptic proteins and synaptic membranes by a nerve cell or brain cell; methods for treating a subject with a memory disorder, memory impairment, a neurological disease or a brain disease or disorder, comprising administering to the subject a composition comprising an omega-3 fatty acid, omega-6 fatty acid, uridine, a metabolic precursor thereof, or a combination thereof.

In one embodiment, the present invention provides a method for increasing the number of synaptic membranes of a nerve cell or brain cell of a subject with an age-related memory disorder, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which results in an increase in the number of synaptic membranes of a nerve cell or brain cell of a subject with age-related memory disorder.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes of a nerve cell or brain cell of a subject with an age-related memory disorder, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which results in an increase in the number of synaptic membranes of a nerve cell or brain cell of a subject with age-related memory disorder.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes of a nerve cell or brain cell of a subject with an age-related memory disorder, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, which leads to an increase in the number of synaptic membranes of a nerve cell or brain cell of a subject with age-related memory disorder.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes of a nerve cell or brain cell of a subject with an age-related memory disorder, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which leads to an increase in the number of synaptic membranes of a nerve cell or brain cell of a subject with an age-related memory disorder.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, memory impairment or memory loss, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances the synthesis of phospholipid by a nerve cell, providing thus treating a subject with Alzheimer's disease, impaired memory, or memory loss.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, impaired memory or memory loss, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances the synthesis of phospholipid by a nerve cell, providing thus treating a subject with Alzheimer's disease, impaired memory, or memory loss.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, memory impairment or memory loss, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing treatment for a subject with Alzheimer's disease, impaired memory, or memory loss.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, memory impairment or memory loss, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing treatment for a subject with Alzheimer's disease, memory impairment or memory loss.

In another embodiment, the present invention provides a method of treating a subject with a memory disorder selected from Peak's disease (Preschool Limited Brain Atrophy), Lewy Body disease, Huntington's disease, and minimal cognitive impairment (MCI) ), comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor increases the synthesis of a phospholipid by a nerve cell, thereby providing treatment for sub An object with memory impairment selected from Peak's disease, Levy's disease, Huntington's disease, and MCI.

In another embodiment, the present invention provides a method of treating a subject with a memory disorder selected from Peak's disease, Levy's disease, Huntington's disease and MCI, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic the precursor enhances the synthesis of phospholipid by the nerve cell, thereby providing treatment for a subject with a memory disorder selected from Pick disease, Lewy body disease, Huntington's disease and MCI.

In another embodiment, the present invention provides a method of treating a subject with a memory disorder selected from Peak's disease, Levy's disease and Huntington's disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing treatment for a memory impaired subject.

In another embodiment, the present invention provides a method of treating a subject with impaired memory, selected from Pick's disease, Lewy body disease and Huntington's disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative uridinfosfat or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of phospholipid by a nerve cell, thereby providing treatment for a memory impaired subject.

In another embodiment, the present invention provides a method for increasing the size or number of synapses in the brain of a subject with age-related memory impairment, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, thereby providing an increase in the size or number of synapses in the brain of a subject with age-related impairment memory.

In another embodiment, the present invention provides a method for increasing the size or number of synapses in the brain of a subject with age-related memory impairment, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, thereby providing an increase in the size or number of synapses in the brain of a subject with age-related impairment memory.

In another embodiment, the present invention provides a method for increasing the size or number of synapses in the brain of a subject with age-related memory impairment, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, thereby providing an increase in the size or number of synapses in the brain of a subject with age-related memory impairment.

In another embodiment, the present invention provides a method of increasing the size or number of synapses in the brain of a subject with age-related memory impairment, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, thereby providing an increase in the size or number of synapses in the brain of a subject with age-related memory impairment.

In another embodiment, any of the methods and any of the compositions of the present invention includes the introduction of omega-3 fatty acids and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering an omega-3 fatty acid and a choline salt. In another embodiment, any of the methods and any of the compositions of the present invention includes the introduction of omega-6 fatty acids and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering an omega-6 fatty acid and a choline salt.

In another embodiment, any of the methods and any of the compositions of the present invention comprises administering a composition comprising an omega-3 fatty acid, uridine and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering a composition comprising an omega-3 fatty acid, uridine, and choline salt. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering a composition comprising an omega-6 fatty acid, uridine and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering a composition comprising an omega-6 fatty acid, uridine, and choline salt.

In another embodiment, any of the methods and any of the compositions of the present invention comprises administering an omega-6 fatty acid and an omega-3 fatty acid. In another embodiment, any of the methods and any of the compositions of the present invention includes the introduction of omega-6 fatty acids, omega-3 fatty acids and uridine. In another embodiment, any of the methods and any of the compositions of the present invention includes the introduction of omega-6 fatty acids, omega-3 fatty acids and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering an omega-6 fatty acid, an omega-3 fatty acid, and a choline salt. In another embodiment, any of the methods and any of the compositions of the present invention includes the introduction of omega-6 fatty acids, omega-3 fatty acids, uridine and choline. In another embodiment, any of the methods and any of the compositions of the present invention comprises administering an omega-6 fatty acid, an omega-3 fatty acid, uridine, and a choline salt.

Brief Description of the Figures

Figure 1: DHA increases the synthesis of phospholipids in PC12 cells. PC12 cells were incubated overnight with fatty acids, then incubated in a medium containing ¹⁴ C-labeled choline. The graph shows the incorporation of the ¹⁴ C label into phosphatidylcholine in decays per minute (dpm) per microgram (μg) of DNA. DHA: docosahexaenoic acid. OA: oleic acid. RA: palmitic acid. * - p <0.05.

Figure 2: The increase in the synthesis of phospholipids under the action of DHA is dose-dependent. * - p <0.05. ** - p <0.001.

Figure 3. A. Arachidonic acid enhances the synthesis of phospholipids by SHSY-5Y cells. DHA: docosahexaenoic acid. AA: arachidonic acid. RA: palmitic acid. *: p <0.05. **: p <0.001. B. An increase in the synthesis of phospholipids by AA is dose-dependent.

Figure 4. DHA and UMF act synergistically to increase levels of phospholipids in the brain when tested on a whole animal. "*": Significantly higher than the control group in a one-way ANOVA analysis. A. pmol phospholipid per milligram (mg) of protein. The sum of UMF + DHA was significantly higher than the control (p <0.05) (one-way ANOVA analysis [F (3.28) = 4.12; p = 0.015]). Two-way ANOVA analysis also revealed a statistically significant effect of DHA with respect to the control group [F (1.28) = 8.78; p = 0.006]. B. picomol phospholipid per μg of DNA. The sum of UMF + DHA was significantly higher than the control (p = 0.020) (one-way ANOVA analysis [F (3.28) = 3.215; p = 0.038]).

Figure 5. The effect of DHA on the levels of CDP-choline (A) and CDP-ethanolamine (B) in the brain. Groups of 8 gerbils received either a control diet or an UMF-containing diet, and through a gastric probe DHA (as a solution in 5% gum arabic) or a solution of 5% gum arabic without additives for 28 days. On day 29, brains were collected and analyzed for CDP-choline content. Data are presented as mean values ± SEM. Statistical analysis was performed using a one-way or two-way ANOVA analysis with the Tukey posterior test. a: P <0.05 when compared with the values for the group with a control diet plus a solution of gum arabic; b: P <0.05 when compared with the values for the group with the UMF-containing diet plus a solution of gum arabic.

Figure 6. The effect of the UMF-containing diet and DHA on the levels of NF-70 (A) and NF-M (B) in the brain. Gerbils received the diets described in the caption to figure 5 for 21 days (left panels) or 28 days (right panels). On days 22 and 29, brains were collected and analyzed for NF-70 content. Data are presented as mean values ± standard error of the mean (SEM). Statistical analysis was performed using a one-way ANOVA analysis followed by a Tukey test. A. **: P <0.01; ***: P <0.001 when compared with the values for the control diet group + gum arabic solution. AT). * P <0.05; ** P <0.01. No significant differences were found between the groups in the content of beta-tubulin cytoskeleton protein.

Figure 7. The effect of the UMF-containing diet and DHA on the levels of PSD-95 and synapsin-1 in the brain. A) Gerbils received either a control diet, plus, through a stomach tube, a 5% solution of gum arabic, or a UMF-containing (0.5%) diet, plus, through a stomach tube, DHA (300 mg / kg) in 5% gum arabic as a carrier within 7 days (left panels) or 21 days (right panels). The next day, brains were collected and analyzed for PSD-95 (A) or Synapsin-1 (B). A. Data are presented as means ± SEM. Statistical analysis was performed using a one-way ANOVA analysis followed by a Tukey test. ** P <0.01; *** P <0.001 when compared with the values for the control diet group plus a solution of gum arabic. AT). * P <0.05; ** P <0.01.

Figure 8. Increased density of dendritic spines in the hippocampus of adult gerbils.

Figure 9. The effect of uridine and / or DHA on learning.

Figure 10. The effect of DHA on the synthesis of phospholipids in the culture of hippocampal neurons. Vertical axis: ¹⁴ C DPM / 50 μl of sample.

The implementation of the invention

The present invention provides methods for increasing or enhancing the synthesis and levels of phospholipids, synapses, synaptic proteins and synaptic membranes by a nerve cell or brain cell; methods for treating a subject with a memory disorder, memory impairment, a neurological disease or a brain disease or disorder, comprising administering to the subject a composition comprising an omega-3 fatty acid, omega-6 fatty acid, uridine, a metabolic precursor thereof, or a combination thereof.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's nerve cell, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the subject's nerve cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment of the methods and compositions of the present invention, omega-3 fatty acid, omega-6 fatty acid, uridine, choline, choline salt, or a combination thereof, are incorporated into a pharmaceutical composition.

In another embodiment of the methods and compositions of the present invention, the composition that is administered increases the synthesis of a phospholipid by a nerve cell or brain cell of a subject. In another embodiment, an omega-3 fatty acid enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject. In another embodiment, an omega-6 fatty acid enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject. In another embodiment, uridine, its acyl derivative, uridine phosphate or CDP-choline enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject. In another embodiment, choline enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject. In another embodiment, a choline salt enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's brain cell, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which provides an increase in the phospholipid level in the subject's brain cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing or enhancing the synthesis of a phospholipid by a nerve cell or brain cell of a subject, comprising administering to the subject an omega-3 fatty acid or a metabolic precursor thereof, which provides an increase or enhancing the synthesis of phospholipid by a nerve cell or brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As provided here, the results presented in Examples 1 and 5 show that the administration of docosahexaenoic acid (DHA), omega-3 fatty acid into nerve cells and brain cells increases their synthesis of phospholipids, as can be seen from the increased incorporation of labeled choline. Administration of omega-3 fatty acid enhances the synthesis of total phospholipids, phosphatidylcholine and phosphatidylethanolamine (Example 2), showing that its effect is not limited to certain phospholipids. PC 12 cells exhibit differentiated nerve cell functions and are commonly used in the art as a model nerve cell line. The results presented in example 11 show that omega-3 fatty acids increase the synthesis of phospholipids in nerve cells in a short-term (surviving) culture.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes in a nerve cell or brain cell of a subject, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which provides an increase in the number of synaptic membranes in a nerve cell or brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the phospholipid, the level of which is increased by the methods and compositions of the present invention, is phosphatidic acid. In another embodiment, the term “phosphatidic acid” is synonymous with the term “phosphatide”. In another embodiment, the phospholipid is phosphatidylcholine (“PF”; Example 1). In another embodiment, the phospholipid is phosphatidylethanolamine (“PE”; Example 2). In another embodiment, the phospholipid is phosphatidylserine (“PS”). In another embodiment, the phospholipid is phosphatidylinositol (“PHI”). In another embodiment, the phospholipid is sphingomyelin. In another embodiment, the phospholipid is a phosphoglyceride. In another embodiment, the phospholipid is any other phospholipid known in the art. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the PI level significantly increased using the methods of the present invention acts as a reservoir of one or more secondary messenger molecules. In another embodiment, the secondary messenger molecule is inositol-1,4,5-trisphosphate (IP ₃ ). In another embodiment, the secondary messenger molecule is diacylglycerol (DAG). In another embodiment, the signaling pathway through protein kinase C (PKC) is enhanced by the methods and compositions of the present invention. In another embodiment, a signaling pathway below the site of action of IP ₃ is activated using the methods and compositions of the present invention. In another embodiment, intracellular calcium levels are increased using the methods and compositions of the present invention. In another embodiment, a signaling pathway below the site of action of the DAG is activated using the methods and compositions of the present invention. In another embodiment, a signaling pathway located below the site of action of the PKC is activated using the methods and compositions of the present invention. In another embodiment, the signaling pathway below the site of action of intracellular calcium is activated using the methods and compositions of the present invention.

In another embodiment, sphingomyelin, the level of which is increased by the methods and compositions of the present invention, acts as a source of ceramide. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the DHA and / or MFIs in the methods and compositions of the present invention act as common precursors of cellular phospholipids. In another embodiment, UMF acts by activating P2Y receptors for UMF derived from uridine. In another embodiment, DHA acts by activating syntaxin-3. In another embodiment, a combination of these mechanisms is used.

In another embodiment, as can be seen from the data presented here, the administration of DHA and / or UMF is effective for the treatment and / or prevention of disorders characterized by insufficient synapse formation or impaired myelination. Each opportunity represents a separate embodiment of the present invention.

As shown here, the administration of PUFAs and / or uridine to gerbils, the exchange of pyrimidines in which is similar to that in humans, increases the levels of neurofibrillar axon proteins of nerve cells NF-70 and NF-M, the protein of the postsynaptic dense sections of PSD-95 and the vesicular protein Synapse-1 ( example 7). Thus, the introduction of PUFA increases the levels of synaptic membranes in the cells of the brain and nerves. In another embodiment, under the conditions used herein, the methods and compositions of the present invention are also useful for enhancing transmission of nerve signals. In another embodiment, under the conditions used herein, the methods and compositions of the present invention are also useful for enhancing the functioning of nerve cells. In another embodiment, under the conditions used herein, the methods and compositions of the present invention are also useful for enhancing the germination of axons of nerve cells. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the omega-3 fatty acid used in the methods and compositions of the present invention is an omega-3 polyunsaturated fatty acid (PUFA). In another embodiment, the omega-3 fatty acid is DHA (examples 1-2). DHA is an omega-3 polyunsaturated fatty acid containing 22 carbon atoms, also known as 4,7,10,13,16,19-docosahexaenoic acid.

In another embodiment, the omega-3 fatty acid is α-linolenic acid (9,12,15-octadecatrienoic acid). In another embodiment, the omega-3 fatty acid is stearidonic acid (6,9,12,15-octadecatetetraenoic acid). In another embodiment, the omega-3 fatty acid is eicosatrienoic acid (ETK; 11,14,17-eicosatrienoic acid). In another embodiment, the omega-3 fatty acid is eicosatetraenoic acid (8,11,14,17-eicosatetraenoic acid). In another embodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA; 5,8,11,14,17-eicosapentaenoic acid). In another embodiment, the omega-3 fatty acid is eicosahexaenoic acid (also known as “EHCs”; 5,7,9,11,14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acid is docosapentaenoic acid (DPA; 7,10,13,16,19-docosapentaenoic acid). In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid (6,9,12,15,18,21-tetracosahexaenoic acid). In another embodiment, the omega-3 fatty acid is any other omega-3 fatty acid known in the art. Each omega-3 fatty acid is a separate embodiment of the present invention.

In another embodiment, the omega-3 fatty acid is an anti-inflammatory PUFA. In another embodiment, the anti-inflammatory PUFA is eicosapentaenoic acid (EPA; 5,8,11,14,17-eicosapentaenoic acid). In another embodiment, the anti-inflammatory PUFA is DHA. In another embodiment, the anti-inflammatory PUFA is any other anti-inflammatory PUFA known in the art. Each opportunity represents a separate embodiment of the present invention.

As shown here, DHA, EPA and AA increase the levels of phospholipids in the brain (Example 8). Thus, the effects described here are not specific for a particular PUFA, but rather, under the conditions used here, are common to the entire family of omega-3 and omega-6 PUFAs.

In another embodiment, the omega-3 fatty acid is a metabolic precursor of DHA. In another embodiment, the metabolic precursor is EPA. In another embodiment, the metabolic precursor is docosapentaenoic acid (DPA; 7,10,13,16,19-docosapentaenoic acid). Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the term “metabolic precursor” refers to a compound that increases the concentration of fatty acid in the bloodstream or in tissues. In another embodiment, the term “metabolic precursor” refers to a compound that is metabolized by a subject tissue or enzyme to a free fatty acid. In another embodiment, the term “metabolic precursor” refers to a compound that is metabolized by the target cell to a free fatty acid. Each opportunity represents a separate embodiment of the present invention.

In another embodiment of the methods and compositions of the present invention, the metabolic precursor of omega-3 fatty acid is alpha-linolenic acid, which acts as a precursor of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). In another embodiment, the metabolic precursor is any other omega-3 fatty acid precursor known in the art. Each omega-3 fatty acid precursor is a separate embodiment of the present invention.

In another embodiment, the term “PUFA” means omega-3 fatty acid. In another embodiment, the term refers to omega-6 fatty acid. In another embodiment, the term refers to a fatty acid with 2 or more double bonds. In another embodiment, the term denotes a fatty acid with 2 double bonds. In another embodiment, the term denotes a fatty acid with 3 double bonds. In another embodiment, the term refers to a fatty acid with more than 3 double bonds. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes in a nerve cell or brain cell of a subject, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides an increase in the number of synaptic membranes in a nerve cell or brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's nerve cell, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the subject's nerve cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's brain cell, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides an increase in the phospholipid level in the subject's brain cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing or enhancing the synthesis of a phospholipid by a nerve cell or brain cell of a subject, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides an increase or enhancing the synthesis of phospholipid by a nerve cell or brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, the results presented in Example 3 demonstrate that the administration of arachidonic acid, an omega-6 fatty acid, increases the synthesis of phospholipid by nerve cells and brain cells, as can be seen from the increased inclusion of labeled choline. SHSY-5Y cells are derived from human neuroblastomas and are used as a model system of neuronal functions. In another embodiment, increased synthesis of phospholipids leads to an increase in their levels.

In another embodiment, the omega-6 fatty acid is an omega-6 polyunsaturated fatty acid (PUFA). In another embodiment, the omega-6 fatty acid is arachidonic acid (Example 3). Arachidonic acid is an omega-6 containing 20 carbon atoms fatty acid, also known as 5,8,11,14-eicosatetraenoic acid. In another embodiment, the omega-6 fatty acid is a metabolic precursor of arachidonic acid. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the omega-6 fatty acid is linoleic acid (9,12-octadecadienoic acid). In another embodiment, the omega-6 fatty acid is conjugated linoleic acid (CLA). In another embodiment, the omega-6 fatty acid is γ-linolenic acid (6,9,12-octadecatrienoic acid). In another embodiment, the omega-6 fatty acid is eicosadienoic acid (11,14-eicosadienoic acid). In another embodiment, the omega-6 fatty acid is homo-γ-linolenic acid (8,11,14-eicosatrienoic acid). In another embodiment, the omega-6 fatty acid is docosadienoic acid (13.16-docosadienoic acid). In another embodiment, the omega-6 fatty acid is docosatetraenoic acid (7,10,13,16-docosatetraenoic acid). In another embodiment, the omega-6 fatty acid is 4,7,10,13,16-docosapentaenoic acid. In another embodiment, the omega-6 fatty acid is dihomo-gamma-linolenic acid (DGLA). In another embodiment, the omega-6 fatty acid is any omega-6 fatty acid known in the art. Each omega-6 fatty acid is a separate embodiment of the present invention.

In another embodiment of the methods and compositions of the present invention, the omega-6 fatty acid metabolic precursor is linoleic acid. In another embodiment, the metabolic precursor is trans-vaccine acid (TVA), which is the source of linoleic acid. In another embodiment, the metabolic precursor is any other omega-6 fatty acid precursor known in the art. Each omega-6 fatty acid precursor is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's nerve cell, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the subject's nerve cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, each of the omega-3 fatty acids and omega-6 fatty acids act synergistically with uridine (e.g., UMF) to increase the synthesis of phospholipids and phospholipid levels. In another embodiment, the uridine phosphate is uridine monophosphate (UMF).

In another embodiment, the present invention provides a method for increasing a phospholipid level in a brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a subject's nerve cell, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the subject's nerve cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing a phospholipid level in a brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides an increase in the level of phospholipid in the brain cell of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing or enhancing the synthesis of a phospholipid in a nerve cell or brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, which provides an increase or an increase in the synthesis of phospholipid in a nerve cell or brain cell of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing or enhancing the synthesis of a phospholipid in a nerve cell or brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides an increase or enhancement of phospholipid synthesis in a nerve cell or brain cell of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes in a nerve cell or brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, which provides an increase in the number of synaptic membranes in a nerve cell or brain cell of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synaptic membranes in a nerve cell or brain cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides an increase in the number of synaptic membranes in a nerve cell or brain cell of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

Methods for measuring the number of synaptic membranes are well known to those skilled in the art and are described, for example, in Oertner TG et a.l (Facilitation at single synapses probed with optical quantal analysis. Nat Neurosci. 2002 Jul; 5 (7): 657-64); Bloodgood BL et al. (Neuronal activity regulates diffusion across the neck of dendritic spines. Science. 2005 Nov 4; 310 (5749): 866-9); El Fakhri G et al. (Generalized five-dimensional dynamic and spectral factor analysis. Med Phys. 2006 Apr; 33 (4): 1016-24); and Pautler RG. Biological applications of manganese-enhanced magnetic resonance imaging. Methods Mol Med. 2006; 124: 365-86). Each method is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell, thereby providing treating a subject with Alzheimer's disease.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell, thereby providing treating a subject with Alzheimer's disease.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, comprising contacting a subject's nerve cell with a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or PCF-choline; and (b) an omega-3 fatty acid or its metabolic precursor, wherein the composition enhances phospholipid synthesis by a nerve or brain cell, thereby providing treatment for a subject with Alzheimer's disease.

In another embodiment, the present invention provides a method of treating a subject with Alzheimer's disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve or brain cell, thereby providing treatment for a subject with Alzheimer's disease.

As shown here, compositions comprising omega-3 fatty acids, omega-6 fatty acids and / or uridine increase the number of synaptic membranes in nerve cells. Thus, the compositions of the present invention are useful for the treatment of Alzheimer's disease.

In another embodiment of the methods and compositions of the present invention, stimulation of phospholipid synthesis increases phospholipid levels in brain target cells or target nerve cells. In another embodiment, sufficient phospholipid levels are important for many aspects of nerve functioning, for example, signal transmission at synapses, neurotransmitter functioning, axon branching and germination, etc., and are also important, in another embodiment, for the proper functioning of the brain.

In another embodiment, Alzheimer's disease is at an early stage of development. In another embodiment, Alzheimer's disease is at a mild stage of development. In another embodiment, Alzheimer's disease is at a moderate stage of development. In another embodiment, Alzheimer's disease is in a late stage of development. In another embodiment, Alzheimer's disease is at a severe stage of development. In another embodiment, Alzheimer's disease is at an undetermined stage of development. In another embodiment, Alzheimer's disease is at any stage in the development of a disease known in the art. Each stage of the disease is a separate embodiment of the present invention.

In another embodiment, the stage of development of Alzheimer's disease is determined using the Functional Assessment Staging (FAST) scale. The FAST scale was developed by Dr. Reisberg and colleagues. She divides the development of Alzheimer's disease into 16 consecutive stages, combined into 7 main groups according to the functional capabilities of a person and the existing disorders. The main groups are as follows. Stage 1 is defined as a normal adult without impairing functionality or memory impairment. Stage 2 is defined as a normal elderly person with certain personal signs of reduced functionality, usually complaining of memory problems and forgetting the names of friends and places. Stage 3 (an early stage of Alzheimer's disease) becomes noticeable in certain life situations. Signs of this stage include one or more of the following: loss of orientation when traveling in unfamiliar places; a message from colleagues about a decrease in performance; difficulties with the selection of names or words; decreased ability to retell the information read in the book, or to remember the name of the person whom he just met; loss of valuables; decrease in concentration. At stage 4 (the mild stage of Alzheimer's disease), the patient may need help with complex tasks, such as scheduling appointments or financial transactions, demonstrating problems associated with recollections of events from his own life, and also has difficulty concentrating and traveling. At stage 5 (mild stage of Alzheimer's disease), the patient needs help with everyday activities, such as choosing the right clothes. There is a loss of orientation in time and an inability to retell important information from one's current life, however, the patient can still recall basic information about himself, his family, and other people. At stage 6 (moderately severe stage of Alzheimer's disease), the patient begins to forget a significant amount of information about himself and his surroundings and requires help when dressing, washing and using the toilet. Urinary incontinence and troubled sleep patterns are observed. Personality changes and emotional changes become clearly noticeable. Because patients cannot remember the information long enough to act in accordance with their thoughts, they lose their willpower (cognitive abulia). At stage 7 (severe Alzheimer's disease), the ability to talk becomes limited to six or seven words, and the vocabulary can be reduced to one word. The patient loses the ability to move, sit upright, smile and, in the end, cannot hold his head. Each stage of Alzheimer's disease is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the level of PF in the brain of a subject, comprising administering to the subject a composition of the present invention, where the composition increases the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the level of PF in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the level of sphingomyelin (CM) in the brain of a subject, comprising administering to the subject a composition of the present invention, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the level of CM in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the level of PHI in the brain of a subject, comprising administering to the subject a composition of the present invention, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the level of PHI in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the level of PV in the brain of a subject, comprising administering to the subject a composition of the present invention, wherein the composition increases the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the level of PV in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the level of PS in the brain of a subject, comprising administering to the subject a composition of the present invention, wherein the composition increases the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the level of PS in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for improving the cognitive function of a subject, comprising administering to the subject a composition of the present invention, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby improving the cognitive function of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, DHA and UMF improve the animals' memory test (Example 10). Thus, the methods and compositions of the present invention are effective in improving and enhancing memory and other cognitive functions.

As shown here, administration of PUFA and / or uridine increases levels and synthesis of phospholipids in the brain, levels of axon neurofibrillar proteins, and the number of synaptic membranes. Thus, the compositions and methods of the present invention enhance and enhance cognitive function, neurological function, mental ability, synaptic transmission and levels, and the activity of neurotransmitters.

In another embodiment, the present invention provides a method for improving the neurological function of a subject, comprising administering to the subject a composition of the present invention, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby improving the neurological function of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, a neurological function that is improved by the method of the present invention is synaptic transmission. In another embodiment, synaptic transmission refers to a motor neuron. In another embodiment, the synaptic transmission refers to an insertion neuron. In another embodiment, synaptic transmission refers to a sensitive neuron. Each type of synaptic transmission is a separate embodiment of the present invention.

In another embodiment, a neurological function that is improved or enhanced is the functioning of the neurotransmitter. In one embodiment, improving or enhancing the functioning of the neurotransmitter is achieved by increasing the level of the neurotransmitter in the synapse. In another embodiment, improving or enhancing the functioning of the neurotransmitter is achieved by increasing the release of the neurotransmitter at the synapse. In one embodiment, improving or enhancing the functioning of the neurotransmitter is achieved without changing the level or releasing the neurotransmitter at the synapse. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, “improving” cognitive or neurological function or cognitive ability means a 10% improvement in a given parameter. In another embodiment, this term means a 20% improvement in this parameter. In another embodiment, the term means a 30% improvement in a given parameter. In another embodiment, the term means a 40% improvement in a given parameter. In another embodiment, this term means a 50% improvement in a given parameter. In another embodiment, this term means 60% improvement of this parameter. In another embodiment, the term means a 70% improvement in a given parameter. In another embodiment, the term means 80% improvement in a given parameter. In another embodiment, this term means 90% improvement of this parameter. In another embodiment, this term means 100% improvement of this parameter. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, an improvement in cognitive or neurological function or cognitive ability is evaluated compared to that function before treatment. In another embodiment, the improvement is evaluated in comparison with the untreated subject. In another embodiment, the improvement is evaluated according to a standardized criterion, such as, for example, a test or the like. Each type of improvement in cognitive activity is a separate embodiment of the present invention.

In another embodiment, an improvement in cognitive or neurological function or mental ability is measured by the number of contacts between neurons in the brain of the subject. In another embodiment, the improvement is measured by the number of capillaries in the brain of the subject, or in a specific area of the brain of the subject. In another embodiment, improvement is judged by neural function. In another embodiment, improvement is judged by linguistic function. In another embodiment, improvement is judged by communication ability. In another embodiment, improvement is measured by measuring the levels of acetylcholine or other neurotransmitters or other brain substances that are correlated with cognitive function. In another embodiment, the improvement is evaluated by scanning a subject's brain using Positron Emission Tomography (PET). In another embodiment, the improvement is measured by scanning the brain of a subject using magnetic resonance imaging (MRI). In another embodiment, improvement is assessed using a Cognitive Abilities Screening Instrument (CAST) (Peila R et al. Stroke. 32: 2882-9,2001). In another embodiment, the improvement is assessed using a test, such as, for example, the test disclosed herein (Example 13). In another embodiment, the Mini-Mental Test (Tsai L et al. The Mini-Mental State Test and computerized tomography. Am J Psychiatry. 1979 Apr; 136 (4A): 436-8) is used. Additional methods for assessing improvement in cognitive function are well known in the art and are described, for example, in Antonova E et al. (Schizophr Res. 2004 Oct 1; 70 (2-3): 117-45) and Cognitive Function analysis (Greenwood Pub Group, 1998). Each method is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting a decrease in the number of synapses in a patient’s brain, comprising administering to a subject a composition of the present invention, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby inhibiting a decrease in the number of synapses in a patient’s brain. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the amount or level of a neurotransmitter in the brain or central nervous system of a subject, the method comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor increases the synthesis of phospholipid in the brain or The central nervous system, thus increasing the amount or level of a neurotransmitter in the brain or central nervous system of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the amount or level of a neurotransmitter in the brain or central nervous system of a subject, a method comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor increases the synthesis of phospholipid in the brain or The central nervous system, thus increasing the amount or level of a neurotransmitter in the brain or central nervous system of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the amount or level of a neurotransmitter in the brain or central nervous system of a subject, a method comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, wherein the composition increases the synthesis of a phospholipid in the brain or central nervous system, thereby increasing the amount or level of a neurotransmitter in the brain or central nervous system of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the amount or level of a neurotransmitter in the brain or central nervous system of a subject, a method comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, wherein the composition increases the synthesis of a phospholipid in the brain or central nervous system, thereby increasing the amount or level of a neurotransmitter in the brain or central nervous system of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In one embodiment, the neurotransmitter whose levels or activity or release is altered by the methods of the present invention is acetylcholine. In another embodiment, the neurotransmitter is glutamate. 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 a GABA. In another embodiment, the neurotransmitter is any other neurotransmitter known in the art. Each type of neurotransmitter is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing or enhancing a subject's nerve cell ability to repeatedly release an effective amount of a neurotransmitter into a synapse, a method comprising administering to a subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor increases synthesis phospholipid by a brain cell, thereby increasing or enhancing the ability of a subject’s brain cell to repeatedly release an effective amount of rotransmittera into the synapse. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, the density of dendritic spines increases in animals that were injected with DHA and / or uridine (Example 9). Thus, the compositions of the present invention increase the number and size of synapses in the brain and the ability of brain cells to transmit signals using neurotransmitters.

In another embodiment, the present invention provides a method for increasing or enhancing a subject's brain cell ability to repeatedly release an effective amount of a neurotransmitter into a synapse, a method comprising administering to a subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor increases synthesis phospholipid by a brain cell, thereby increasing or enhancing the ability of a subject's brain cell to repeatedly release an effective amount of neurons Transmitter into the synapse. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing or enhancing a subject's brain cell ability to repeatedly release an effective amount of a neurotransmitter into a synapse, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a brain cell, thereby increasing or enhancing the ability of a subject's brain cell to repeatedly release an effective amount of a neurotransmitter to a synapse. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing or enhancing a subject's brain cell ability to repeatedly release an effective amount of a neurotransmitter into a synapse, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a brain cell, thereby increasing or enhancing the ability of a subject's brain cell to repeatedly release an effective amount of a neurotransmitter into a synapse. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, administration of PUFA and / or uridine increases levels and synthesis of phospholipids in the brain, levels of neurofibrillary proteins in axons, and the number of synaptic membranes. Thus, the compositions and methods of the present invention increase and enhance the release and amount of neurotransmitters.

In another embodiment, the present invention provides a method for improving or enhancing a subject’s cognitive ability, comprising administering to the subject a composition of the present invention, wherein the composition enhances the synthesis of a phospholipid by a subject nerve or brain cell, thereby improving or enhancing the subject’s cognitive ability. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of dendritic spines in the brain or a specific area of the brain of a subject, comprising administering to the subject a composition of the present invention, wherein the composition increases the synthesis of a phospholipid by a nerve cell or brain cell of the subject, thereby increasing the number of dendritic spines in the brain or a specific area of the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, a cognitive ability that is improved or enhanced by the methods and compositions of the present invention is a language ability. In another embodiment, a thinking ability is a musical ability. In another embodiment, mental ability is the ability to navigate in space. In another embodiment, a thinking ability is a physical ability. In another embodiment, mental ability is the ability for interpersonal communication. In another embodiment, mental ability is the ability to self-esteem. In another embodiment, mental ability is the ability for interpersonal communication. In another embodiment, mental ability is a logical-mathematical ability. In another embodiment, mental ability is any other type of mental ability known in the art. Each type of mental ability is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of facilitating or enhancing brain recovery, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which facilitates or enhances brain recovery. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of facilitating or enhancing brain recovery, comprising administering to a subject an omega-6 fatty acid or its metabolic precursor, which facilitates or enhances brain recovery. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of facilitating or enhancing brain recovery, comprising administering to a subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, which facilitates or enhances brain recovery. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of facilitating or enhancing brain recovery, comprising administering to a subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which facilitates or enhances brain recovery. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, brain repair is facilitated or enhanced after a stroke. In another embodiment, brain repair is facilitated or enhanced after brain damage. In another embodiment, brain repair is facilitated or enhanced after any other event, disease, or disorder known in the art that requires brain repair. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, a subject whose cognitive function, neurological function, cognitive ability, synaptic transmission, or levels and activity of neurotransmitters are enhanced or improved by the composition or method of the present invention, has a cognitive impairment or impaired memory. In another embodiment, the subject is old. In another embodiment, the subject does not have a cognitive impairment or impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing the sensitivity of a neuron to a stimulus, comprising contacting the neuron with the composition of the present invention, which provides an increase in the sensitivity of the neuron to the stimulus. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

As shown here, administration of PUFA and / or uridine increases levels and synthesis of phospholipids in the brain, levels of neurofibrillary proteins in axons, and the number of synaptic membranes. Thus, the compositions and methods of the present invention increase and enhance the sensitivity of neurons to stimuli and the size and number of synapses in the brain and central nervous system (CNS).

In another embodiment, the present invention provides a method of increasing the average size of the synapse in the brain of a subject, comprising administering to the subject the composition of the present invention, which provides an increase in the average size of the synapse in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synapses in the brain of a subject, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, which provides an increase in the number of synapses in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synapses in the brain of a subject, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides an increase in the number of synapses in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synapses in the brain of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, which provides an increase in the number of synapses in the brain of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method for increasing the number of synapses in the brain of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides an increase in the number of synapses in the brain of the subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

Methods for measuring and determining the average synapse size, the number of synapses, and the level of synaptic activity and release of neurotransmitters in the brain and central nervous system of a subject are well known in the art and are disclosed, for example, in Wheeler DW et al. (Estimating use-dependent synaptic gain in autonomic ganglia by computational simulation and dynamic-clamp analysis. J Neurophysiol. 2004 Nov; 92 (5): 2659-71), Viele K et al. (Estimating the number of release sites and probability of firing within the nerve terminal by statistical analysis of synaptic charge. Synapse. 2003 Jan; 47 (1): 15-25), and DeFelipe J et al. (Estimation of the number of synapses in the cerebral cortex: methodological considerations. Cereb Cortex. 1999 Oct-Nov; 9 (7): 722-32). Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of stimulating or enhancing the formation of membranes in a brain cell or in a nerve cell of a subject, comprising administering to the subject an omega-3 fatty acid or a metabolic precursor thereof, which stimulates or enhances the formation of membranes in a brain cell or in a nerve cell of the subject . In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of stimulating or enhancing the formation of membranes in a brain cell or in a nerve cell of a subject, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, which provides stimulation or enhancement of membrane formation in a brain cell or in a nerve cell of a subject . In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of stimulating or enhancing the formation of membranes in a brain cell or nerve cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, which provides stimulation or enhancement of membrane formation in a brain cell or in a subject's nerve cell. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of stimulating or enhancing the formation of membranes in a brain cell or nerve cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, which provides stimulation or enhancement of membrane formation in a brain cell or in a nerve cell of a subject. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the methods of the present invention increase phospholipid levels, while substantially maintaining ratios of 2 or more phospholipids in the target brain cell or target nerve cell. In another embodiment, the methods of the present invention increase phospholipid levels, while substantially maintaining the ratio of 3 or more phospholipids in the brain target cell or target nerve cell. In another embodiment, the methods of the present invention increase phospholipid levels, while substantially maintaining ratios of 4 or more phospholipids in the brain target cell or target nerve cell. In another embodiment, the phospholipids are selected from PF, PE, PS and sphingomyelin (CM). In another embodiment, maintaining substantially these ratios is important for the above aspects of nerve and brain functioning. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the term “substantially retains” means that the deviation from the previous ratio is less than 10%. In another embodiment, the term “substantially retains” means that the deviation is less than 15%. In another embodiment, the deviation is less than 20%. In another embodiment, the deviation is less than 25%. In another embodiment, the deviation is less than 30%. In another embodiment, the deviation is less than 35%. In another embodiment, the deviation is less than 40%. In another embodiment, the deviation is less than 45%. In another embodiment, the deviation is less than 50%. In another embodiment, the deviation is less than 55%. In another embodiment, the deviation is less than 60%. In another embodiment, the deviation is less than 65%. In another embodiment, the deviation is less than 70%. In another embodiment, the deviation is less than 75%. In another embodiment, the deviation is less than 80%. In another embodiment, the deviation is less than 85%. In another embodiment, the deviation is less than 90%. In another embodiment, the deviation is less than 95%. In another embodiment, the deviation is less than 90% (100%?). Each opportunity represents a separate embodiment of the present invention.

In another embodiment of the methods of the present invention, stimulation of the synthesis of phospholipids enhances axon branching. In another embodiment, stimulation of phospholipid synthesis enhances axon germination. In another embodiment, stimulating the synthesis of phospholipids increases the pool of phospholipid residues that can be released through activation of phospholipases. Some of the phospholipid residues have biological activity, for example, such as inositol-1,4,5-trisphosphate (IP ₃ ), diacylglycerol (DAG) and platelet activation lyso-factor (lyso-PAF), which during the subsequent metabolism provides the formation of bioactive lipid - platelet activation factor (PAF, 1-O-alkyl-2-acetyl-sn-3-glycerol-3-phosphocholine).

In another embodiment, stimulation of phospholipid synthesis protects the synaptic membranes from stress. In another embodiment, stress is oxidative stress. In another embodiment, stress is any other type of stress known in the art.

In another embodiment, each of these phospholipid synthesis stimulation effects enhances neurotransmitter-mediated signaling, thereby slowing down memory loss. In another embodiment, memory loss or impairment is stopped due to one of the effects described above. In another embodiment, lost memory is recovered through one of the effects described above. Each of the above-described effects of stimulating the synthesis of phospholipids and each of the above-described effects of this stimulation is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with memory impairment or memory loss, comprising contacting a subject's nerve cell with an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve cell or a brain cell that provides treatment for a subject with impaired memory or memory loss.

In another embodiment, the present invention provides a method of treating a subject with memory impairment or memory loss, comprising contacting a subject's nerve cell with an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve cell or a brain cell that provides treatment for a subject with impaired memory or memory loss.

In another embodiment, the present invention provides a method of treating a subject with memory impairment or memory loss, comprising contacting a subject's nerve cell with a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of phospholipid by a nerve cell or brain cell, which provides treatment for a subject with memory impairment or memory loss.

In another embodiment, the present invention provides a method of treating a subject with memory impairment or memory loss, comprising contacting a subject's nerve cell with a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of phospholipid by a nerve cell or brain cell, which provides treatment for a subject with memory impairment or memory loss.

In another embodiment, a memory impairment is an age-related (senile) impaired memory. In another embodiment, memory impairment is secondary to cerebrovascular disease. In another embodiment, memory impairment is secondary to stroke. In another embodiment, the stroke is a clinical stroke. In another embodiment, the stroke is a non-observational stroke. In another embodiment, memory impairment is the result of multiple microstrokes not established by clinical observations. In another embodiment, a memory impairment is secondary to cardiovascular disease. In another embodiment, memory impairment is secondary to age-related cardiovascular disease.

In another embodiment, the impaired memory is secondary to depression, for example, is the result of depression. As is well known to those skilled in the art, depressed people often exhibit memory impairment, often to the extent that older people with depression are mistakenly diagnosed with Alzheimer's disease. In another embodiment, the methods and compositions of the present invention will increase the release of neurotransmitters that are known to be involved in both the development of depression (e.g., dopamine, norepinephrine and serotonin) and the development of memory impairment (e.g., acetylcholine).

In another embodiment, impaired memory is secondary to insomnia.

In another embodiment, memory impairment is a consequence of any other cause of memory impairment known in the art. Each cause of memory impairment or memory loss is a separate embodiment of the present invention.

In another embodiment, the term “age (senile)” refers to the effects of old age. In another embodiment, it is over 65 years old. In another embodiment, it is over 60 years old. In another embodiment, it is over 55 years old. In another embodiment, it is over 68 years old. In another embodiment, this is an age older than 70 years. In another embodiment, it is over 72 years old. In another embodiment, it is over 75 years old. In another embodiment, it is over 78 years old. In another embodiment, it is over 80 years old. In another embodiment, it is over 85 years old. In another embodiment, it is over 90 years old. In another embodiment, the term "age (senile)" refers to the consequences of any age associated with impaired memory as a result of any disease or disorder, including impaired memory. Each opportunity represents a separate embodiment of the present invention.

The subject of the methods of the present invention, in one embodiment, is a human. In another embodiment, the subject is a woman. In another embodiment, the subject is a man. In another embodiment, the subject is a pregnant woman. In another embodiment, the subject is a breastfeeding woman. In another embodiment, the subject is a newborn. In another embodiment, the subject is a child. In another embodiment, the subject is a small child. In another embodiment, the subject is an adult. In another embodiment, the subject is an elderly person. In another embodiment, the term "elderly" refers to any embodiment described above. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the term "newborn" means a subject under the age of 1 year. In another embodiment, the term refers to a subject under the age of 18 months. In another embodiment, the term refers to a subject under the age of 6 months. In another embodiment, the term refers to a subject under the age of 7 months. In another embodiment, the term refers to a subject under the age of 8 months. In another embodiment, the term refers to a subject under the age of 9 months. In another embodiment, the term refers to a subject under the age of 10 months. In another embodiment, the term refers to a subject under 11 months of age. In another embodiment, the term refers to a subject under the age of 13 months. In another embodiment, the term refers to a subject under the age of 14 months. In another embodiment, the term refers to a subject under the age of 16 months. In another embodiment, the term refers to a subject under the age of 20 months. In another embodiment, the term refers to a subject under the age of 2 years. In another embodiment, the term refers to a subject who is not yet excommunicated. In another embodiment, the term refers to a subject who is already excommunicated but has an age within the above boundaries. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the term "child" means a subject under the age of 18 years. In another embodiment, the term refers to a subject under the age of 17 years. In another embodiment, the term refers to a subject under the age of 16 years. In another embodiment, the term refers to a subject under the age of 15 years. In another embodiment, the term refers to a subject under the age of 14 years. In another embodiment, the term refers to a subject under the age of 13 years. In another embodiment, the term refers to a subject under the age of 12 years. In another embodiment, the term refers to a subject under the age of 11 years. In another embodiment, the term refers to a subject under the age of 10 years. In another embodiment, the term refers to a subject under the age of 9 years. In another embodiment, the term refers to a subject under the age of 8 years. In another embodiment, the term refers to a subject under the age of 7 years.

In another embodiment, the term "small child" refers to a subject under the age of 7 years. In another embodiment, the term refers to a subject under the age of 6 years. In another embodiment, the term refers to a subject under the age of 5 years. In another embodiment, the term refers to a subject under the age of 4 years. In another embodiment, the term refers to a subject under the age of 3.5 years. In another embodiment, the term refers to a subject under the age of 3 years. In another embodiment, the term refers to a subject under the age of 2.5 years. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the term “adult” refers to a subject of one of the ages indicated above that is above the age limit of the child. In another embodiment, the term refers to a subject of one of the ages indicated above that is above the age limit of a young child. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with Peak disease, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell of the subject, providing such thus, treatment of a subject with Pick disease. In another embodiment, the present invention provides a method of treating a subject with Levy body disease, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, providing thus treating a subject with Levy's disease. In another embodiment, the present invention provides a method of treating a subject with Huntington’s disease, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by a nerve or brain cell of the subject, providing such thus, treatment of a subject with Huntington's disease. In another embodiment, the present invention provides a method of treating a subject with mild cognitive impairment (MCI), comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by a nerve cell or a brain cell of a subject, thereby providing treatment for a subject with MCI. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with Peak disease, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell of the subject, providing such thus, treatment of a subject with Pick disease. In another embodiment, the present invention provides a method of treating a subject with Levy body disease, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell of the subject, providing thus treating a subject with Levy's disease. In another embodiment, the present invention provides a method of treating a subject with Huntington's disease, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by a nerve cell or brain cell of the subject, providing such thus, treatment of a subject with Huntington's disease. In another embodiment, the present invention provides a method of treating a subject with MCI, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell of the subject, thereby providing treating a subject with MCI. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with Peak disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with Pick disease. In another embodiment, the present invention provides a method of treating a subject with a Levy body disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with Levy's disease. In another embodiment, the present invention provides a method of treating a subject with Huntington's disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with Huntington's disease. In another embodiment, the present invention provides a method of treating a subject with MCI, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with MCI. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with Peak disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with Pick disease. In another embodiment, the present invention provides a method of treating a subject with a Levy body disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with Levy body disease. In another embodiment, the present invention provides a method of treating a subject with Huntington's disease, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, wherein the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for a subject with Huntington's disease. In another embodiment, the present invention provides a method of treating a subject with MCI, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell of a subject, thereby providing treatment for the subject with MCI. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a subject with vascular dementia, comprising administering to the subject a composition of the present invention, where the omega-3 fatty acid or its metabolic precursor enhances phospholipid synthesis by a nerve or brain cell of the subject, thereby providing treatment for the subject with vascular dementia.

In another embodiment, the present invention provides a method of treating a subject with an age-related neurological disorder, comprising contacting a subject's nerve cell with a composition of the present invention, wherein the composition enhances phospholipid synthesis by a nerve or brain cell, thereby providing treatment for a subject with an age-related neurological impairment.

In another embodiment, the present invention provides a method of treating a subject with a symptom of depression, comprising contacting a nerve cell of a subject with a composition of the present invention, where the composition enhances the synthesis of a phospholipid by a nerve cell or brain cell, thereby providing treatment for a subject with a symptom of depression. In another embodiment, the subject has a memory impairment. In another embodiment, a memory impairment is an age-related (senile) impaired memory. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the symptom of depression is sadness. In another embodiment, the symptom of depression is a decreased activity. In another embodiment, the symptom of depression is fatigue. In another embodiment, the symptom of depression is weakness. In another embodiment, the symptom of depression is mood variability. In another embodiment, the symptom of depression is a reduced concentration. In another embodiment, the symptom of depression is insomnia. In another embodiment, the symptom of depression is weight fluctuation. In another embodiment, the symptom of depression is a decreased libido. In another embodiment, the symptom of depression is suicidal thoughts. Each symptom is a separate embodiment of the present invention.

In another embodiment, the methods and compositions of the present invention, omega-3 fatty acid, omega-6 fatty acid, their metabolic precursor or composition of the present invention provides one of the effects listed here by increasing the synthesis of phospholipid. In another embodiment, a visible effect is achieved without increasing the synthesis of phospholipid. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the methods and compositions of the present invention include a source of uridine. In another embodiment, the methods and compositions of the present invention include a choline source. In another embodiment, the term “source” refers to a compound that increases the concentration of the desired compound (eg, uridine, choline, etc.) in the bloodstream or in tissues. In another embodiment, the term “source” refers to a compound that is metabolized by the tissues or enzyme of the subject to form the desired compound. In another embodiment, the term “source” refers to a compound that is metabolized in the target cell to form the desired compound. In another embodiment, the uridine source is cytidine, which is converted to uridine in the human liver. In another embodiment, the uridine source is cytidine-5'-monophosphate. In another embodiment, the uridine source is cytidine-5'-diphosphate. In another embodiment, the source of uridine is cytidine-5'-triphosphate. In another embodiment, the uridine source is any other cytidine phosphate known in the art. In another embodiment, the uridine source is CDP-choline. In another embodiment, the uridine source is any other uridine source known in the art. Each source of uridine is a separate embodiment of the present invention. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the uridine phosphate used in the methods of the present invention is uridine-5'-monophosphate. In another embodiment, the uridine phosphate is uridine-5'-diphosphate. In another embodiment, the uridine phosphate is uridine-5'-triphosphate. In another embodiment, uridine phosphate is any other uridine phosphate known in the art. Each opportunity represents a separate embodiment of the present invention.

In other embodiments, uridine-based compounds other than uridine itself act as sources of uridine or uridine precursors. In other embodiments, these include uridine-rich foods or diet foods, such as algae, salts of uridine-like uridine phosphates, acylated uridine, and the like. In another embodiment, acylated uridine derivatives or mixtures thereof, for example, such as those disclosed in U.S., are also introduced. Pat. No. 5,470,838. Each uridine precursor is a separate embodiment of the present invention.

In another embodiment, the method of the present invention further comprises administering choline. In another embodiment, the method further comprises administering a choline salt. In another embodiment, the method further comprises administering a compound that is metabolized to form choline. In another embodiment, the method further comprises administering a choline source. In another embodiment, administration of one of the above compounds enhances the effect of omega-3 or omega-6 fatty acid and / or uridine on the synthesis of membrane phospholipids. As shown here (examples), the administration of choline and omega-3 or omega-6 fatty acids demonstrates an unexpected increase in the levels of phospholipids, synaptic proteins and synaptic membranes in neurons and brain tissue and an improvement in memory, cognitive activity and cognitive and neurological functions.

In another embodiment, any of the methods and compositions of the present invention includes the introduction of omega-3 fatty acids and choline. In another embodiment, any of the methods and compositions of the present invention comprises administering an omega-3 fatty acid and a choline salt. In another embodiment, any of the methods and compositions of the present invention includes the introduction of omega-6 fatty acids and choline. In another embodiment, any of the methods and compositions of the present invention comprises administering an omega-6 fatty acid and a choline salt.

In another embodiment, any of the methods and compositions of the present invention includes administering a composition comprising omega-3 fatty acid, uridine, and choline. In another embodiment, any of the methods and compositions of the present invention includes administering a composition comprising an omega-3 fatty acid, uridine, and choline salt. In another embodiment, any of the methods and compositions of the present invention includes administering a composition comprising omega-6 fatty acid, uridine, and choline. In another embodiment, any of the methods and compositions of the present invention includes administering a composition comprising an omega-6 fatty acid, uridine, and choline salt.

In another embodiment, any of the methods and compositions of the present invention comprises administering an omega-6 fatty acid and an omega-3 fatty acid. In another embodiment, any of the methods and compositions of the present invention includes the introduction of omega-6 fatty acids, omega-3 fatty acids and uridine. In another embodiment, any of the methods and compositions of the present invention includes the administration of omega-6 fatty acids, omega-3 fatty acids and choline. In another embodiment, any of the methods and compositions of the present invention comprises administering an omega-6 fatty acid, an omega-3 fatty acid, and a choline salt. In another embodiment, any of the methods and compositions of the present invention includes the administration of omega-6 fatty acids, omega-3 fatty acids, uridine and choline. In another embodiment, any of the methods and compositions of the present invention comprises administering an omega-6 fatty acid, an omega-3 fatty acid, uridine, and a choline salt.

In another embodiment, the anti-inflammatory PUFA is included in the methods and compositions of the present invention. In another embodiment, 2 different omega-3 fatty acids are included. In another embodiment, 1 of 2 omega-3 fatty acids is an anti-inflammatory PUFA. In another embodiment, 1 of 2 omega-3 fatty acids is DHA. In another embodiment, 1 of 2 omega-3 fatty acids is EPA. In another embodiment, the 2 omega-3 fatty acids are EPA and DHA.

In another embodiment, the ratio of 2 omega-3 fatty acids is 0.05: 1. In another embodiment, the ratio is 0.1: 1. In another embodiment, the ratio is 0.15: 1. In another embodiment, the ratio is 0.2: 1. In another embodiment, the ratio is 0.3: 1. In another embodiment, the ratio is 0.4: 1. In another embodiment, the ratio is 0.5: 1. In another embodiment, the ratio is 0.6: 1. In another embodiment, the ratio is 0.7: 1. In another embodiment, the ratio is 0.8: 1. In another embodiment, the ratio is 0.9: 1. In another embodiment, the ratio is 1: 1. In another embodiment, DHA and EPA are included in one of the above ratios (DHA: EPA). In another embodiment, DHA and EPA are included in one of the above ratios (EPA: DHA).

In another embodiment, 2 different omega-6 fatty acids are included in the methods and compositions of the present invention.

In another embodiment, the ratio of omega-3 fatty acid to omega-6 fatty acid in the method or composition of the present invention is 1: 1. In another embodiment, the ratio is 1.5: 1. In another embodiment, the ratio is 2: 1. In another embodiment, the ratio is 3: 1. In another embodiment, the ratio is 4: 1. In another embodiment, the ratio is 5: 1. In another embodiment, the ratio is 6: 1. In another embodiment, the ratio is 8: 1. In another embodiment, the ratio is 10: 1. In another embodiment, the ratio is 12: 1. In another embodiment, the ratio is 15: 1. In another embodiment, the ratio is 20: 1. In another embodiment, the ratio is 30: 1. In another embodiment, the ratio is 40: 1. In another embodiment, the ratio is 50: 1. In another embodiment, the ratio is 60: 1. In another embodiment, the ratio is 80: 1. In another embodiment, the ratio is 100: 1.

Each combination of omega-3 fatty acid, omega-6 fatty acid, uridine, choline and / or choline salt is a separate embodiment of the present invention. Each combination of different omega-3 fatty acids is a separate embodiment of the present invention. Each combination of different omega-6 fatty acids is a separate embodiment of the present invention. Each ratio represents a separate embodiment of the present invention.

In another embodiment, the choline source is lecithin. In another embodiment, the choline source is lecithin. In another embodiment, the choline source is acetylcholine. In another embodiment, the choline source is cyticholine or alpha-glycerophosphorylcholine. In another embodiment, the choline source is CDP-choline. In another embodiment, the choline source is any other choline source known in the art. Each source of choline is a separate embodiment of the present invention.

In another embodiment, the choline salt is a sulfonate, for example a long chain alkyl sulfonate salt. In another embodiment, the choline salt is choline chloride. In another embodiment, the choline salt is choline bitartrate. In another embodiment, the choline salt is choline citrate. In another embodiment, the choline salt is choline tartrate. In another embodiment, the choline salt is an iron-choline nitrate complex. In another embodiment, the choline salt is any other choline salt known in the art. Each choline salt is a separate embodiment of the present invention.

In another embodiment, the present invention provides a composition for the treatment of Alzheimer's disease, consisting of any compositions disclosed in the methods of the present invention. Each composition is a separate embodiment of the present invention.

In another embodiment, the present invention provides a composition for treating memory impairment or memory loss, consisting of any compositions disclosed in the methods of the present invention. Each composition is a separate embodiment of the present invention. In addition, each type of memory impairment or memory loss is a separate embodiment of the present invention.

In another embodiment, the present invention provides a composition for the treatment of Pick disease, consisting of any compositions disclosed in the methods of the present invention. In another embodiment, the present invention provides a composition for treating Levy body disease consisting of any of the compositions disclosed in the methods of the present invention. In another embodiment, the present invention provides a composition for the treatment of Huntington's disease, consisting of any compositions disclosed in the methods of the present invention. Each composition and each of the above diseases represent a separate embodiment of the present invention.

In another embodiment, the methods and compositions of the present invention show their effects even in subjects who are not deficient in omega-3 fatty acids or omega-6 fatty acids. In another embodiment, a pharmacological dose of PUFA is used in the methods and compositions of the present invention. In another embodiment, a therapeutic dose is used. In another embodiment, the pharmacological dose is higher than what is usually received with food in the case of a diet rich in PUFA. In another embodiment, membrane levels in a non-PUFA deficient subject increase with the administration of pharmacological doses of PUFA and / or uridine. In another embodiment, the results of the present invention demonstrate that PUFAs have a biochemical effect on the brain, which speaks in favor of the use of pharmacological doses of PUFA. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, the dosage of omega-3 fatty acid to be included in the methods and compositions of the present invention is in the range of about 400-2000 mg / day. In another embodiment, the dosage is in the range of about 500-2000 mg / day. In another embodiment, the range is approximately 600-2000 mg / day. In another embodiment, the range is approximately 800-2000 mg / day. In another embodiment, the range is approximately 1000-2000 mg / day. In another embodiment, the range is approximately 1200-2000 mg / day. In another embodiment, the range is approximately 1500-2000 mg / day. In another embodiment, the range is approximately 400-3000 mg / day. In another embodiment, the dosage is in the range of about 500-3000 mg / day. In another embodiment, the range is approximately 600-3000 mg / day. In another embodiment, the range is approximately 800-3000 mg / day. In another embodiment, the range is approximately 1000-3000 mg / day. In another embodiment, the range is approximately 1200-3000 mg / day. In another embodiment, the range is approximately 1500-3000 mg / day. In another embodiment, the range is approximately 2000-3000 mg / day. In another embodiment, the range is approximately 400-4000 mg / day. In another embodiment, the dosage is in the range of about 500-4000 mg / day. In another embodiment, the range is approximately 600-4000 mg / day. In another embodiment, the range is approximately 800-4000 mg / day. In another embodiment, the range is approximately 1000-4000 mg / day. In another embodiment, the range is approximately 1200-4000 mg / day. In another. the embodiment, the range is approximately 1500-4000 mg / day. In another embodiment, the range is approximately 2000-4000 mg / day. In another embodiment, the range is approximately 3000-4000 mg / day. In another embodiment, the range is approximately 400-1000 mg / day. In another embodiment, the range is approximately 500-1000 mg / day. In another embodiment, the range is approximately 600-1000 mg / day. In another embodiment, the range is approximately 800-100 mg / day.

In another embodiment, the dosage of omega-3 fatty acid is at least 400 mg / day. In another embodiment, the dosage is at least 500 mg / day. In another embodiment, the dosage is at least 600 mg / day. In another embodiment, the dosage is at least 700 mg / day. In another embodiment, the dosage is at least 800 mg / day. In another embodiment, the dosage is at least 900 mg / day. In another embodiment, the dosage is at least 1 g / day. In another embodiment, the dosage is at least 1200 mg / day. In another embodiment, the dosage is at least 1.5 g / day. In another embodiment, the dosage is at least 2 g / day.

In another embodiment, the dosage of omega-3 fatty acid is approximately 400 mg / day. In another embodiment, the dosage is approximately 500 mg / day. In another embodiment, the dosage is approximately 600 mg / day. In another embodiment, the dosage is approximately 700 mg / day. In another embodiment, the dosage is approximately 800 mg / day. In another embodiment, the dosage is approximately 900 mg / day. In another embodiment, the dosage is about 1 g / day. In another embodiment, the dosage is approximately 1200 mg / day. In another embodiment, the dosage is approximately 1.5 g / day. In another embodiment, the dosage is approximately 2 g / day.

In another embodiment, pregnant women receive a specific dosage to achieve the desired result. In another embodiment, the range is approximately 200-2000 mg / day. In another embodiment, the range is approximately 400-1700 mg / day. In another embodiment, the range is approximately 600-1500 mg / day. In another embodiment, the range is approximately 800-1300 mg / day. In another embodiment, the range is approximately 200-3000 mg / day. In another embodiment, the range is approximately 400-3000 mg / day. In another embodiment, the range is approximately 600-3000 mg / day. In another embodiment, the range is approximately 800-3000 mg / day. In another embodiment, the range is approximately 1000-3000 mg / day. In another embodiment, the range is approximately 2000-3000 mg / day. In another embodiment, the dosage for pregnant women is approximately 1000 mg / day. In another embodiment, the dosage is approximately 1500 mg / day. In another embodiment, the dosage is approximately 2000 mg / day. In another embodiment, the dosage is approximately 3000 mg / day.

In another embodiment, subjects with high cholesterol receive a certain dosage to achieve the desired result. In another embodiment, the dosage for subjects with high cholesterol is in the range of about 200-4000 mg / day. In another embodiment, the dosage for subjects with high cholesterol is in the range of about 400-3500 mg / day. In another embodiment, the dosage for subjects with high cholesterol is in the range of about 600-3000 mg / day. In another embodiment, the dosage for subjects with high cholesterol is in the range of about 1000-2500 mg / day. In another embodiment, the dosage for subjects with high cholesterol is in the range of about 1500-2300 mg / day. In another embodiment, the dosage for subjects with high cholesterol is approximately 2000 mg / day.

In another embodiment of the methods and compositions of the present invention, DHA is included in one of the above doses. In another embodiment, the dosage of DHA is 1-50 mg / kg / day. In another embodiment, the dosage of DHA is 400-1000 mg / day. In another embodiment, EPA is included in one of the above doses. In another embodiment, the dosage of EPA is 1-50 mg / kg / day. In another embodiment, the dosage of EPA is 400-1000 mg / day. Each dosage represents a separate embodiment of the present invention.

In other embodiments, the dosage of omega-6 fatty acid to be included in the methods and compositions of the present invention corresponds to any of the dosages of omega-3 fatty acid indicated above. In another embodiment, arachidonic acid is included in one of the above doses. In another embodiment, the dosage of arachidonic acid is 1-50 mg / kg / day. In another embodiment, the dosage of arachidonic acid is 400-1000 mg / day. Each dosage represents a separate embodiment of the present invention.

In another embodiment, eicosahexaenoic acid (EPA) is administered together, or additionally, with another omega-3 or omega-6 fatty acid. In another embodiment, EPA is added at a dosage of about 200 mg / day. In another embodiment, the dosage is 100-300 mg / day. In another embodiment, the range is 150-250 mg / day. In another embodiment, the range is 170-230 mg / day. In another embodiment, the range is 100-1000 mg / day. In another embodiment, the range is 150-800 mg / day. In another embodiment, the range is 200-600 mg / day. In another embodiment, the range is 300-500 mg / day. In another embodiment, the dosage is approximately 300 mg / day. In another embodiment, the dosage is approximately 400 mg / day. In another embodiment, the dosage is approximately 500 mg / day. In another embodiment, the dosage is approximately 600 mg / day. In another embodiment, the dosage is approximately 800 mg / day. In another embodiment, the dosage is approximately 1000 mg / day.

In another embodiment, a higher dose of EPA is administered to pregnant women. In another embodiment, the dosage is approximately 1200 mg / day. In another embodiment, the dosage is approximately 1500 mg / day. In another embodiment, the dosage is approximately 1800 mg / day. In another embodiment, the dosage is approximately 2000 mg / day. In another embodiment, the dosage is approximately 2500 mg / day. In another embodiment, the dosage is approximately 3000 mg / day. In another embodiment, the dosage is 500-3000 mg / day. In another embodiment, the dosage is 800-3000 mg / day. In another embodiment, the dosage is 1000-3000 mg / day. In another embodiment, the dosage is 1500-3000 mg / day. In another embodiment, the dosage is 2000-3000 mg / day. In another embodiment, the dosage is 500-2000 mg / day. In another embodiment, the dosage is 800-2000 mg / day. In another embodiment, the dosage is 1000-2000 mg / day. In another embodiment, the dosage is 1500-2000 mg / day.

Each dosage of omega-3 fatty acid, omega-6 fatty acid or additionally added EPA is a separate embodiment of the present invention.

In another embodiment, the dosage of uridine to be included in the methods and compositions of the present invention is in the range of 10-500 mg / day (inclusive). In another embodiment, the dosage is 20-500 mg / day. In another embodiment, the dosage is 30-500 mg / day. In another embodiment, the dosage is 50-500 mg / day. In another embodiment, the dosage is 100-500 mg / day. In another embodiment, the dosage is 150-500 mg / day. In another embodiment, the dosage is 200-500 mg / day. In another embodiment, the dosage is 300-500 mg / day. In another embodiment, the dosage of uridine is in the range of 10-400 mg / day. In another embodiment, the dosage is 20-400 mg / day. In another embodiment, the dosage is 30-400 mg / day. In another embodiment, the dosage is 50-400 mg / day. In another embodiment, the dosage is 100-400 mg / day. In another embodiment, the dosage is 150-400 mg / day. In another embodiment, the dosage is 200-400 mg / day. In another embodiment, the dosage of uridine is in the range of 10-300 mg / day. In another embodiment, the dosage is 20-300 mg / day. In another embodiment, the dosage is 30-300 mg / day. In another embodiment, the dosage is 50-300 mg / day. In another embodiment, the dosage is 100-300 mg / day. In another embodiment, the dosage is 150-300 mg / day. In another embodiment, the dosage is 200-300 mg / day. In another embodiment, the dosage is approximately 50 mg / day. In another embodiment, the dosage is approximately 70 mg / day. In another embodiment, the dosage is approximately 100 mg / day. In another embodiment, the dosage is approximately 150 mg / day. In another embodiment, the dosage is approximately 200 mg / day. In another embodiment, the dosage is approximately 300 mg / day. In another embodiment, the dosage is approximately 400 mg / day. In another embodiment, the dosage is approximately 500 mg / day.

Each dosage of uridine is a separate embodiment of the present invention.

In another embodiment, the dosage of choline included in the methods and compositions of the present invention is in the range of 100 mg-10 g / day (inclusive). In another embodiment, the dosage is 1 g to 3 g. In another embodiment, the dosage is 150 mg to 8 g. In another embodiment, the dosage is 200 mg to 6 g. In another embodiment, the dosage is 300 mg to 5 g. In another in an embodiment, the dosage is 400 mg to 4.5 g. In another embodiment, the dosage is 500 mg to 4 g. In another embodiment, the dosage is 600 mg to 4 g. In another embodiment, the dosage is 800 mg to 3.5 g. In another embodiment, the dosage is 1.2 g - 3 g. In another embodiment, the dosage is 1.5 g - 2.5 g. In another embodiment, the dosage a is approximately 0.5 g. In another embodiment, the dosage is approximately 0.7 g. In another embodiment, the dosage is approximately 1 g. In another embodiment, the dosage is approximately 1.2 g. In another embodiment, the dosage is approximately 1 , 5 g. In another embodiment, the dosage is approximately 2 g. In another embodiment, the dosage is approximately 2.5 g. In another embodiment, the dosage is approximately 3 g. In another embodiment, the dosage is approximately 4 g. Each of the decree The above dosage is the number of choline equivalents; thus, the actual dosages of a choline compound (e.g., choline chloride or choline tartrate) will be correspondingly higher.

Each dosage of choline represents a separate embodiment of the present invention.

In another embodiment, the composition of the present invention is administered chronically. In another embodiment, the term “chronically” refers to administration for at least 1 week. In another embodiment, the term refers to administration for at least 2 weeks. In another embodiment, the time period is at least 10 days. In another embodiment, the period of time is at least 3 weeks. In another embodiment, the period of time is at least 4 weeks. In another embodiment, the period of time is at least 5 weeks. In another embodiment, the period of time is at least 6 weeks. In another embodiment, the period of time is at least 2 months. In another embodiment, the period of time is at least 3 months. In another embodiment, the period of time is at least 4 months. In another embodiment, the period of time is at least 5 months. In another embodiment, the period of time is at least 6 months. In another embodiment, the period of time is at least 1 year. In another embodiment, the period of time is at least 2 years. In another embodiment, the period of time is at least 3 years. In another embodiment, the period of time is at least 5 years. In another embodiment, the period of time is at least 10 years.

In another embodiment, the time period is 1 week. In another embodiment, the term means administration within 2 weeks. In another embodiment, the time period is 10 days. In another embodiment, the time period is 3 weeks. In another embodiment, the time period is 4 weeks. In another embodiment, the time period is 5 weeks. In another embodiment, the period of time is 6 weeks. In another embodiment, the time period is 2 months. In another embodiment, the time period is 3 months. In another embodiment, the time period is 4 months. In another embodiment, the time period is 5 months. In another embodiment, the period of time is 6 months. In another embodiment, the time period is 1 year. In another embodiment, the period of time is 2 years. In another embodiment, the period of time is 3 years. In another embodiment, the period of time is 5 years. In another embodiment, the time period is 10 years.

In another embodiment, the PUFA component of the composition of the present invention is administered over one of the above time periods. In another embodiment, the omega-3 fatty acid component of the composition of the present invention is administered over one of the above time periods. In another embodiment, the omega-6 fatty acid component of the composition of the present invention is administered over one of the above time periods. In another embodiment, the uridine component of the composition of the present invention is administered over one of the above time periods. In another embodiment, the choline or choline salt component of the composition of the present invention is administered over one of the above time periods.

Each time period is a separate embodiment of the present invention.

In another embodiment, the term “contacting” means direct administration to a subject of a composition of the present invention. In another embodiment, the term “contacting” means the indirect administration to a subject of a composition of the present invention. Thus, in another embodiment, the methods of the present invention include methods in which the subject comes into contact with a compound or composition that is metabolized to form omega-3 or omega-6 fatty acid in cerebrospinal fluid, bloodstream, etc., after wherein the omega-3 or omega-6 fatty acid comes into contact with the brain cell due to diffusion or due to any other active or passive transport process known in the art by which the compounds circulate in the body e. In another embodiment, the compound is metabolized by target cells to omega-3 or omega-6 fatty acid. Each opportunity represents a separate embodiment of the present invention.

In another embodiment, an omega-3 or omega-6 fatty acid derivative is used in the methods and compositions of the present invention. In another embodiment, the derivative is an omega-6 fatty acid derivative of gamma-linolenic acid. In another embodiment, the derivative is any other omega-3 or omega-6 fatty acid derivative known in the art. Each derivative is a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of increasing axon branching of a nerve cell of a subject, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances phospholipid synthesis by the nerve cell, thereby providing an increase axon branching of a nerve cell. In another embodiment, the present invention provides a method for increasing axon branching of a subject's nerve cell, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by the nerve cell, thereby providing an increase axon branching of a subject’s nerve cell.

In another embodiment, the present invention provides a method for increasing axon branching of a subject's nerve cell, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing an increase in axon branching of the nerve cell. In another embodiment, the present invention provides a method for increasing axon branching of a subject's nerve cell, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing an increase in axon branching of the subject's nerve cell.

In another embodiment, the present invention provides a method for increasing the axon germination of a nerve cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-3 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing an increase in axon germination of the nerve cell. In another embodiment, the present invention provides a method for increasing the axon germination of a nerve cell of a subject, comprising administering to the subject a composition comprising: (a) uridine, its acyl derivative, uridine phosphate or CDP-choline; and (b) an omega-6 fatty acid or its metabolic precursor, where the composition enhances the synthesis of a phospholipid by a nerve cell, thereby providing an increase in the axon germination of the nerve cell of the subject.

In another embodiment, the present invention provides a method for increasing the axon germination of a nerve cell of a subject, comprising administering to the subject an omega-3 fatty acid or its metabolic precursor, where the omega-3 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by the nerve cell, thereby providing an increase germination of an axon of a nerve cell. In another embodiment, the present invention provides a method for increasing the axon germination of a nerve cell of a subject, comprising administering to the subject an omega-6 fatty acid or its metabolic precursor, where the omega-6 fatty acid or its metabolic precursor enhances the synthesis of a phospholipid by the nerve cell, thereby providing an increase germination of the axon of the nerve cell of the subject.

In another embodiment, the present invention provides a kit comprising compounds or a composition useful in carrying out the method of the present invention.

In another embodiment, the term “pharmaceutical composition” means a dietary supplement. In another embodiment, the term refers to a nutritional supplement. In another embodiment, the term refers to foods of any kind that are rich in omega-3 fatty acid. In another embodiment, the term refers to foods that are rich in omega-6 fatty acid. In another embodiment, the term refers to foods that are enriched in uridine. In another embodiment, the term refers to foods that are enriched in choline. In another embodiment, the term refers to foods that are enriched in a choline salt.

In another embodiment, the term "food" means solid food. In another embodiment, the term refers to a beverage. In another embodiment, the term refers to a powder mixture for preparing a beverage. In another embodiment, the term refers to food-based preparations, functional foods, dietary supplements, or nutraceuticals.

In another embodiment, the food products can be in various forms, including liquid, suspension, powder, semi-solid and solid products. Semi-solid foods include sweet creams, dessert puddings, greasy creams, mousses, ice cream (sundae), yoghurts and sweet gelatin products. Not limited to specific embodiments, the solid form can be prepared in the form of a bar, like a nutritious bar, chips, buns, crackers, pasta or foam, for example popcorn or products like rice muffins. Some embodiments require an individual approach to dissolving, suspending or soaking a food product.

Each type of pharmaceutical composition is a separate embodiment of the present invention.

In another embodiment, the present invention relates to the use of an omega-3 or omega-6 fatty acid and / or its analogue, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, or a combination thereof. Thus, in another embodiment, the methods of the present invention comprise administering a PUFA analogue. In another embodiment, the methods of the present invention comprise administering a PUFA derivative. In another embodiment, the methods of the present invention comprise administering a PUFA isomer. In another embodiment, the methods of the present invention comprise administering a PUFA metabolite. In another embodiment, the methods of the present invention comprise administering a pharmaceutically acceptable PUFA salt. In another embodiment, the methods of the present invention comprise administering a PUFA pharmaceutical product. In another embodiment, the methods of the present invention comprise administering a PUFA hydrate. In another embodiment, the methods of the present invention comprise administering a PUFA N-oxide. In another embodiment, the methods of the present invention comprise administering any combination of an analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, PUFA hydrate or N-oxide.

In another embodiment of the methods and compositions of the present invention, PUFA is administered as a triglyceride.

In another embodiment, the term “isomer” includes, but, in another embodiment, is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.

In another embodiment, the present invention further includes PUFA derivatives. The term “derivatives” includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In addition, the present invention further includes hydrates of PUFA compounds. The term "hydrate" includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

The present invention further includes metabolites of PUFA compounds. The term "metabolites" refers to any substance obtained from another substance during metabolism or as a result of a metabolic process.

The present invention further includes pharmaceutical products of PUFA compounds. The term “pharmaceutical products” means compositions suitable for pharmaceutical use (pharmaceutical compositions), as defined herein.

In addition, the present invention includes the pure (Z) and (E) isomers of the PUFA compounds defined herein and mixtures thereof, as well as the pure (RR, SS) and (RS, SR) enantiomeric pairs and mixtures thereof .

Pharmaceutical compositions and methods of administration

In another embodiment, pharmaceutical compositions containing a PUFA and / or uridine can be administered to a subject by any means known to a person skilled in the art, for example parenterally, paracancerally (inside a cancerous tumor), transmucosally (through mucous membranes), transdermally (through skin), intramuscularly , intravenously, intradermally, subcutaneously, intraperitoneally, inside the ventricle, inside the skull, intravaginally or inside the tumor.

In another embodiment of the methods and compositions of the present invention, the pharmaceutical compositions are administered orally and, therefore, are prepared in a form suitable for oral administration, i.e. in the form of a solid or liquid dosage form. Suitable solid dosage forms for oral administration include tablets, capsules, pills, granules, pads and the like. Suitable for oral administration liquid dosage forms include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is inside the capsule. According to this embodiment, the compositions of the present invention comprise, in addition to the active compound and an inert carrier or diluent, a hard gelatin capsule.

In another embodiment, the pharmaceutical compositions are administered intravenously, intraarterially or intramuscularly by injection of a liquid dosage form. Suitable liquid dosage forms include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and, therefore, are prepared in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and, therefore, are prepared in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly and, therefore, are prepared in a form suitable for intramuscular administration.

In another embodiment, the pharmaceutical compositions are administered locally on the surface of the body and, therefore, are prepared in a form suitable for topical application. In another embodiment, suitable topical dosage forms include gels, ointments, creams, lotions, drops, and the like.

In another embodiment, the pharmaceutical composition is administered as a suppository, for example a rectal suppository or urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pill. In another embodiment, such a pill provides a controlled release of PUFA and / or uridine over a period of time.

In another embodiment, the active compound is delivered in a vesicle, for example, inside a liposome.

In other embodiments, carriers or solvents used in the methods of the present invention include, but are not limited to, resin, starch (e.g., corn starch, peptized starch), sugar (e.g., lactose, mannitol, sucrose, dextrose), cellulosic material ( for example, microcrystalline cellulose), acrylate (e.g. polymethyl acrylate), calcium carbonate, magnesium oxide, talc or mixtures thereof.

In other embodiments, pharmaceutically acceptable carriers for liquid dosage forms are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and organic esters for injection, such as ethyl oleate. Aqueous carriers include water, alcohol-water solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are oils of animal, vegetable or synthetic origin, for example peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oil or lipid from milk or eggs.

In another embodiment, parenteral carriers (for subcutaneous, intravenous, intraarterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose and the like. Examples are sterile fluids, such as water and oils, with or without surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous solutions of dextrose and related sugars, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, in particular for injection solutions. Examples of oils are oils of animal, vegetable or synthetic origin, for example peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oil or lipid from milk or eggs.

In other embodiments, the compositions further include binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. corn starch, silica, potato dihydrate croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCl, acetate, phosphate) with different pH and ionic strengths, additives such as albumin or and gelatin, to prevent absorption on surfaces, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), penetration enhancers, solubilizing agents (e.g. glycerin, polyethylene glycerol) , antioxidants (e.g. ascorbic acid, sodium metabisulfite, butyl hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hydroxypropyl methyl cellulose), viscosity increasing agents (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose cu, guar gum), sweeteners (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 regulators (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 agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and / or excipients. Each of the above excipients is a separate embodiment of the present invention.

In another embodiment, the pharmaceutical compositions provided herein are controlled release compositions, that is, compositions in which PUFA and / or uridine are released over a period of time after administration. Controlled or sustained release compositions include dosage forms in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate release composition, that is, a composition in which all PUFA and / or uridine are released immediately after administration.

In another embodiment, the pharmaceutical composition is administered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be 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. J. Med. 321: 574 (1989). In another embodiment, polymeric materials are used, for example, in microspheres or in an implant. In yet another embodiment, a controlled release system is placed in close proximity to a therapeutic target, such as a brain, that only part of the systemic dose is required (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990).

In another embodiment, the compositions also include introducing the active material into or on the surface of certain forms of polymeric compounds, such as polylactic acid, polyglycolic acid, hydrogels, etc., or into liposomes, microemulsions, micelles, unilamellar or multilayer vesicles, erythrocyte shadows or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate and in vivo clearance rate.

The present invention also includes certain compositions coated with polymers (for example, poloxamers or poloxamines) and a compound associated with antibodies against tissue-specific receptors, ligands or antigens, or associated with ligands of tissue-specific receptors.

The present invention also encompasses compounds modified by covalent attachment to water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline. Modified compounds are known to exhibit significantly longer blood half-lives after intravenous injection than the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications can also increase the solubility of compounds in aqueous solutions, eliminate aggregation, increase the physical and chemical stability of the compound, and significantly reduce the immunogenicity and reactivity of the compound. As a result of this, the desired in vivo biological activity can be achieved by introducing such adducts of the compound with the polymer at a lower frequency or in lower doses than when the unmodified compound is introduced.

The preparation of pharmaceutical compositions that contain an active ingredient, for example, by mixing, granulating, or a tabletting process, is well known in the art. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, PUFAs and / or uridine or their physiologically acceptable derivatives, such as salts, esters, N-oxides and the like, are mixed with additives customary for this purpose, such as delivery vehicles, stabilizers or inert solvents, and are converted using conventional methods in suitable administration forms, such as tablets, coated tablets, hard or soft gelatine capsules, aqueous, alcoholic or oily solutions. For parenteral administration, PUFAs and / or uridine or their physiologically tolerable derivatives, such as salts, esters, N-oxides and the like, are converted into solutions, suspensions or emulsions, if desired, together with substances conventional and suitable for this purpose , for example, solvents or other substances.

In another embodiment, the active component is introduced into the composition in the form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include additional acid salts (formed with free amino groups of a polypeptide or antibody molecule) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic and the like . Salts formed with free carboxyl groups can also be derivatives of inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. like that.

Each / each of the above additives, excipients, dosage forms and methods of administration is a separate embodiment of the present invention.

Experimental Section

Example 1

Treatment of pc-12 omega-3 fatty acids increases phospholipid synthesis

Materials and experimental methods

Reagents

¹⁴ C-labeled choline chloride was obtained from Perkin-Elmer (Boston, MA). DHA, oleic acid or palmitic acid were obtained from Biomol, (Plymouth Meeting, PA). ¹⁴ C-choline was obtained from Amersham Biosciences Corp (Piscataway, NJ).

Cell culture

PC-12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For experiments, cells were grown in five times collagen-coated plates measuring 35 millimeters (mm). Cells were incubated for 18 hours (hour) in serum-free DMEM containing 28 μM choline +/- 5 μM DHA, oleic acid or palmitic acid. The cells were then labeled for 2.5 hours with ¹⁴ C-choline — 0.5 microcurie (μCi) / ml in serum-free DMEM containing 10 μM choline.

Quantification of labeled phospholipids

Cells were homogenized in 100 volumes of ice-cold deionized water using a tissue shredder (Polytron PT 10-35, Kinematica AG, Switzerland); then 1 ml aliquots were mixed with 3 ml of a mixture of chloroform + methanol (2: 1 by volume, v / v) and vortexed vigorously for 30 seconds. After cooling on ice for 1 hour, the mixture was successively mixed with 3 ml of a mixture composed of a mixture of chloroform + methanol (2: 1 by volume) and 1 ml of ice-cold deionized water. The mixture was vortexed vigorously and kept overnight in a cold room (18 hours). The organic (lower) and aqueous (upper) phases of the mixtures were separated by centrifugation (10 min at 4 ° C; 1000 × g). An aliquot (2 ml) of the upper phase was used to determine the content of CDP-choline (see below), and aliquots of the lower phase with a volume of 0.1-0.4 ml were dried under vacuum for analysis of phospholipids. Precipitation of 0.1 ml aliquots of the lower phase was used to determine the total phospholipid content by determining phosphorus. Precipitation of 0.4 ml aliquots of the lower phase was dissolved in 40 μl of methanol and analyzed by thin layer chromatography using silica gel plates (silica G plates, Adsorbosil Plus-1®, Alltech), and a system consisting of the following components - chloroform / ethanol / triethylamine / water (30: 34: 30: 8) as the mobile phase. The phospholipid standards were used to identify the corresponding bands in ultraviolet light after spraying the plates with a 0.1% solution of diphenylhexatriene in petroleum ether. The bands corresponding to the individual classes of phospholipids (PF, FE, SM, FS and FI) were scraped off the plates, and the phospholipids were extracted with 1 ml of methanol; the extract was dried under vacuum and the phosphorus content was measured. The total phosphorus content was determined by comparing the data with the data of the calibration graphs obtained using the KH ₂ PO ₄ standard for each measurement. 0.5 ml of 4.5% HClO ₄ /27% H ₂ SO _{4 was} added to each sample, and the tubes were kept at 180 ° C for 3 hours. After the samples were cooled to room temperature, 5 ml of color reagent was added (dilution in a 10: 1 ratio of solutions containing 2.5 mg / ml of ammonium molybdate, 8.2 mg / ml of sodium acetate and 100 mg / ml of ascorbic acid, respectively) and tubes incubated for 2 hours at 37 ° C. The optical density was measured spectrophotometrically at 820 nm. The mass of phospholipids was determined by multiplying the phosphorus content by 25.

Statistical analysis

Data was analyzed using a one-way ANOVA analysis (difference analysis) followed by Student's t-test.

results

To determine the effect of omega-3 fatty acids on phospholipid synthesis, PC-12 cells were incubated with ¹⁴ C-labeled choline after 18 hours of preincubation in the presence or absence of DHA. Then the incorporation of the label into phospholipids was measured. Oleic acid and palmitic acid, which are not omega-3 fatty acids, were used as negative controls. The synthesis of phosphatidylcholine (PF) significantly increased as a result of pre-incubation with DHA, but not with oleic acid or palmitic acid, as evidenced by an increase in the inclusion of labels in PF (Figure 1). Thus, treatment with omega-3 fatty acids increases cellular synthesis of phospholipids.

Example 2

Omega-3 fatty acids increase the synthesis of a number of phospholipids in a dose-dependent manner

To further characterize the stimulation of the synthesis of omega-3 fatty acid phospholipids, PC-12 cells were pretreated with different doses of DHA and incubated with labeled choline as described in Example 1, then the incorporation of ¹⁴ C-labels into phospholipids was measured. Pretreatment of DHA led to an increase in the synthesis of phospholipids (figure 2). The increase in synthesis was dose dependent. Thus, omega-3 fatty acids stimulate the synthesis of phospholipids in a dose-dependent manner.

Example 3

Treatment of shsy-5y omega-6 cells with fatty acids enhances phospholipid synthesis

Materials and experimental methods

Cell culture

SHSY-5Y cells were grown almost to confluence in DMEM supplemented with 10% FBS in 35 mm dishes. Cells were incubated for 18 hours in serum-free DMEM supplemented with 1% FBS containing 30 μM choline +/- 10 μM DHA, arachidonic acid, or palmitic acid. Then the cells were labeled, and the amount of labeled phospholipids was determined as described in example 1.

Obtaining complex DHA-BSA

DHA was dissolved in ethanol to a final concentration of 100 μM and frozen in 10 μl aliquots at -80 ° C. For each experiment, one aliquot was diluted with ethanol to a concentration of 10 μM; the volume giving the desired final solution in the incubation medium was mixed with an equal volume of BSA solution (1 g / ml).

results

The effect of omega-6 fatty acids on phospholipid synthesis was then tested on SHSY-5Y cells, a human neuroblastoma cell line. In this case, the synthesis of phospholipids increased with the use of arachidonic acid, an omega-6 fatty acid, but not DHA or palmitic acid (Figure 3A).

Example 4

Omega-6 fatty acids increase the synthesis of a number of phospholipids in a dose-dependent manner

The effect of arachidonic acid on the synthesis of phospholipids in SHSY-5Y cells was further characterized as described for omega-3 fatty acids and PC-12 cells in Example 2. Arachidonic acid increased the overall synthesis of phospholipids, PX and phosphatidylethanolamine in a dosage range used in a dose-dependent manner, as shown for DHA (Figure 3B). Thus, omega-6 fatty acids stimulate the synthesis of a number of phospholipids in a dose-dependent manner.

Example 5

The introduction of PUFA increases the levels of phospholipids in the brain, and the addition of uridine leads to an additional synergistic increase

Materials and experimental methods

Diets

The standard control diet (Table 4) consisted of Teklad Global 16% protein rodent diet (Harlan Teklad, Madison, WI), which contained 0.1% choline chloride (CC), which corresponds to a daily dose of 50 mg / kg / day. MFIs were added to a control diet, also developed by Harlan Teklad, in an amount of 0.5% MFI · 2Ta ⁺ by weight, corresponding to 240 mg / kg / day MFI. DHA was administered in an amount of 300 mg / kg / day in a volume of 200 μl / day as a solution in 5% gum arabic, while groups not receiving DHA were given only a carrier substance (5% gum arabic solution without additives). DHA was provided by Nu-Chek Prep (Elysian, MN), and MFIs were provided by Numico (Wagenigen, NL). None of the groups showed significant changes in body weight during the experiment.

Table 4. Standard control diet. Express analysis (%) Protein 16.7% Carbohydrate 60.9% Fat, fiber, ash 13.7% Choline 0.1% Fatty Acids (g / kg) Saturated 7.34 Unsaturated C18: 1n-9 oleic acid 8.96 C18: 2n-6 linoleic acid 23.12 C18: 3n-3 linolenic acid 1,53

Getting brain

Gerbils were anesthetized using ketamine and xylazine (80 and 10 mg / kg body weight, intraperitoneally), the animals were killed by immersing their head in liquid nitrogen for 2 minutes and then beheaded. Brains were immediately and quickly removed (30 sec) using bone nippers and stored at -80 ° C.

Measurement of phospholipids in the brain

Frozen hemispheres of the brain were weighed and homogenized in 100 volumes of ice-cold deionized water using a tissue shredder (Polytron PT 10-35, Kinematica AG, Switzerland), then analyzed as described in Example 1.

Determination of DNA and protein

Protein in whole brain homogenate samples was measured using bicinchoninic acid (Perkin Elmer, Norwalk, CT, USA) as a regent for protein. DNA content was determined by measuring the emission of samples at 460 nm on a fluorimeter in the presence of bis-benzimidisole, a fluorescent dye known as Hoechst H 33258 (American Hoechst Corporation), which has a maximum excitation at 356 nm and an maximum emission at 458 nm when it binds to DNA .

results

Male gerbils weighing 80-100 g were divided into 4 groups of 8 gerbils, animals were introduced additives, are presented in table 1:

Table 1. Study groups. Group Additive Quantity / Method one Control diet + carrier (5% gum arabic) 2 Na-UMF + vehicle (5% gum arabic) Na-UMF 0.5% as a meal. 3 DHA 300 mg / kg per day through a gastric tube four DHA + Na-UMF As mentioned above

After 4 weeks, animals were killed and 1 hemisphere of the brain, without cerebellum and brain stem, was used to determine the total content of phospholipids and the content of FC, phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylserine (PS). Omega-3 fatty acids (DHA) increased total phospholipid levels to significantly higher levels than the control group (Figure 4 and Tables 2 and 3). The combination of DHA with UMF led to an additional increase (26%), which was synergistic (i.e., the increase in phospholipid content in this case was greater than the sum of the increases observed in the groups treated with DHA (12%) or UMF (5%)). Similar results were observed for each individual phospholipid (tables 2 and 3). Statistically reliable data were obtained in either of two cases - when the phospholipid content was normalized to the protein content (Figure 4A and Table 2) or DNA (Figure 4B and Table 3).

Table 2. Effect of DHA, UMF, or the use of both compounds on brain phospholipids levels normalized to protein levels. Data are presented as mean values +/- standard error of the mean (SEM). Statistical analysis was performed using two-way ANOVA analysis and Tukey test. "*" Means that P <0.05; "**" - P <0.01; "***" - p <0.001 when compared with the values for the control group.

Processing / Lipid Total phospholipid content FH FE CM FS FI The control 351 ± 8 152 ± 6 64 ± 4 45 ± 2 33 ± 3 21 ± 2 UMF 367 ± 22 171 ± 8 * 84 ± 8 * 52 ± 5 35 ± 3 31 ± 2 ** DHA 392 ± 20 185 ± 12 * 78 ± 5 * 56 ± 3 * 39 ± 3 32 ± 2 ** UMF + DHA 442 ± 24 *** 220 ± 12 *** 113 ± 6 *** 73 ± 4 *** 46 ± 6 *** 36 ± 6 ***

Table 3. The effect of DHA, UMF, or both compounds on brain phospholipids levels normalized to DNA levels. Statistical analysis and presentation of the data obtained, as described in the signature to table 2.

Processing / Lipid Total phospholipid content FH FE CM FS FI The control 885 ± 45 332 ± 12 176 ± 13 112 ± 5 79 ± 8 54 ± 5 UMF 878 ± 18 368 ± 10 * 195 ± 9 111 ± 4 86 ± 7 78 ± 6 ** DHA 909 ± 77 366 ± 13 * 196 ± 18 126 ± 8 98 ± 7 84 ± 13 ** UMF + DHA 1058 ± 25 *** 462 ± 26 *** 261 ± 30 *** 169 ± 11 *** 110 ± 5 *** 85 ± 10 ***

These data confirm the results of the above examples, showing that both omega-3 fatty acids and omega-6 fatty acids increase brain phospholipid synthesis and brain phospholipid levels, both general and individual phospholipid levels. Moreover, these data show that the combination of PUFA with uridine leads to additional synergistic increases. In addition, these data show that the stimulation of synthesis under the action of PUFAs is a common phenomenon and is not limited to a specific phospholipid or experimental model.

The proportional increase in the levels of 4 structural phospholipids, which make up the bulk of the cell membranes in the brain (4 phosphatides: PC, PE, PS, and sphingomyelin), was approximately the same, and the levels of each of these four compounds increased by about 20%. Thus, the proportional ratios of 4 structural phospholipids in the membranes were preserved. As a result of this, the membrane mass increased without disturbing the normal structure and function of the membrane. These data confirm the data of the previous examples, providing additional evidence that the compositions of the present invention improve and enhance brain activity.

Example 6

Introducing omega-3 fatty acids to gerbils reduces brain levels of CDP-choline but increases levels of phospholipids in the brain

Materials and experimental methods

Determination of CDP-Choline

Aliquots (2 ml) of the upper (aqueous) phase were dried under vacuum, re-dissolved and introduced into the HPLC apparatus. The dried samples were dissolved in 100-200 μl of water and analyzed by HPLC on an anion exchange column (Alltech Hypersil APS-2.5 mm, 250 × 4.6 mm). CDP-choline was eluted using a linear gradient of buffers A (H ₃ PO ₄ , 1.75 mM, pH 2.9) and B (NaH ₂ PO ₂ , 500 mM, pH 4.5) from 0 to 100% for 30 min When using this solvent system, CDP-choline was well separated from compounds eluting next to it, such as UMF, in isocratic mode for 40 minutes. The retention time for CDP-choline was 9.5 minutes. The column was washed with buffer B at the end of each experiment and every few days to remove tightly bound nucleotides. Peaks of individual nucleotides were detected by absorption in the ultraviolet region at 280 nm and identified by comparison with the position of authentic standards, as well as by adding nucleotide standards to the analyzed sample.

results

To determine the effect of PUFA administration on CDP-choline levels, CDP-choline levels in the brain were measured in animals from the previous example. Administration of DHA and / or UMF decreased levels of CDP-choline (Figure 5A) and levels of CDP-ethanolamine (Figure 5B). DHA lowered the levels of CDP-choline by 26% (compared to levels in animals that received only the control diet and carrier substance DHA), and in gerbils treated with UMF by 21% (compared to levels in animals treated with UFM-containing diet and carrier substance DHA) (in both cases, P <0.05). Two-way ANOVA analysis revealed a significant effect of DHA [F (1.28) = 31.7; P <0.001].

In another experiment, administering UMF to a standard diet without concomitant administration of DHA significantly increased levels in the brain of PF, FE, and PI by 13%, 29%, and 48%, respectively (Table 5A). Administration of DHA without UMF also significantly increased brain levels of these phosphatides (by 22%, 20% and 52%, respectively), as well as sphingomyelin (by 24%). UMF + DHA increased the levels of all phospholipids more than the total increased UMF or DHA used individually.

Next, the time dependence of this increase was investigated. After 1 week of treatment, the UMF did not provide a significant effect, while the UMF + DHA caused a small but significant increase in the content of PF (21%) and PS (38%) in the brain. Treatment with UMF + DHA for 3 weeks caused a significant increase (21-48%) of all 5 phospholipids; one MFI caused a smaller, but nonetheless significant increase (Table 5B).

Table 5. The effect of UMF and / or PUFA on brain phospholipids. Processing (groups) Total phospholipids (nmol / mg protein) PF (nmol / mg protein) PV (nmol / mg protein) CM (nmol / mg protein) FS (nmol / mg protein) PI (nmol / mg protein) BUT Control diet + carrier 351 ± 8 152 ± 6 65 ± 4 45 ± 2 33 ± 3 21 ± 2 Diet with UMF + carrier 367 ± 22 171 ± 8 * 84 ± 8 * 52 ± 5 35 ± 3 31 ± 2 ** Control diet + DHA 392 ± 20 185 ± 12 * 78 ± 5 * 56 ± 3 * 39 ± 3 32 ± 2 ** Diet with UMF + DHA 442 ± 24 *** 220 ± 12 *** 113 ± 6 *** 73 ± 4 *** 46 ± 6 *** 36 ± 3 *** AT Control diet + carrier 403 ± 23 155 ± 8 69 ± 3 47 ± 3 34 ± 1 20 ± 2 One week UMF + carrier 390 ± 21 154 ± 6 63 ± 4 49 ± 3 39 ± 1 20 ± 4 UMF + DHA 435 ± 15 188 ± 8 * 79 ± 6 57 ± 6 47 ± 1 *** 23 ± 1 Three weeks UMF + carrier 479 ± 5 * 199 ± 5 *** 87 ± 4 * 70 ± 4 * 42 ± 2 * 25 ± 1 UMF + DHA 502 ± 12 *** 217 ± 5 *** 102 ± 4 *** 73 ± 5 ** 41 ± 1 * 27 ± 1 *

Thus, under these conditions, the effect of PUFA administration on brain phospholipids is realized through an increase in the conversion of CDP-choline to PF and related phosphatides.

Example 7

Introducing gerbils with omega-3 fatty acids and / or uridine increases synaptic protein levels

Materials and experimental methods

Measurement of synoptic protein levels

Synaptic proteins were determined by the method of Slot blot and Western blotting. For Western blotting, aliquots of brain homogenates were mixed with 2 × KFL application buffer and boiled for 5 min before gel electrophoresis. Equal amounts of protein were applied to the gel and separation was performed using SDS-PAGE (4-20%; Bio-Rad, Hercules, CA, USA) polyacrylamide gel with sodium dodecyl sulfate. Proteins were then transferred onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA). The remaining free binding sites were blocked using 4% skimmed milk powder (Vamation, Glendale, CA, USA) for 30 min in a Tris-buffered saline-Tween (TBST) solution. 2 × KFL application buffer was prepared by mixing 3.76 ml of 1M TRIS, pH 6.8; 6 ml of 20% sodium dodecyl sulfate; 6 ml of glycerin; 1.5 ml mercaptoethanol; 2 ml of 1% bromphenol blue paint; and 10.74 ml of water.

For Slot blotting, two sets of aliquots (18-21 μl; containing 20 μg protein) of brain homogenates in deionized water were transferred directly to polyvinylidene difluoride membranes (PVDF, Immobilon-P, Millipore, Billerica, MA, USA) using vacuum filtration using an instrument for microfiltration slot blots [Minifold® II Slot Blot System (SCR 072/0); Schleicher & Schuell, Inc., Keene, NH, USA]. The remaining free binding sites were blocked using 4% skimmed milk powder (Vamation, Glendale, CA, USA) in TBST for 30 minutes. Membranes (after slot blotting and Western blotting) were then washed 5 times in TBST buffer and incubated in a TBST solution containing the desired antibodies (mouse anti-NF-70, rabbit anti-NF-M, mouse anti-PSD-95 and mouse anti-synapsin-1). After overnight incubation and five washes in TBST buffer, blots were incubated for 1 hour with suitable secondary peroxidase-associated antibodies. The blots were then washed five times in TBST buffer, and protein-antibody complexes were detected and visualized using an ECL system (Amersham Biosciences, Piscataway, NJ, USA) and Kodak X-AR film. The films were digitized using a Supervista S-12 scanner with a transillumination adapter (UMAX Technologies, Freemont, CA, USA). Immunoreactive bands were compared by density using the Public Domain NIH Image program. The area under the absorption curve was normalized as a percentage of the area obtained for control groups on the same blot. Protein levels were expressed as a percentage of the levels of the same proteins in control animals.

results

Levels of 4 synaptic proteins of the brain were measured in animals (n = 8) treated with both UMF and DHA in the amounts indicated in Example 5. After 3 or 4 weeks of treatment, the number of neurofibrillar axon proteins NF-70 and NF-M increased by 43 % (P <0.01) or 102% (P <0.001), and 19% (P <0.05) or 48% (P <0.01), respectively (figure 6). PSD-95 postsynaptic dense protein and synapsin-1 vesicular protein levels increased by 38% and 41% after 3 weeks (in both cases P <0.001) and 35% (P <0.01) or 25% (P <0 05) after 1 week (Figure 7).

These data provide additional evidence that the introduction of PUFAs and uridine increases the number of synaptic membranes. These increases were similar to those observed for phospholipid levels, indicating an increase in the number of synapses in the brain.

Example 8

DHA, EPA, and arachidonic acid (aa) increase brain phospholipids

Materials and experimental methods

Adult gerbils received a standard control diet (table 4) with or without the addition of 0.5% UMF and / or 300 mg / kg / day of DHA, EPA or AA. For groups not treated with DHA, only the vehicle (5% gum arabic solution) was administered.

results

Gerbils were injected with UMF and / or PUFA for 3 weeks, after which they were sacrificed and levels of various phospholipids in the brain were measured. As shown in Table 6, DHA, EPA, and AA all increased phospholipid levels.

Table 6. Levels of brain phospholipids after administration of PUFA and / or uridine. Treatment Total phospholipid content FH FE CM FS FI Control + Media 333 ± 9 113 ± 6 63 ± 4 19 ± 1 25 ± 2 15 ± 1 UMF + carrier 332 ± 5 131 ± 2 70 ± 1 22 ± 1 29 ± 1 16 ± 1 Control + DHA 344 ± 16 133 ± 6 77 ± 2 24 ± 2 34 ± 3 18 ± 2 Control + EPA 347 ± 19 125 ± 8 76 ± 4 26 ± 3 31 ± 1 22 ± 2 UMF + DHA 374 ± 17 147 ± 6 88 ± 3 28 ± 3 39 ± 2 22 ± 2 UMF + EPA 407 ± 22 148 ± 3 91 ± 4 30 ± 1 41 ± 2 26 ± 2 UMF + AA 389 ± 28 127 ± 8 88 ± 10 25 ± 2 31 ± 3 22 ± 2

Example 9

Omega-3 fatty acids and uridine increase the number of dendritic spines in the brain of adult and developing gerbils and rats

Ordinary adult gerbils received a control diet or a diet enriched in UMF (240 mg uridine / kg) and DHA (300 mg / kg, via a gastric tube) daily for 2 weeks. Animals were decapitated, after which fixed sections of the hippocampus were stained with carbocyanine stain of DiI (C18) 3 membranes (”DiI,” Molecular Probes, Eugene, OR) at the end of treatment. Images of hippocampal neurons were obtained using two-photon microscopy. In animals treated with DHA + UMF, the density of dendritic spines (the number of processes per unit length of the dendrite) significantly increased in the pyramidal neurons CA1 of the hippocampus (an increase of 27%, p = 0.001 compared with the control group; Figure 8).

In another experiment, pregnant rats were given ad libitum (without limitation) either a choline-containing control diet or the same diet enriched with uridine in the form of UMF for 10 days before birth and 20 days of feeding; half of each group also received DHA or its solvent daily through a gastric tube. The pups were then sacrificed and brain sections were examined to determine the number of dendritic spines in the hippocampus. By themselves, both UMF and DHA increased the number of dendritic spines, while their combination provided an additional increase in their number (Table 7).

Table 7. An increase in the number of dendritic spines in developing animals in response to the administration of UMF and DHA. Treatment Number of spines The excess of control (%) The control 53 - UMF 68 28% DHA 70 32% UMF + DHA 75 42%

Example 10

DHA and UMF improve learning ability

Materials and experimental methods

The animals were provided with ad libitum food and water until the day of testing, after which the gerbils were first kept hungry for 17 hours during the night and then fed with food from 11 a.m. to 6 p.m. Gerbils received UMF-enriched food and / or 300 mg / kg of DHA, starting from the age of 3 months (n = 8 in each group) for 4 weeks before the start of behavior training and until the end of the training period. At first, within 4 days the animals got used to the test conditions. Over the next 4 days, they got used to the labyrinth, in the compartments of which pieces of food were placed, and the animals were given 3 minutes to find food. Gerbils were given 1 attempt per day, and between attempts all surfaces were treated with 10% ethanol. The training consisted in the fact that pieces of food were placed at the far end of the maze always in the same 2 compartments in all attempts. A gerbil was placed in the center of the maze and given 2 min in order to find food. A working memory error occurred if the gerbil re-entered the compartment that contained a piece of feed, and which it had already visited during this attempt. A long-term memory error occurred if the gerbil entered a compartment in which there had never been food during previous attempts. The percentage of feed pieces found was recorded.

results

To determine the effect of uridine and / or DHA on learning, the animals were given a uridine and / or DHA-containing diet and then a memory test was performed. DHA and UMF increased the percentage of animals able to perform the test (figure 9).

Example 11

Omega-3 fatty acids increase the synthesis of phospholipids in neurons in a surviving culture

Materials and experimental methods

Rat hippocampal cells were cultured for 3 weeks in Neutrobasal plus B27 medium until complete maturation was achieved. On the day of the experiment, cells were incubated in DMEM + choline, with or without DHA. Then, ¹⁴ C-choline was added, and the cells were incubated for another 2 hours, then the newly formed FC labeled with ¹⁴ C was extracted and its amount was determined as described in Example 1.

results

To determine the effect of DHA on phospholipids in neurons in a surviving culture, neurons were pretreated with a mixture of DHA + choline. DHA increased the synthesis of phospholipids by more than 2 times compared with cells pretreated with choline alone (“control”) (Figure 10; P = 0.04). These data confirm that DHA increases phospholipid levels in brain cells and nerve cells.

Claims (15)

1. A method of increasing the level of synaptic proteins in a nerve cell or brain cell of a subject, comprising administering to said subject a) omega-3 fatty acid, omega-6 fatty acid, or a combination thereof, and b) uridine or c) a choline salt. 2. The method according to claim 1, where the named subject suffers from impaired memory. 3. The method according to claim 2, where the impaired memory is age-related. 4. The method according to claim 1, where the specified introduction increases the synthesis of phospholipid in a nerve cell or brain cell of the named subject. 5. The method according to claim 4, where the named phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine or sphingomyelin. 6. The method according to claim 1, wherein said omega-3 fatty acid is docosahexaenoic acid or its metabolic precursor, eicosapentaenoic acid or its metabolic precursor, docosapentaenoic acid or its metabolic precursor, alpha-linolenic acid or its metabolic precursor or a combination thereof. 7. The method according to claim 1, where the named omega-6 fatty acid is arachidonic acid or its metabolic precursor, linoleic acid or its metabolic precursor, or a combination thereof. 8. The method according to claim 1, where the named uridine is an acyl derivative of uridine, uridine phosphate or cytidine-5'-diphosphocholine (CDP-choline). 9. The method of claim 8, where the named uridine phosphate is uridine-5'-monophosphate (UMP), uridine-5'-diphosphate (UDP), uridine-5'-triphosphate (UTP) or is a salt of the named UMP, UDP or UTP 10. The method according to claim 9, where the named uridine phosphate is a uridine-5'-monophosphate (UMP). 11. The method according to claim 1, where the named salt of choline is choline chloride, choline bitartrate or choline stearate. 12. The method according to claim 1, where the introduction takes place in the form of a medicinal product or nutritional supplement. 13. The method according to claim 9, where the subject is administered from about 200 to about 500 mg of uridine, its acyl derivative, uridine phosphate or CDP-choline. 14. The method of claim 1, wherein about 200 to about 800 mg of choline salt is administered. 15. The method according to claim 1, wherein uridine-5'-monophosphate (UMP), a choline salt and docosahexaenoic acid are administered.

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