The present invention refers to a combination comprising D-aspartic acid and L-arginine or salts thereof for the improvement of cognitive activities and memory in dementia such as dementia senile as Alzheimer's disease.

Dementia senile is a loss of brain function of degenerative origin due to a series of causes leading to vascular damage (vascular dementia) and to Alzheimer's disease. Most of the dementia causes are not avoidable. Usually dementia occurs with age. The main symptoms are problems with language, memory, perception, personality change, cognitive abilities, confusion on persons, places or times, motory problems, changes in personality; anxiety, decreased ability to take care of themselves; decreased level of interest in daily life activities; depression; irritability; egocentrism; incontinence, problems in eating.

Alzheimer's disease was detected for the first time by the German scientist Alois Alzheimer at the beginning of the past century. It is due to the onset in the brain of amyloid plaques which surround and disrupt neurons, so that people suffering from this disease lose their memory and cognitive functions, do not recognize places where they are and also lose the knowledge of persons around them, including their family members.

In the most advanced forms, the Alzheimer's patients become completely inactive and inefficient in self-management (they do not cure their own person, do not eat or wash and do not feel the excretory stimuli). The people affected by Alzheimer's disease represent approximately 3-5% of male and female population between 55 and 65 years of age with an in- crease of about 5-8% in people between 65- and 75 years of age and about 15-20% in people between 75 and 85 years of age.

The course of the disease includes a first mild phase, then an intermediate stage and finally a severe stage. The disease is often antic- ipated by the so-called mild cognitive impairment (MCI), a slight decrease in performance in different cognitive (memory, orientation and verbal skills) together with progressive amnesia and other cognitive impairments. The memory impairment is primarily circumscribed to sporadic episodes in dai- ly life, or disorders of the so-called on-going memory (remember what you ate for lunch, what was done during the day) and of prospective memory (which concerns the organization of the near future such as remembering to go to an appointment). Then, the deficit progressively increases, also the backward episodic memory (concerning the facts of one's own life or public events of the past) and the semantic memory (the foreground) are affected, while the procedural memory (which concerns the automatic execution of actions) is relatively spared until the late stages of the disease. Starting from the mild and intermediate stages, increasing difficulties in speech production may then occur, with inability in definition of people or objects names.

In advanced stages, behavioral problems may occur (wandering, compulsion to repeat movements or actions, inconsistent behavioral reactions) or psychiatric problems (confusion, anxiety, depression and occasionally, deliria and hallucinations). As the disease progresses, people have not only memory impairment, but also a loss in instrumental functions mediated by the associative cortex, and may therefore present aphasia and apraxia.

From the epidemiological standpoint, apart from rare genetic familiar forms wherein the person is affected by the disease at a young age, the main factor related to the incidence of the disease is age. Typically, after age 65, the incidence of the disease increases progressively with age, reaching a significant spread in population over age 85. Biological and biochemical causes of the disease are due to a widespread disruption of neurons, caused mainly by a small protein (or polypeptide) of 42 amino acids, i.e., β-amyloid. Such peptide is originated from a membrane protein P T/IT2011/000411

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with molecular weight of about 90,000 Da, the β-Amyloid Precursor Protein (β-ΑΡΡ). β-ΑΡΡ is a protein present on the neuronal plasmatic membrane and is partly located within the neuron membrane and partly outside the neuron (transmembrane protein). When this protein is hydrolyzed in two specific points by the action of two enzymes, β-secretase and γ- secretase, the 42 amino acids peptide is released. The latter becomes insoluble over time and precipitates on neurons forming tangled plaques, i.e. the amyloid plaques, which enwrap and disrupt the active neurons (1). This results in a blockage of synaptic transmission and progressive brain atrophy. A strong reduction of acetylcholine - a neurotransmitter essential to the brain function - was also observed in the brain, thus reducing memory and any other intellectual faculty (2).

In healthy subjects the β-ΑΡΡ (β-Amyloid Precursor Protein) is degraded in situ by the action of two digestive enzymes. The a-secretase and β-secretase (a variant of a-secretase) enzymes cleave a big size precursor protein (a transmembrane protein) called β-Amyloid Precursor Protein (β-ΑΡΡ), thus generating a peptide fragment of 42 amino acids which is β-amyloid. The β-amyloid, once generated, becomes insoluble and settles as extracellular aggregates on neurons membranes, thus forming the amyloid plaques. These are cell clusters which over time disrupt the healthy neurons and also give rise to an inflammatory process that triggers an immune response by recalling macrophages and neutrophils with production of cytokines, interleukins which contribute to neuronal damage. Other studies have found that in people with Alzheimer's disease an addi- tional pathological mechanism occurs. Within the neurons, a protein called "Tau", abnormally phosphorylated, accumulates in the so-called "neurofibrillary tangles" disrupting the cholinergic neurons of the cortical areas, subcortical and hippocampal areas. The hippocampus is a brain structure that plays a crucial role in learning and memory processes and conse- quently the disruption of neurons in these regions is believed to be the main cause of memory loss. There is no specific cure for Alzheimer's disease. Decreased levels of acetylcholine have been observed in the brain affected by Alzheimer's disease, a chemical neurotransmitter in the central and peripheral nervous system. Inhibitors of cholinesterase (the enzyme which catabolizes acetylcholine) as, for example, physostigmine, galanta- mine, rivastigmine and neostigmine are used in therapy to mitigate the loss of acetylcholine (3). However, we have to refer that these drugs have only a temporary effect in time. Another set of drugs are those that directly affect the glutamatergic system, as memantine and donepezil. These drugs have also a limited efficacy in the treatment period (4). Since the formation of amyloid plaques in the brain affected by Alzheimer's are accompanied by an inflammation in situ, drugs having an anti-inflammatory effect are often used. Finally, for the treatment of the Alzheimer's disease, Gingko biloba (an extract from roots of tropical plants) is also used, based on the hypothesis that extracts of this plant are able to improve cerebral blood flow (5) increasing the oxygen supply. A new approach for the treatment of the Alzheimer's disease is based on the use of β-secretase inhibitors. Some synthetic compounds have been proposed belonging to the group of dichlorophenol derivatives (6). However these compounds, though have produced encouraging results in laboratory animals, they have never been used on humans in the treatment for Alzheimer's disease because when administered orally they are unable to cross the blood brain barrier. Finally, a cure based on immunotherapy has been proposed using vaccines based on antibodies anti-Beta-amyloid. Also in this case the treatment was not successful because the antibody is not able to cross the blood brain barrier (7).

In addition to Alzheimer's disease, there are other kinds of dementia senile, which are all included in a clinical framework of vascular dementia, characterized by insufficient blood flow to the brain and therefore by a reduced intake of oxygen and nutrients. The most common form of de- mentia is atherosclerosis caused by brain stroke or by any cause of blood flow interruption.

In view of the foregoing, there appears to be a clear need of providing new therapies for the treatment of dementia such as dementia senile and Alzheimer's disease which do not present the disadvantages of known therapies.

D-aspartic acid is a naturally occurring amino acid having the same chemical structure of L-aspartic acid (an amino acid naturally present in free form in animal tissues and is also a constituent of proteins), but a dif- ferent spatial configuration (Fig.1). The D-aspartic acid has been discovered for the first time in 1977 in the brain of the sea mollusc Octopus vulgaris (8) and was later found in the endocrine and nervous tissues of several animal species (9) including rat (10-13) and humans (14-15). High levels of D-aspartic acid are found in the nervous system of the embryo of mammals including the rat (12.13) and humans (15). On the other hand, in adults high levels of D-aspartic acid are found in the endocrine glands (16- 20). Recent studies about some marine molluscs have shown that D- aspartic acid is present in the retina and has a role in the visual system (21-22). Moreover, experiments on hippocampal slices of mice have shown that D-aspartic acid is involved in synaptic plasticity of neurons (23) and, finally, water maze experiments (an experimental method to assess the spatial learning of rats and mice) carried out on young rats have shown that there is a direct correlation between D-aspartic acid concentration in the brain and spatial memory (24). It is interesting to note that in humans, it has been shown that, the concentration of D-aspartic acid is significantly reduced in brains of patients affected by Alzheimer's disease (brain tissue obtained post-mortem) compared to brains of persons not affected by Alzheimer's disease (25). In the white matter of a brain with Alzheimer's, D-aspartic acid concentration is 10.5 nmoles/g tissue and in the brain of healthy subjects is 22.4 nmoles/g tissue. Similar values are 1

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also found in gray matter (25). In addition, subsequent studies on the brains of people suffering from Alzheimer's disease and healthy brains, have shown that also in other regions of the brain (frontal cortex, hypothalamus, hippocampus, occipital and parietal cortex, cerebellum, etc.) the concentration of D-aspartic acid in Alzheimer's was found significantly reduced compared to that found in corresponding areas of healthy brains (26) (Fig. 2).

The author of the present invention has recently discovered that D- aspartic acid is an endogenous neurotransmitter in the rat and human brain neurons. It is specifically located in synaptic vesicles (neuronal structures containing chemical neurotransmitters) and is involved in learning and memory.

Studies carried out show that D-Aspartic acid in humans is able to improve learning and memory for receptor activation of D-Aspartic present in the cerebral cortex and hippocampus that represent areas of the brain more involved in learning and memory. Therefore, D-Aspartic acid in humans is an aid to improving memory, for example, in cases of Alzheimer's disease and dementia senile. The author of the present invention has also conducted a study in rats to evaluate the effectiveness of the treatment of D-Aspartic acid, L-Aspartic acid and L-arginine alone or the combination consisting of D-Aspartic acid and L-Arginine or a combination consisting of D-Aspartic acid and L-Aspartic acid in appropriate molecular ratios, on the improvement of cognitive activities and memory of rats and to evaluate the efficacy of the compound formed by the combination D-Aspartic acid and L-Arginine on improvement of cognitive activities and memory in patients with dementia senile and Alzheimer's. First, The study was conducted on different experimental groups of rats and then on a group of demented patients. Rats were subjected to oral treatment of certain amino acids alone and in combinations with each other. After a period of 30 days of treatment, the rats were tested for using the Learning System for Water- Maze (R-Morris, 1984), worldwide recognized as a method suitable for the purpose. The results showed that D-aspartic acid or L-arginine or the combination of D-Aspartic acid and the L-Aspartic acid induce a good positive effect on increasing learning and memory in rats. Rats treated with these compounds improve memory by about 2-2.5 times compared with the control group. Moreover, this study showed that, among the various combinations of amino acids tested, the compound consisting of 20 mM D-Aspartic acid and 10 mM L-arginine was found to be the most efficient way to improve memory in rodents by improving cognitive activities and memory by about 10 fold compared to control rats. In addition, also the compound consisting of 20 mM D-Aspartic acid and 20 mM L-arginine has efficiently responded to the increase of memory both in animals and humans.

The study on humans showed that in all patients treated with the combination of D-aspartic acid and L-arginine there was a memory recovery of 60-70%.

Therefore, it is a specific object of the present invention a combination comprising, or consisting of, D-Aspartic acid and L-Arginine and/or L- Aspartic acid or their salts for simultaneous, separate or sequential use in the treatment for restoring of and/or improving cognitive activities and memory in short and long period.

The invention also concerns a pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or their salts, as active ingredients, in combination with one or more exci- pients and/or pharmaceutically acceptable adjuvants, for use in the treatment for restoring of and/or improving cognitive activities and memory in short and long period.

The combination and the pharmaceutical composition according to the present invention can be used both in the treatment of dementia such as dementia senile and Alzheimer's disease in order to restore and im- P T/IT2011/000411

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prove cognitive activities and memory and as supplements to increase the memory, for example in case of school exams, competitions etc.. The dementia may be by vascular origin or by different origin. Moreover, since L- Aspartic acid is one of the main amino acids of the nervous system and since the L-Arginine has properties on the restoration of memory, the invention also concerns the association consisting of a combination of L- aspartic acid and L-arginine or salts thereof for simultaneous, separate or sequential use in the treatment to restore and/or improve cognitive activities and memory in the short and long period, for example in the treatment of dementias such as dementia senile and Alzheimer's disease.

The combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may contain a percentage of D-Aspartic acid or L-arginine or L-aspartic acid or salts thereof, which varies from 0.1% to 99.9% compared to the total of said D-aspartic acid and L-arginine or said D-aspartic acid and L-aspartic acid or salts thereof.

The combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention, can be administered in one or more daily doses, each dose comprising from 0,1 g to 10 g of D- Aspartic acid, 0.1 g to 10 g of L-Arginine and/or 0.1 g to 10 g L-Aspartic acid or salts thereof.

The combination or pharmaceutical composition comprising or con- sisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may further comprise one or more basic or acidic substances in suitable amounts to bring the pH to neutrality.

The combination or pharmaceutical composition comprising or con- sisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may contain salts of D- Aspartic acid or L-Aspartic acid selected from the group consisting of salts of Sodium, Potassium, Calcium, Magnesium, Zinc, Selenium, Copper, Manganese, Iodine.

Moreover, the combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may further comprise at least an amino acid selected from the group consisting of D- or L-glutammic acid; D- or L-asparagine; D- or L-glutamine; D- or L- serine; D- or L-alanine, D- or L-threonine; D- or L-hystidine, D-arginine; D- or L-cysteine; D- or L-cystine; D- or L-valine; D- or L-methionine; D- or L-tyrosine; D- or L-phenylalanine; D- or L-isoleucine; D- or L-leucine; D- or L-tryptophan; D- or L-carnitine; propionyl-L or D-carnitine; D- or L- acetyl-carnitine; L-carnitin fumarate; carnosine; melatonine; theanine, or salts thereof. The amino acids may be employed as such or as compounds salified with one of the following basic ions: Na, K, Ca, Mg and carbonate, or acid ions. CI, bicarbonate ion, etc.

The combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may further comprise a ketoacid, such as, e.g., for example, alpha-oxalacetic acid, alpha- ketoglutaric, etc, or a salt thereof.

According to a further aspect, the combination or pharmaceutical composition of the invention may include at least one water-soluble and/or liposoluble vitamin.

Among the water-soluble vitamins the following may be mentioned:

Vitamin B1 (thiamine),

Vitamin B2 (riboflavin)

Vitamin B3 or Vitamin PP (niacin or nicotinic acid) Vitamin B5 or Vitamin W (pantothenic acid)

Vitamin B6 or Vitamin Y (pyridoxine or pyridoxamine or pyridox- al)

Vitamin B8 or Vitamin H (biotin)

Vitamin B9 or Vitamin M (folic acid)

Vitamin B12 (cobalamin)

Vitamin C (ascorbic acid)

Among the liposoluble vitamins the following may be mentioned:

Vitamin A (retinol and analogues)

Vitamin D (ergocalcipherol D2 and colecalcipherol D3)

Vitamin E (tocopherol)

Vitamin K (naphthoquinone and derivatives).

The combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may further comprise at least one antioxidant selected the group consisting of coenzyme Q-10, ascorbic acid, folic acid, alpha-lipoic acid, glutathione, inosidol, tocopherols, tocotrienols (Vitamin E), superoxide dismutase, lycopene, ellagic acid, melatonin.

In addition, the combination or pharmaceutical composition comprising or consisting of D-Aspartic acid and L-Arginine and/or L-Aspartic acid or salts thereof for use according to the present invention may further comprise at least one metal ion selected from the group consisting of magnesium, zinc, selenium, sodium, potassium, calcium, in the form of chlorides, sulphates, fluorides, or in the form of metal oxides.

The present invention will be now described by way of non- limitative illustration according to some preferred embodiments thereof, with particular reference to the figures of the annexed drawings wherein:

Figure 1 shows the composition and the stereo-chemical configu- IT2011/000411

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ration of D-Aspartic acid (D-Asp) and L-Aspartic acid (L-Asp) molecule.

Figure 2 shows the concentration of D-aspartic acid in various regions of brain with Alzheimer's disease and normal brain (control). The values of the figure refer to the concentration of D-Aspartic acid assessed in the total homogenate of the brain tissue.

Figure 3 shows an example of separation of D-Aspartic acid and L-Aspartic acid from other amino acids by HPLC. The figure shows a typical chromatogram run by HPLC of a mixture of standard amino acids consisting of 20 pmoles of D-aspartic acid and 100 pmoles di L- Aspartic acid and other L-amino acids (L-Glu, L-Ser, L-His, L-Thr, Gly, L-Arg, L-Ala, L-Tyr, L-Val, L-Met, L-lle, L-Leu, L-Phe and L-Lys), deriva- tized with OPA-NAC (ortho-phthaldialdehyde-N-acetyl-L-Cysteine). D- Aspartic acid is separated from other amino acids and can be quantified on the basis of the peak area.

Figure 4 shows synaptosomes and synaptic vesicles from human brain. The figure shows a typical example of synaptosomes and synaptic vesicles purified from human brain of a healthy person of 30 years. The synaptosomes (0.8-1.5 m in size) represent the peripheral ends of the neuron and are filled with synaptic vesicles (40-70 nm in size) con- taining the chemical neurotransmitters.

Example 1: Study on the role of D-Aspartic acid and L-Aspartic acid in restoring memory in patients with Alzheimer's disease and dementia senile

The study carried out is divided into the following phases:

1) Demonstrating that in human brain D-Aspartic acid is present in synaptic vesicles (subcellular particles of nerve endings containing chemical neurotransmitters which are involved in synaptic transmission), there is a mechanism of D-Aspartic acid synthesis in neurons starting from L- Aspartic acid; neurons possess receptors specific for D-Aspartic acid, and which are responsible for communication between neurons.

2) Proving that the use of D-Aspartic acid in patients with Alzheimer's disease increases cognitive skills and memory.

1.1 Method for determining D-Aspartic acid by HPLC

The determination of D-Aspartic acid and other amino acids in the human brain was carried out by modifying the method described in (27) as follows. 0.5 g of the human brain (Alzheimer's or normal) of the frontal region was homogenized in 0.2 M perchloric acid in a 1 :10 ratio, then centri- fuged at 13,000 rpm for 5 min. 10 μΙ of the supernatant (sample) are mixed with 200 μΙ of 0.2 M borate buffer, pH 10.0 and 20 μΙ of OPA-NAC (OPA- N-acetyl-cysteine, consisting of 20 mg of OPA and 10 mg of N-acetyl- cysteine in 2 ml of 50% methanol), 2 min after add 900 I of distilled H2O and then 100 μΙ of this mixture is injected on the HPLC column (High Liquid Chromatography) using a running program consists of a solution A (ci- trate phosphate, 0.03 M pH 5.3 containing 5% acetonitrile) and solution B (comprising 90% acetonitrile). The run program was: 0-10% B in 10 min, then 10-100% B in 7 min, then 100% B in 5 min and 0% B in 1 min. With this program, D-Asp acid exits at an elution time of 5.5 min followed by L- Asp at 6.2 min and L-Glu at 8.0 min and then by other amino acids. As standard was used a mixture consisting of 0.1 mM D-aspartic acid and 0.2 mM of other amino acids instead of the sample. The same procedure was carried out to evaluate the concentration of D-Asp and other amino acids in the homogenate of the whole human brain with Alzheimer's and control and in the synaptic vesicles isolated from these brain tissues. In this case, 100 μΙ of a suspension of synaptic vesicles obtained as described in the next section were homogenized with 100 μΙ of 0.2 M perchloric acid and centrifuged as above and the supernatant used as a sample for the determination of amino acids. The brains of Alzheimer's patients and normal brains were obtained post-mortem by organ bank from Loyola University of Chicago (USA). In Figure 3 is shown a typical example of the evaluation of D-Asp acid and other amino acids (Fig. 3).

1.2 Determination of D-Aspartic acid in human brain and in synaptic vesicles from brains of Alzheimer's patients and healthy persons

The purpose of this experiment is to see if D-Aspartic acid is present as a neurotransmitter in the synaptic vesicles (subcellular particles of neuron endings of 40-60 nm in size) and if there is a difference in concentrations between Alzheimer's brain and control brain. In order to isolate synaptic vesicles, the isolation of synaptosomes was carried out, 0.8-1.5 pm circular biological structures which represent the terminal part of nerve endings (dendrites). They are obtained by homogenization of nerve tissue with a solution of iso-osmotic sucrose. The synaptosomes contain synaptic vesicles that are rich in chemical neurotransmitters such as acetylcholine, L-glutamic acid, L-Aspartic acid, serotonin, GABA, etc.. The preparation of synaptosomes was performed as follows: 1 g of brain tissue was homogenized in 20 ml of 0.32 M sucrose in 0.05 M Tris-HCI, pH 7.5 with a potter homogenizer with a piston - glass walls gap of 15-20 A. The homogenate was then centrifuged at 1 ,000 g for 10 min and the supernatant was centri- fuged at 22,000 g for 20 min. The resulting precipitate was taken up in 4 ml of sucrose 0.32 M and layered on a sucrose gradient consisting of 20 ml of 1.2 M sucrose and 20 ml of 0.8 M sucrose and then centrifuged at 22,000 g for 2 hours. The synaptosomal fraction of the visible in the interface between the solution 1.2 M and 0.8 M sucrose was collected and diluted with an equal volume of physiological buffer (125 mM NaCI, 3 mM KCI, 1.2 mM MgS0 ₄ , 1.2 mM CaCI 2, 1.2 mM NaH ₂ P0 ₄ , 22 mM glucose, 10 mM Na2HC0 ₃ pH 7.4) and centrifuged for 90 min at 22,000 g. The pellet (synaptosomes) was taken up in 400 μΙ of 0.32 M sucrose, pH 7.4 and layered on a second gradient consisting of 5 ml of 1.2 M sucrose, 5 ml of sucrose 0.95 M sucrose and 5 ml of 0.75 M, respectively, and centrifuged at 22,000 g for 90 min. The synaptosomal fraction located in the interface between 0.95 M and 0.75 M sucrose was collected and diluted in 4 ml of physiological buffer and analyzed by electron microscopy. For the preparation of synaptic vesicles (SV), 3 ml of the suspension of synaptosomes were diluted with 30 ml of distilled H20 to break the synaptosomes by os- mosis (vesicles being too small will not break) and allowed to stir for 30 min. Then centrifugation at 22,000 g for 20 min was carried out and the pellet consisting of purified synaptic vesicles was taken up in 0.5 ml of

0.9% NaCI. A portion of the pellet was used for morphology with electron microscopy and part was used for the analysis of D-Aspartic acid content. For this purpose, the pellet of synaptic vesicles was homogenized with a double volume of 0.2 M perchloric acid (to disrupt the vesicles and recover the amino acids contained therein) and centrifuged for 5 min at 22,000 g. The supernatant was then analyzed by HPLC for the content of D-aspartic acid and other amino acids. Fig 4 shows an example of a preparation of synaptosomes and synaptic vesicles from normal human brain, while Table 1 shows the concentration of D-aspartic acid and other amino acids in the homogenate of the brain as a whole and synaptic vesicles obtained from the brains of Alzheimer's patients and healthy patients. The results of this experiment shows that D-Aspartic acid is present in the brain in very small amounts compared to other amino acids, but in synaptic vesicles it presents high concentrations in comparison with other amino acids present in the vesicles (Table 1). Thus, this fact means that D-aspartic acid which is found in the brain is all concentrated in the synaptic vesicles,

1. e. in those sub-cellular structures containing the chemical neurotransmit- ters. This concentration pattern is similar to what occurs for acetylcholine and unlike the L-glutamic acid. For instance, acetylcholine in the brain is also located mainly in the synaptic vesicles and as such serves as a neurotransmitter, on the contrary L-glutamic acid has high concentration throughout the nervous tissue including synaptic vesicles. Clearly, L- glutamic acid plays a metabolic role as well as a role of neurotransmitter. In conclusion, D-aspartic acid can be considered a new chemical neurotransmitter involved in learning and memory processes, because as is shown later, the assumption of an integration based on D-Aspartic acid improves memory in Alzheimer's patients (see below). To confirm this hy- pothesis it is demonstrated that D-Aspartic acid content in the brain of Alzheimer's patients is significantly reduced compared to the brains of control patients. Table 1 shows the concentration of D-aspartic acid (D-Asp), L- aspartic acid (L-Asp) and L-glutamic acid (L-Glu) in the frontal area of the brain of Alzheimer's and normal patients and within synaptic vesicles ob- tained by the same brains

Table 1. D-Aspartic acid content of the frontal zone of human brain and in the synaptic vesicles of healthy people and persons

affected by Alzheimer's disease.

D-Asp L-Asp L-Glu Total

Amino acids

Normal Human Brain

Brain

nmoles/g brain ^* 35.6+6.4 4.650±450 6.100±740 55.000+5.240

% in respect of total amino acids* 0.064±0.01 8.45±0.82 11.1±1.1 100

nmoles/mg proteins ^*** 0.45±0.15 54.3±4.5 64.5±6.8 665±50

Synaptic vesicles

nmoles/mg proteins* ^** 280±25 350±32 420±45 3.300±310

% in respect of total amino acids 8.5+1.0 10.611.2 12.7H . 7 100

Ratio amino acids/mg ** ^* *

proteins 622 6.44 6.51

Human Brain with Alzheimer's

Brain

nmoles/g brain ^** 18.5+5.2 3.900±450 5.750+740 47.300+5.240

% in respect of total amino acids 0.039+0.01 8.2410.92 12.2H .3 100

nmoles/mg proteins* ^** 0.34+0.15 36.4+4.1 48.4±4.8 540+50

Synaptic vesicles

nmoles/mg proteins ^** * 205±18 270+30 330+41 2.650+250

% in respect of total amino acids 7.7±1.0 10.1+1.1 12.411.4 100

Ratio amino acids/mg 602 7.41 6.81

proteins

^* indicates nmoles of D-Asp, L-Asp, L-Glu and total free amino acids contained in 1 g of homogenate of human brain (obtained from the University of Chiga organ bank). * ^* Indicates the percent ratio of the concentration of single amino acid in relation to total amino acids. *** indicates nmoles of amino acids relat to 1 mg of total protein (soluble and insoluble) of vesicles sample. ** ^* * Indicates the ratio of amino acids expressed as nmol / mg of proteins in the sam used among those contained in purified synaptic vesicles and those contained in the brain homogenate.

1.3 Synthesis of D-Aspartic acid in normal and Alzheimer's human brain.

Since D-Aspartic acid is synthesized in the brain by a D-Aspartate racemase, an enzyme found in some mollusks (28) which transforms L- aspartic in D-aspartic (EC 5.1.1.13), in this study it was investigated whether the isaid enzyme was present or not in the human brain and if there is any difference in enzyme activity between the human brain affected by Alzheimer's disease and human control brain.

The enzymatic assay is based on the following reactions:

1) L-Aspartic acid + D-Aspartate racemase = D-Aspartic acid

2) D-Aspartic acid + 02 + H ₂ O + D-Aspartate oxidase = o oxaloacetate + H2O2 + NH3

3) oc-oxaloacetate + 2,4-dinitrophenilhydrazine = a-oxaloacetate- dinitrophenyl hydrazine (yellow color)

4) a-oxaloacetate-dinitrophenylhydrazine + NaOH= -oxaloacetate- dinitrophenilidrazone complex (strong purple red color).

The process of the enzymatic assay was as follows

0.5 g of Alzheimer's brain or control brain was homogenized (1 :5) in 0.02 M phosphate buffer pH 8.0. Then 500 μΙ of supernatant were mixed with 100 μΙ I of Na L-aspartate 1 M (sample) or 100 μΙ of distilled H ₂ 0 (blank sample) and 10 μΙ of purified D-aspartate oxidase from rabbit kidney (22) and incubated for 2 hours at 37°C. Then the test mixture was added with 50 μΙ of perchloric acid 5 M, mixed and centrifuged at 13,000 rpm for 5 min. Then 500 μΙ of supernatant was mixed with 100 μΙ of 2.4- dinitrophenylhydrazine (1 mM in 1 M HCI) and left to stand for 20 min at room temperature. Then 200 μΙ of 4 M NaOH was added, mixed and centrifuged at 13,000 rpm for 5 min. The supernatant is read at 445 nm against distilled H ₂ 0. Finally, the D.O. of the blank sample are subtracted from the D.O. of the sample and D.O. net are index of racemase activity in the brain tissue. For the measurement of enzyme activity it was assumed that the enzyme activity that produces a color intensity corresponding to 1 OD at 445 nm and in the conditions of enzymatic assay is equal to 1 Enzymatic Unit. As in 100 μΙ of the assay mixture there is an enzyme amount from 20 mg of tissue it follows that 1 g of tissue contains 50 enzymatic units.

Table 2 shows the enzyme units obtained from 1 g of the brains of people suffering from Alzheimer's and from 1 g of the brains of healthy people (control). As it can seen the racemase enzymatic activity in the Alzheimer's brain is significantly reduced and this phenomenon is in agreement with the fact that in the Alzheimer's brain also the content of D- Asp acid is present at concentrations significantly lower than in controls.

Table 2 shows the enzymatic activity of D-aspartate racemase (synthesis of D-Asp acid) in the brains of healthy persons and of patients suffering from Alzheimer's disease.

Table 2. Activity of D-Aspartate racemase in normal and Alzheimer's brains

Enzymatic Units /g of brain tissue

Normal brain Alzheimer's brain

E.U./g E.U./g

Patient n. 1 74 Patient n. 1 55

Patient n. 2 68 Patient n. 2 41

Patient n. 3 59 Patient n. 3 39

Patient n. 4 72 Patient n. 4 45

Patient n. 5 45 Patient n. 5 28

Average±SD 63.6111.8 Average±SD 41.6±9.8

The results were obtained from 5 patients suffering from Alzheimer's disease and 5 persons not affected from this disease. Both groups of people were aged between 70 and 80 years. T-tests (T di Student) between Alzheimer's patients and normal person is P <0.000234

1.4 D-Aspartic acid receptors in human healthy brain and in Alzheimer's brains The receptors for the chemical neurotransmitter in the nervous system are membrane proteins of the neuron postsynaptic, and constitute the essential elements to capture the input transmitted by the neurotransmitter and then turn them into physiological signals to the cell (visual signals, memory signals, etc.). In the present study it was ascertained whether in the Alzheimer's brain receptors of Aspartic acid were present and if there is a difference of these receptors in Alzheimer's brains compared to control brains. For this purpose affinity (binding) experiments were carried out using radioactive D-Aspartic (D-[ ³ H] aspartic acid) and in accordance with the procedures described in the literature (29), amended as follows: 0.5 g of brain tissue was homogenized 1 :10 in NaCI 0.9%. Centrifuged at 13,000 rpm for 10 min. the supernatant was removed and the precipitate was taken up with 10 ml of 0.9% NaCI. It was centrifuged as above and the washing operation was repeated for 4 more times. The final precipitate was taken up in 2 ml of NaCI 0.9% and the binding assay was carried out on the latter as follows: 100 μΙ of membrane suspension were mixed with 100 μΙ I of D-[ ³ H] aspartate (100 nM) and 300 μΙ 0.05M of phosphate buffer pH 7.4. Then 5.0 pi of each amino acid to be tested at concentrations between 0 and 0.1 mM was added to the assay mixture and incubated for 60 min at 20°C under stirring. Then the sample was centrifuged for 5 min at 13,000 rpm, it was washed 2 more times in phosphate buffer 0.05 m pH 7.4 and then the precipitate was dissolved in 400 μΙ of 0.1 M NaOH. Finally, it was mixed with 3 ml of scintillation fluid and radioactivity was assessed (CPM counts) and the affinity calculated the membranes affinity has been calculated according to the described methods (29). The results obtained are shown in Table 3. As it can seen from the values reported in this table, human brain possesses high affinity receptors towards D- Aspartic acid. D-aspartic acid binds to these specific receptors for this amino acid and not to glutamate receptors. This consideration derives from the fact that the radioactive D-Aspartic acid, which was previously linked to the membrane, detaches from the same membrane when used in the binding mixture to non-radioactive D-Aspartic acid (cold) and this fact occurs at a very low concentration of D-Asp. On the contrary, the L- glutamic acid and L-Aspartic acid do not have a low affinity in displacing D-Aspartic-membranes bond, thereby indicating that the receptors to which D-Asp is linked are specific receptors for this amino acid. It worth noting that the lower the concentration of cold amino acid displacing the radioactive, the more binding affinity is present. NMDA and others, even at concentrations of 100 mM, are not able at all to move D-Aspartic radioactive binding. This would indicate that these amino acids have no capacity on the D-Asp receptors.

Finally, it is important to note that in Alzheimer's and control brains, the Alzheimer's brain has a significant reduction of these receptors compared to normal brain. In fact, as shown in Table 3, Ki (inhibition constant or affinity constant), which indicates the concentration at which 50% of the binding ties are displaced by cold amino acid, is of 0.45 ± 0.04mM with membranes obtained from Alzheimer's brain and 0:22 ± 0.02 mM in case of membranes obtained from the control brain.

Table 3 shows the activity of D-aspartic acid receptors (displacement of specific bonds for D-[ ³ H] Asp by various amino acids and amino acids analogues on the postsynaptic membranes (PSMS) obtained from the brains of Alzheimer's patients and normal persons).

Table 3

Displacement of D-[ ³ H]Asp binding by various chemical competitors

Brain from Brain from

Control patients Alzheimer's patients

(frontal area) (frontal area)

Binding competitors Κ/ (μΜ) Ki (μΜ)

D-Aspartate 0.22±0.05 0.45±0.04

L-Glutamate 2.4±0.4 3.5±0.4

L-Aspartate 3.5±0.5 5.4±0.6

D-Glutamate 43±8.6 54±10

NMDA >100 >100

Quisqualate >100 >100

Kainate >100 >100

DL-Homocisteate >100 >100

L-Alanine >100 >100

L-Serine >100 >100 The concentration of D-[ ³ H] Asp in the binding assay was 100 nM, and the amino acids or amino acids analogues were 0 to 100 μΜ. The inhibition constant (Ki) indicates the amino acids concentration capable of displacing 50% of the binding ties. The values indicate the mean ± SD obtained from 4 experiments, each carried out on plasma membranes from Alzheimer's or control brains. Values > 00 indicate that the amino acid tested for displacement was completely inactive. The amino acid affinity to membrane receptors is as stronger (specific) as is lower the amino acid concentration which displaced 50% of the bonds of (D-[ ³ H] Asp) to the membranes. 1.5 Accumulation of D-Aspartic acid and L-Aspartic acid in brain and synaptic vesicles in rat after treatment with exogenous D- Aspartic acid

The purpose of this experiment was to know whether D-Aspartic ac- id orally administered enters or not in the brain and if it is transported into synaptic vesicles in order to explicate its action as a neurotransmitter. The experiment was carried out on adult rats of the Wistar strain (kept in cages and in a suitable environment according to the ministerial regulations). The rats of the first group, consisting of 5 animals, were given to drink a solution of 20 mM sodium D-aspartate. A second group of 5 rats was given to drink sodium L-aspartate at a concentration of 20 mM and finally the third group of 5 rats was given to drink tap water. The duration of treatment was 15 days. At the end of the treatment the rats were sacrificed, the brains were removed and homogenized 1:10 in sucrose 0,32. A portion (1/10) of each homogenate was used for the analysis of amino acids (see section 1.1), and the remainder was used for the preparation of synaptic vesicles according to the procedure described above (see section 1.2). Finally, the content of D-aspartic acid was evaluated in the whole homogenate and in the synaptic vesicles. The results obtained from this experiment are shown in Table 4, and indicate that when a rat is administered orally by sodium D-aspartate at a concentration of 20 mM, the concentration of this amino acid in the brain increases of 2.42 times compared to control (rat who drank tap water). It is interesting to note that at least half (56%) of D- Aspartic acid present in the whole brain is carried into the synaptic vesicles, which represent, in terms of cell mass, about a thousandth of brain tissue mass. Therefore these data show that D-Aspartic acid has a specific role in the synaptic vesicles, namely the role of neurotransmitter. In addition to these data, the results presented in Table 4 show that if the animal drinks the L-aspartic acid in place of D-Aspartic acid, a significantly increased concentration of D-Aspartic acid is also accumulated in the brain and in the synaptic vesicles, compared to control (Table 4). Therefore, This result shows that exogenous L-Aspartic acid as well as the endogenous turns in D-Aspartic acid by an endogenous racemase.

Table 4 shows the concentration of D-Aspartic acid in whole brain and in synaptic vesicles in rats after the intake of 20 mM D-Asp or 20 mM L-Asp or H ₂ 0.

Table 4. Concentration of D-Aspartic acid in whole brain and in the synaptic vesicles in rats after the intake of 20 mM D-Asp or 20 mM L-Asp or H ₂ 0.

Rats groups which drunk 20 mM sodium D-Aspartate

nmoles/g of brain nmoles/mg of proteins

Whole brain* 85±10.5 0.65±0.2

Synaptic vesicles ^** 48±5.6 620±55

Rats groups which drunk 20 mM sodium L-Aspartate

nmoles/g of brain nmoles/mg of proteins

Whole brain * 48±4.5 5±0.04

Synaptic vesicles ^** 24±4.0 ±35

Rats groups which drunk tap water (control)

nmoles/g of brain nmoles/mg of protein

Whole brain * 35±4.2 0.18±0.02

Synaptic vesicles ** 18±3.4 150±18

The values represent the mean ± SD obtained from 5 adult rats treated with 20 mM sodium D-aspartate or 20 mM sodium L-aspartate or tap water (control). * Indicates nmoles of D-Asp contained in 1 g of brain homoge- nate or nmoles referred to the content of 1 mg of the total proteins (soluble and insoluble). ^** Indicates nmoles of D-Asp contained in synaptic vesicles prepared from 1 g of brain or the nmoles referred to 1 mg of total proteins (soluble and insoluble).

1.6 Use of D-Aspartate in the recovery of learning and memory in Alzheimer's disease patients

In the light of the results obtained above, it was intuitive to think that oral administration of sodium D-aspartate to persons affected by Alzheimer's disease could improve memory and cognitive status in these patients. Therefore, an experiment was carried out on a group of 6 patients diagnosed with Alzheimer's disease in clinical stage between moderate and severe. These patients were not under any treatment of Alzheimer's drugs.

Each patient was given orally a solution comprising 10 ml of sodium D-aspartate at a concentration of 2 M (2.66 g of D-Aspartic acid, corresponding to 3.12 g of sodium D-aspartate) mixed in half a glass of water or juice after lunch or dinner. The solution of sodium D-aspartate given to the patient consisted of a dietary supplement approved by the Ministry of Health and covered by a patent and marketed under the trademark DADAVIT ® and used as a supplement for other purposes. The experiment had a duration of 30 days and was carried out under medical control. After this period, some specific symptoms of the Alzheimer's disease (below) have been evaluated and compared with those of the same patients before therapy.

Symptoms used for the current study were as follows and the obtained results are presented in Table 5.

Amnesia: Patients with dementia due to Alzheimer's have good memory of past things but cannot remember recent essential things as, for example, they forget the place where they are, or they do not, for example, remember where is the toilet of their house, etc.

Apraxia. Inability to perform common actions: whistling, making coffee, cooking, doing drawings

Agnosia: The inability to recognize common things e.g. fruit, pets, etc..

Anomia: Inability to name an object, while recognizing it.

Disorientation in time and space: Inability to remember the day or month or season or year, as well as inability to remember the place where he is.

Agraphia: The subject has difficulty in writing. In the most severe forms he cannot even write his own signature.

.Changes in mood: The mood tends to change abruptly in the sub- ject. Depression, euphoria, tears also associated with depression, anxiety, insomnia, agitation.

Language problems: The subject finds difficulty to name an object. Loss of initiative: Loss of each initiative and interest.

The results obtained in this study, which are presented limited only to 6 patients, give a clear indication that the use of sodium D-aspartate in Alzheimer's patients improved, in all subjects, learning and memory with a recovery of cognitive activities at least of 40-60%. Table 5 shows the evaluation of the recovery status of learning and memory improvement in Alzheimer's patients (moderate to severe) treated with sodium D-aspartate.

Table 5

Amnesia Apraxia Agnosia Anomia Disorientation Agraphia Changes in Language Loss of in time and space mood problems initiative

Patient n. 1 recovery recovery recovery recovery recovery recovery recovery recovery recovery

Male 77 years 40-50% 40-50% 40-50% 50-60% 60-70% 30^0% 50-60% 40-50% 30-40% seriousness =moderate

Patient n. 2 recovery recovery recovery recovery recovery recovery recovery recovery recovery

Male 80 years 45-55% 45-55% 40-50% 45-65% 55-65% 35-45% 60-665% 35-45% 45-55% seriousness =moderate

Patient n. 3 recovery recovery recovery recovery recovery recovery recovery recovery recovery

Male 74 years 30-40% 25-35% 35-45% 35-45% 45-55% 25-35% 40-50% 30-40% 25-35% seriousness =severe

Patient n. 4 recovery recovery recovery recovery recovery recovery recovery recovery recovery

Female 81 years 55-65% 55-65% 55-65% 55-65% 50-60% 45-55% 45-55% 55-65% 35-45% seriousness =moderate

Patient n. 5 recovery recovery recovery recovery recovery recovery recovery recovery recovery

Male 79 years 35-45% 35-45% 35-45% 55-65% 45-55% 45-55% 65-75% 70-80% 35-45% seriousness =moderate

Patient n. 6 recovery recovery recovery recovery recovery recovery recovery recovery recovery Male 81 years 20-30% 20-30% 20-30% 35-45% 35-45% 0-30% 30-40% 35-45% 25-35% seriousness =severe

The evaluation is expressed in terms of recovery of that specific function in comparison with same age persons not affected by Alzheimer's (value=10

Conclusions on the studies carried out

In conclusion, the experimental results obtained so far show that D- Aspartic acid is present in endogenous form in human and rat brains and that there is an enzyme system for the synthesis of D-Aspartic and mem- brane receptors for this amino acid and that D-aspartic acid is able to induce improvement in learning and memory in persons affected by Alzheimer's disease.

EXAMPLE 2: Study on the effects of amino acids and their combination on learning and memory in rats

For these experiments, rats were used as an animal model as they possess a biological system which is considered very similar to that of humans. So, it is believed that such biological effects induced in rats by a substance will be present also in humans.

In this specific case a study was carried out on rats in order to evaluate:

1) The effect of treatment with the following amino acids on rats: (i) D-Aspartic acid, (ii) L-Aspartic acid, (iii) L-Arginine, (iv) a combination consisting of D-Aspartic acid and L -Aspartic acid and (iv) a combination consisting of D-Aspartic acid and L-Arginine, on increasing learning activity and memory in rats.

2) The effect of treatment with the following amino acids on rats: (i) D-Aspartic acid, (ii) L -Aspartic acid, (iii) L-Arginine, (iv) a combination consisting of D-Aspartic acid and L -Aspartic acid and (iv) a combination consisting of D-Aspartic acid and L-Arginine, on the accumulation of the same in the brain and in the synaptic vesicles.

3) The effect of treatment with the following amino acids on rats: (i) D-Aspartic acid, (ii) L -Aspartic acid, (iii) L-Arginine, (iv) a combination consisting of D-Aspartic acid and L -Aspartic acid and (iv) a combination consisting of D-Aspartic acid and L-Arginine, on any side effects.

For D-aspartate is meant the salt of D-aspartic acid. For L-aspartate is meant the salt of L-aspartic acid.

Principles and Methodology (Test di Water-Maze).

For the study of learning and memory adult rats were used of about 12 months of age, males and females (Wistar). Rats were hold 2-3 per cage, separated by sex, in an environment controlled for temperature (23- 24°C) and humidity (60%), with cycles of daylight and night. The hygienic conditions of housing were in line with international standards of mainten- 1 000411

29

ance of rats. For the experiment in learning and memory the Morris method Water Maze-1984 (R-Morris, 1984) has been used. It consists in the evaluation of learning activity and memory in rats in remembering the position of the floating and not visible platform in a tank water. This method is officially considered the most suitable, because the rat is subjected to a type of learning and memory that involves temporal and spatial memory. In practice, the rat, after finishing the course of treatment with the substance the efficiency of memory of which is to be tested, is IMMERSO into a circular pool of 2 meters in diameter and 50 cm high, filled with water (made opaque wit an addition of milk) and containing a floating platform (10x10 cm) at the center of the tank below the surface of water. Just placed in water, the rat swims randomly to seek support for salvation. After reaching the platform, the rat is left on the platform for about 30 seconds allowing it to store the spatial position of the platform in the tank. Then it is dried and put it in his cage. In the first attempts, the rat takes a long time to find the platform (1.5-2 min), then gradually learns the location of the platform and takes less time to find it. If the drug being tested has an activity on learning and memory, the rat will learn quicker the position of the platform compared to a control rat. In this case, the complete experiment lasted 5 days with 2 sessions per day: one in the morning and evening. For each exercise of Water-Maze time in seconds that the rat employed to reach the platform is relieved. Then, the data were statistically processed (using the test "ANOVA" one-way and two-way) to know the significance of the treatment effect of the compound versus placebo.

1st Experiment: Effect of D-Aspartic acid on learning and memory in rats with Water-Maze.

The experiment was carried out as follows: 5 rats males and 5 rats females drank a solution of 20 mM D-aspartic acid (dissolved in 20 mM NaOH to pH 7.0) for 30 consecutive days. Throughout the treatment the rats were free to drink and eat (food for rats) without limitation. At the thir- 2011/000411

30

ty-first day, each rat was subjected to the experiment of learning and memory with the "Water-Maze" by making 2 attempts per day (10 hours and at 17 hours). To this purpose it was used a circular pool of 2 meters in diameter and 50 cm in height containing water made opaque (by adding milk). At the center of the tank a floating platform was put at 1 cm below the water surface to avoid the rat from seeing. Every day for 5 consecutive days, each rat of the group was placed in the tank (in the same place) and left free to swim until it found the platform on which to lean. After finding the platform the rat was left for 20 sec on the platform allowing him to store the position of the platform. Then the rat was dried and put back in the cage. After each exercise the time that the rat had employed to reach the platform was collected. The Water-Maze experiment was carried out every day at 10 and 18.

2nd Experiment: Effect of L-Aspartic acid in learning and memory in rats with the Water - Maze method.

This experiment of learning and memory using the Water-Maze method has been carried out in the same manner as above, but on other 5 male and 5 female rats which drank, for 30 days, a solution of 20 mM L- Aspartic acid (neutralized at pH 7.0 with 0.1 M NaOH).

3rd Experiment: Effect of the L-Arginine acid in learning and memory in rats with the Water-Maze method.

This experiment of learning and memory using the Water-Maze method has been carried out in the same manner as above, but on other 5 male and 5 female rats which drank, for 30 days, a solution of 20 mM L- Arginine (neutralized at pH 7.0 with 0.1 M HCI).

4th Experiment: Effect of using a combination constituted by D- Aspartic acid and L-Aspartic acid in learning and memory in rats with the Water-Maze method.

This experiment of learning and memory using the Water-Maze me- thod has been carried out in the same manner as above, but on other 5 male and 5 female rats which drank, for 30 days, a solution of 20 mM of D- Aspartic acid and 20 mM L-Aspartic acid (neutralized at pH 7.0 with 0.1 M NaOH).

5th Experiment: Effect of using a combination constituted by 20 mM D-Aspartic acid and 10 mM L-Arginine in learning and memory in rats with the Water-Maze method.

This experiment of learning and memory using the Water-Maze method has been carried out in the same manner as above, but on other 5 male and 5 female rats which drank, for 30 days, a solution of 20 mM of D-Aspartic acid and 10 mM L-Arginine (neutralized at pH 7.0 with sodium bicarbonate 0.1 M).

6th Experiment: Effect of using a combination constituted by 20 mM acido D-Aspartic and 20 mM L-Arginine in learning and memory in rats with the Water-Maze method.

This experiment of learning and memory using the Water-Maze method has been carried out in the same manner as above, but on other 5 male and 5 female rats which drank, for 30 days, a solution of 20 mM of D-Aspartic acid and 20 mM L-Arginine (neutralized with each other).

7th Experiment: Control

This experiment was carried out with the Water-Maze in the same conditions as above but using a different group of rats, 5 males and 5 females of 12 months of age. In this case, the rats drank only tap water. The time in seconds used to reach the platform of this group of control rats was used as time reference for the assessment of learning and memory in the other groups of rats treated with amino acids.

RESULTS OBTAINED FROM LEARNING AND MEMORY EXPERIMENTS.

The results obtained on the effect of the amino acids and their compounds are shown in Tables 6-11. In each table is shown the time taken (in seconds) from each rat of each experimental group and in each 0411

32

of the 5 days and in each period of days to reach the platform in the Water-Maze system. As it has not been observed a significant difference in the time necessary to reach the platform between male and female, the values obtained from both male and female rats have been taken together. The mean and the standard deviation have been evaluated for each exercise of the 10 control rats and of those subjected to different treatments with different amino acids. Finally, the value of Student's T (T test) has been calculated on each experimental group of rats versus the control group. This statistical study revealed the following:

1) L-Aspartic acid alone does not improve learning and memory in rats. Indeed, the comparison of the average time in seconds to reach the platform and of the value P of StudentT, shows that the rats improve the time to reach the platform in the progressive days of exercise, but this improvement is not different from that of controls (Table 6). For each day of experiment, the value of P is always greater than 0.1 (P> 0.1). As to be significant, the P value should be less than 0.05 (P <0.05) (Table 7).

Table 6. Learning and memory experiment of rats treated with tap water (control) with the Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav 2 ^nd dav 3 ^rd dav 4 ^th dav 5 ^th dav

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 172 160 148 156 130 122 102 98 44 42

2 142 148 134 128 107 99 69 51 38 31

3 150 144 152 124 109 102 76 64 52 34

4 162 144 154 133 111 96 78 72 44 45

5 155 143 132 123 96 92 68 65 56 44

Female 1 165 160 143 145 125 112 99 102 65 54

2 169 161 154 133 114 115 102 91 65 53

3 163 162 133 129 104 98 99 87 51 44

4 176 154 154 162 134 131 111 93 66 55

5 122 151 102 98 98 88 91 82 61 31

Average 157 152 141 133 123 106 90 81 55 44

±SD ±16 ±8 ±16 ±18 ±13 ±14 ±15 ±17 ±9 ±8

In this experiment rats was used which drank tap water (control) instead of an amino acid (see experimental part, section 1.7). Then the learning and memory experiment began using the Water-Maze system. This method consisted in placing the rat in a tank of 2 m in diameter with a central platform containing opaque water and the time that the rats employed to reach the platform were collected. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, the average and SD of the time in seconds for the 10 rats at each water-maze experiment was calculated.

Table 7. Learning and memory experiment of rats treated with L- aspartic acid with the Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav day 3 ^rd dav 4 ^th dav 5 ^th dav

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 160 175 160 155 130 120 60 55 35 30

2 155 150 145 135 110 105 75 65 44 50

3 180 172 144 132 124 114 86 74 42 40

4 162 148 142 150 108 100 96 90 64 44

5 180 175 165 142 124 120 98 86 45 35

Female 1 134 132 125 115 110 110 90 85 54 45

2 174 180 175 145 115 95 78 64 46 35

3 158 156 135 124 110 88 85 76 55 42

4 145 152 135 120 98 86 44 56 28 26

5 164 154 132 144 120 110 86 64 52 48

Average 161 159 146 136 115 105 80 72 47 40

±SD ±14 ±15 ±16 ±13 ±9 ±12 ±16 ±12 ±10 ±8

Student's t test (P) >0.5 >0.5 >0.4 >0.4 >0.4 >0.3 >0.4 >0.2 >0.1 >0.3

The rats drank a solution of 20 mM L-Aspartic acid for 30 days (see experimental part, section 1.7). Then the learning and memory experiment began using the Water-Maze system. This method consisted in placing the rat in a tank of 2 m in diameter with a central platform and containing opaque water and the time that the rats employed to reach the platform were collected. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, the average and SD of 10 rats for each experiment was calculated and the significance of differences of time in seconds to reach the platform was calculated using the Student's T. A Student's T value, P <0.05 was assumed statistically valid.

2) L-Arginine alone produces a good improvement in learning and memory in rats. Indeed, from a comparison of the average time in seconds to reach the platform and the value P of Student-T obtained by comparing the times of rats treated with L-arginine and those treated with placebo, it is evident that rats improve the time to reach the platform only on day 5 after which they were subjected to the exercise of Water-Maze (values in Table 8 against values in Table 6). On Day 5 the value P of Student-T is <0,001 both at the cycle at 10 a.m. and 18 p.m. (Table 8).

Table 8 Learning and memory experiment of rats treated with L-Arginine with the Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav 2 ^nd dav 3 ^rd dav 4 ^th dav 5 ^th dav

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 174 164 144 150 110 102 82 75 25 22

2 144 140 130 124 105 96 66 48 26 18

3 155 160 155 135 85 86 55 54 30 13

4 172 155 162 154 112 100 80 74 25 25

5 140 134 123 115 98 102 96 68 28 26

Female 1 155 164 146 155 110 102 98 96 41 30

2 178 166 175 143 1 12 110 96 97 38 27

3 143 132 124 125 86 98 90 90 33 20

4 155 150 160 172 140 136 87 90 20 15

5 146 152 132 121 1 12 106 97 88 25 10

Average 156 152 145 139 107 104 82 78 24.9 20.6

±SD ±13 ±12 ±17 ±18 ±15 ±13 ±17 ±17 ±7.7 ±6.5

Student's t test (P) >0.5 >0.5 >0.4 >0.4 >0.4 >0.3 >0.2 >0.1 <0.001 <0.001

The rats drank a solution of 20 mM L-Arginine for 30 days (see experimental part, section 1.7). Then the learning and memory experiment began using the Water-Maze system. This consisted in placing the rat in a tank of 2 m in diameter with a central platform and containing opaque water and the time that the rats employed to reach the platform. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, the average and SD of 5 male and 5 female rats for each experiment was calculated. Finally, it has been calculated the average and SD of 10 rats for each experiment and the significance of differences of time in seconds to reach the platform was calculated using the Student's T.. A Student's T value, P <0.05 was assumed statistically valid.

3) D-Aspartic acid produces a good improvement in learning and memory in rats. Indeed, a comparison of the average time in seconds to reach the platform, and the value of P of Student'T, shows that up to the first attempt of the 4 ^th day of testing with Water-Maze system there were no significant improvements in learning and memory compared with rats in the control group (P value of Student'T is always greater than 0.05 , P> 0.05). However, the second attempt of 4 ^th day, it has been observed that the rats reached the platform with times statistically significant compared to the same day of the controls (see values from Table 9 compared to the values of Table 6). This value of such significant learning is also confirmed in the two sessions of Day 5 ^th where, for both sessions, the value P is always less than 0.001 (P <0.001) (Table 9).

Table 9. Learning and memory experiment of rats treated with D-Aspartic acid with the Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav day 3 ^rd day 4 ^th day 5 ^th day

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 150 150 140 130 112 85 75 35 9 8

2 145 160 150 130 120 97 99 44 14 13

3 155 160 155 135 1 15 05 101 50 19 15

4 170 175 165 160 104 1 13 103 46 14 14

5 160 150 145 133 110 102 87 44 26 23

Female 1 150 145 130 135 125 98 64 35 18 15

2 170 165 155 130 131 103 74 40 26 23

3 135 155 114 1 15 105 90 71 30 23 19

4 190 185 150 130 110 102 98 32 26 24

5 135 150 120 110 100 100 78 30 28 23

Average 156 159 142 131 113 99 85 38 20 18

±SD ±17 ±12 ±16 ±18 ±10 ±8 ±14 ±7 ±6 ±5

Student's t iest (P) >0.3 >0.1 >0.3 >0.3 >0.4 >0.1 >0.29 <0.001 <0.001 <0.001

The rats drank a solution of 20 mM D-Aspartic acid for 30 days (see experimental part, section 1.7). Then the learning and memory experiment began using the Water-Maze system. This consisted in placing the rat in a tank of 2 m in diameter with a central platform and containing opaque water; the time that rats employed to reach the platform has been measured. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, it was calculated the mean and SD of 5 male and 5 female rats for each experiment. Finally, it has been calculated the average and SD of 10 rats for each experiment and the significance of differences of time in seconds to reach the platform was calculated using the Student's T.. A Student's T value, P <0.05 was assumed statistically valid.

4) The composition consisting in D-Aspartic acid and L-Aspartic acid produces a good improvement on learning and memory in rats, but less than using D-Aspartic acid alone (see the P values of Student's T- on the fifth day of experiment). In the first 4 days of exercise of rats in the Water-Maze any significant change in the times to reach the platform does not occur in these rats compared to control group (Table 6). However, even in this case, the times that the rats used on 5th day in reaching the platform were significantly different from the control group value of a Student's T, P <0.001 both in morning test at 10am and in the evening test at 6 pm (Table 10).

Table 10. Learning and memory experiment of rats treated with a combination of D-Aspartic acid + L-Aspartic acid with Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav 2 ^nd dav 3 ^rd dav 4 ^th dav 5 ^th day

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 155 145 134 135 90 75 34 32 20 16

2 164 160 155 144 97 77 44 42 25 22

3 166 155 165 143 112 100 76 56 33 30

4 172 187 174 166 130 133 122 90 46 44

5 161 149 140 143 121 119 90 88 61 39

Female 1 165 144 138 138 98 97 64 65 46 48

2 130 131 158 169 130 132 98 91 55 53

3 138 135 124 135 122 1 10 76 80 61 55

4 121 142 151 148 130 121 110 96 56 45

5 134 133 110 102 98 81 64 56 43 41

Average 150 148 149 142 1 13 104 78 69 45 39

±SD ±18 ±16 ±19 ±18 ±16 ±22 ±27 ±22 ±14 ±13

Student's T test (P) >1 >1 >1 >0.5 >0.5 >0.5 >0.1 >0.1 <0.01 <0.01

The rats drank a solution of 20 mM D-Aspartic acid + 20 mM L-Aspartic acid for 30 days (see experimental part, section .7). Then the learning and memory experiment began using the Water-Maze system. This test consisted in placing the rat in a tank of 2 m in diameter with a central platform and containing opaque water; the time that rats employed to reach the platform has been measured. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, was calculated the mean and SD of 5 male and 5 female rats for each experiment. Finally, it has been calculated the average and SD of 10 rats for each experiment and the significance of differences of time in seconds to reach the platform was calculated using the Student's T.. A Student's T value, P <0.05 was assumed statistically valid.

5) The composition consisting of D-Aspartic acid, 20 mM and 10 mM L-arginine improves very significantly the learning and memory in rats and the efficiency of this compound is much higher even than the improvement induced by D Aspartic acid or by the compound including D- Aspartic acid or by L-Arginine or by L-Aspartic acid. In fact, as shown in Table 11 , after the rats were treated with the compound consisting of D- Aspartic acid and L-Arginine, the rat has learned a lot before the other treatments the position of the platform. The comparison of times for rats to reach the platform after treatment with the composition consisting of D- Aspartic acid and L-arginine (Table 11) with those of the times of the control rats (Table 6) is observed already in the session of 3 ^rd day of the Water-Maze times are significantly reduced so that the P value of Student's T is highly significant (P <0.001) in respect of the times spent by control rats (See the results of Table 11 in respect of the results of Table 6, control). In addition, the improvement in learning and memory was always higher by increasing the number of the attempts so that on the 5 ^th day of testing in the Water Maze system, rats employ an average of just 5 seconds to reach the platform.

Much more specifically, the group of rats that were treated with D- Aspartic acid or with the combination D-Aspartic acid and L-Aspartic acid, on the fourth day of the test still show no increase in memory compared to control: only at the fifth day of the test there was an increase of the memory of 2-2.5 times (expressed as the ratio of times to reach the platform). Instead, the group of rats treated with the compound consisting of D- Aspartic acid and L-Arginine, on the fourth day of test showed an improvement of memory by about 4 times on the control and on the fifth day showed an improvement of about 9 -10 times compared with the control group (expressed as the ratio of times to reach the platform). Table 11. Learning and memory experiment of rats treated with a combination of D-Aspartic acid + L-Arginine with Water-Maze method

Time in seconds employed by the rat to reach the platform

Day of experiment 1 ^st dav dav 3 ^rd dav 4 ^th day 5 ^th dav

Time of experiment 10 18 10 18 10 18 10 18 10 18

Male 1 150 150 140 130 80 50 20 10 3 3

2 145 160 150 130 75 55 15 8 3 4

3 155 160 155 135 85 50 25 15 7 5

4 170 175 165 160 75 70 15 5 4 4

5 160 150 145 133 110 75 22 12 6 6

Female 1 150 145 130 135 90 70 30 22 6 5

2 170 165 155 130 80 65 20 16 4 3

3 135 155 114 115 70 40 10 6 3 3

4 190 185 150 130 100 74 22 14 6 4

5 135 150 120 110 100 84 18 16 6 6

Average 156 160 142 131 87 63 20 12 5 5

±SD ±17 ±12 ±16 ±13 ±13 ±14 ±6 ±5 ±1.7 ±1.5

Student's T test (P) >0.4 >0.40 >0.3 >0.3 <0.001 <0.001 <0.001 <0.001 <0.001 <0.0(

The rats drank a solution of 20 mM D-Aspartic acid + 10 mM L-Arginine for 30 days (see experimental part, section 1.7). Then the learning and memory experiment began using the Water-Maze system. The test consisted in placing the rat in a tank of 2 m in diameter with a central platform and containing opaque water; the time that rats employed to reach the platform has been measured. This experiment was carried out 2 times a day (at 10.00 and at 18.00) and for 5 consecutive days. Finally, the average and SD of 10 rats for each experiment it has been calculated and the significance of differences of times in seconds to reach the platform was calculated using the Student's T.. A Student's T value, P <0.05 was assumed statistically valid

P T/IT2011/000411

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6) The composition consisting of 20 mM D-Aspartic acid and L- arginine 20 m ^' M also very significantly improves learning and memory in rats with efficiency comparable to the composition consisting of D-Aspartic acid, 20 mM and 10 mM L-arginine (for the latter composition the times of the rats for reaching the platform are not reported in the table, because the P values of Student's T are in practice the same as those shown in Table 11).

CONCLUSION ON THE STUDIES CARRIED OUT ABOVE

The conclusion of the experimental studies carried out on rats, about the effect of D-Aspartic acid, L-Aspartic Acid, L-Arginine, and the combinations consisting of D-Aspartic acid and L-Aspartic acid and the composition consisting of D-Aspartic and L-arginine on learning and memory is as follows:

a) L-Aspartic acid alone did not show any effect on learning and memory.

b) D-Aspartic acid or L-arginine or the combination of D-Aspartic acid with L-Aspartic acid induce a good positive effect on increasing learning and memory in rats. Rats treated with these compounds improve memory by about 2-2.5 times compared with the control group.

c) The combination consisting of D-Aspartic acid and L-Arginine, both the association of 20 mM D-Aspartic acid and 10 mM L-arginine and the association of 20 mM D-Aspartic acid and 20 mM L-arginine, induces a very high increase in learning and memory which is about 10 times compared with the control group. This result indicates, therefore, that these last two are by far the best associations of compounds for the treatment of memory in rats and therefore also on human. EXAMPLE 3: Study on the effects of the treatment with D-Aspartic acid, L-Aspartic Acid, L-Arginine, with a combination consisting of D- Aspartic acid and L-Aspartic acid, and with a combination consisting of D- Aspartic Acid and L -arginine about their build up in the brain and in the synaptic vesicles

This study was conducted in order to know the concentration of D- Aspartic acid, L-Aspartic acid and L-arginine in the brain and in the synaptic vesicles after treatment of rats for 30 days by each of these amino acids and the concentration of the combination consisting of D-Aspartic acid and L-Aspartic acid, and of the combination consisting of D-Aspartic acid and L-arginine. To this aim, after several experimental groups of rats had finished the learning and memory experiment using the-Water-Maze system, they were sacrificed and their brains have been collected. Then a mid-portion of the brain has been used for the determination of amino ac- ids by HPLC (Ref. 27 and 28) (see section 1.1) and the other half portion was used for the preparation of synaptic vesicles (see paragraph 1.2 ). The results obtained from this study are shown in Table 12. This table shows the concentrations and SD obtained from the 10 rats for each experimental group made by 5 males and 5 females.

As it can be seen from the same table, D-Aspartic acid is usually present in the endogenous form in the brain and in synaptic vesicles of rats. In the brains of control rats (i.e. rats that drank only tap water) the concentration of D-aspartic acid is on average 35 ± 5 nmoles / g of brain (4.65 ± 0.66 pg / g). However, a substantial amount of D-Aspartic acid, corresponding approximately to 10-15% of the total amount that is located in synaptic vesicles, i.e., in the cell structures that are rich in neurotransmitters and that are localized at the nerve endings of neurons (dendrites). This is very important because in these structures are located the neurotransmitters from the neuron cell body. Neurotransmitters are molecules that following nerve or electrical stimulus are transferred from pre-synaptic neuron to post-synaptic neuron creating cell information which are at the base of learning and memory processes. Normally in synaptic vesicles, the P T/IT2011/000411

45 concentration of D-Aspartic reported as the content of amino acid/mg of protein is about 300 times higher than the brain (Table 12).

Table 12. Concentration of D-Aspartic acid, L-Aspartico acid and L- Arginine in the brain and in the synaptic vesicles in rats treated with control

Concentration in the brain Concentration in the

synaptic vesicles

(nmoles/g of brain) (nmoles/mg of proteins)

Rats treated D-Asp L-Asp L-Arg D-Asp L-Asp L-Ar£ with:

Tap water 35 5600 1600 285 510 20

±5 ±460 ±210 ±30 ±55 ±5

L-Aspartate 45 5.800 1550 310 520 25

±6 ±490 ±180 ±55 ±54 ±8

L-Arginine 34 5530 1580 265 490 27

±7 ±450 ±205 ±35 ±60 ±6

D-Aspartate 85± 5750 1530 565 520 15

±12 ±530 ±195 ±46 ±45 ±5

D-Aspartate 95 5750 1760 510 515 18

+L-Aspartate ±13 ±495 ±160 ±52 ±52 ±7

D-Aspartate 120 5340 1730 585 515 19

+L-Arginine ±18 ±410 ±182 ±59 ±48 ±8

The concentration of amino acids in synaptic vesicles is expressed in terms of nmoles/mg of vesicular protein. The determination of amino acids was carried out using HPLC methods described in section 1.1. The preparation of synaptic vesicles from rat brain was performed as described in section 1.2. The results refer to the average ± SD obtained from 5 adult males and 5 adult females. When rats were treated with D-Aspartic acid (in the form of sodium D-aspartate), this amino acid accumulates significantly in the brain and synaptic vesicles and its concentration in these tissues increases the value of about 2.8 times compared to that in the brain and vesicles from con- trol rats. This fact indicates therefore that D-Aspartic acid ingested can pass the blood brain barrier, enter the neurons of the brain and move into synaptic vesicles where acts as a neurotransmitter chemical (Table 12). By a comparative study of the maximum concentration of D-Aspartic synaptic acid in the brain and in vesicles occurs when the animal ingests the compound consisting of D-Aspartic acid and L-arginine, where the concentration may be as high as 120 nmol / g in whole brain and 585 nmol /mg protein in synaptic vesicles. Therefore, this result indicates that the combination of D-Aspartic acid and L-Arginine is the most efficient compound to introduce D-Aspartic acid in the brain and therefore also the compound that gives more brain activity and that validates the results obtained in experiments on memory above mentioned.

With regard to the concentration of other amino acids tested in the rat, any significant difference is observed in growth after the animal has been treated with L-Aspartic acid or L-arginine both in the brain as a whole and in the vesicles. This is probably due to the amount, already high, of these two amino acids found in the brain and to their metabolism.

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ESEMPIO 4: Study on the effects of the treatment with D-Aspartic acid L- Aspartic Acid, L-Arginine, with an association consisting in D-Aspartic acid and L-Aspartic acid, and an association consisting in D-Aspartic Acid and L -arginine on the health of rats

In order to exclude possible damage to the health of rats following the treatment with amino acids or associations thereof as above reported, after each rat had completed the cycle of exercises for learning and memory with the Water-Maze system, the rat was weighed and then decapitated by guillotine. Blood was collected as well as the serum from the blood. Then different types of clinical investigations were carried out on the blood and serum with the aim to verify if a damage in the tissues occurs. Analyses carried out were as follows: complete blood count (with the instrumewnt: HBxL80, HORIBA Medical, 34184 Montpellier, France), glucose, urea, ammonia, cholesterol, triglycerides, creatinine, uric acid, elec- trolytes: sodium, potassium, calcium, magnesium, iron, and copper. Then the following enzymes: transaminase, alkaline phosphatase, cholineste- rase and lactate dehydrogenase (using the instrument: LaB Taurus Instrument Laboratory SpA, Viale Monza 338, 20128 Milan), d) the following hormones: testosterone, T3, T4, (using the instrument: UnicellTM Dx1800, with Beckman Counter, Inc., 2505 Kraemer Blvd, Brea, CA 92821 Germany), and plasma protein electrophoresis (performed with the instrument: INTERLAB G26, Interlab Ltd., 00155 Rome). The result obtained from this study indicated that there was no significant variation in the values of the clinical analysis among the experimental groups and control group about each type of clinical investigation mentioned above (one-way ANOVA ") nor differences in their body weight.

In conclusion, the treatment with all compounds used for testing the rats have not any negative effect on the health of the animal. EXAMPLE 5: Study on the effects of an association consisting of D- Aspartic acid and L-Arginine to improve memory in patients with dementia senile and Alzheimer's disease.

Based on the results obtained in rats about the improving of learn- ing and memory with the use of the compound consisting of D-Aspartic acid and L-Arginine, we were encouraged to test the same compound to increase the cognitive activities and memory in elder persons suffering from dementia senile in general and in particular suffering from Alzheimer's disease. For this purpose we conducted a pilot study of 6 elder pa- tients, three men and three women aged between 75 and 82 years, diagnosed as suffering from dementia senile with a dominant component of Alzheimer's disease.

Each patient was given a daily dose consisting of 2.5 g of D- Aspartic acid, 1.5 g of L-arginine. The mixture of the two amino acids was added 1.1 g of sodium bicarbonate in order to dissolve D-Aspartic acid and L-arginine in water. The patient was given to drink a solution made as above dissolved in half a glass of spring water or juice, preferably with food (lunch or after dinner). The treatment lasted for 30 consecutive days. After this time, for each patient the cognitive activities and memory, before and after treatment, have been assessed. The improvement or restoration of memory was evaluated using the following clinical parameters: Amnesia, Apraxia, Agnosia, Anomy, disorientation in time and space, agraphia, mood changes, language problems and loss of initiatives.

The results obtained in this study are showed in Table 13. As can be seen in said table, in all 6 patients who underwent the study, recovery of cognitive activities and memory occurs (Table 13). In fact, as can be seen in said table, the percentage of recovery of each parameter for the evaluation of the memory is large enough that each patient has recovered overall around 60-70% of the lost memory. Table 13: Effect of an association of D-Aspartic acid and L-Arginine on cognitive activities and memory in dementia senile with a tendency to Alzheimer's

Recovery of cognitive activities and memory assessed by each symptom

Amnesia Apraxia Agnosia Anomy Disorientation Agraphy Mood Language Loss of

In time and space changes problems initiative

% of recovery of cognitive activities and memory

Female of 82 years 60-70 60-75 55-70 70-82 70-80 50 60-70 70-82 60-70

Female of 80 years 55-65 65-75 60-80 64-74 55-65 65-75 60-74 65-75 65-75

Female of 75 years 60-70 65-75 65-75 55-65 65-75 50-60 60-70 65-70 55-70

Male of 81 years 65-75 65-75 60-70 60-70 75-80 65-75 65-75 55-75 60-70

Male of 79 years 45-55 50-60 55-65 65-75 65-75 65-75 70-80 75-85 45-60

Male of 75 years 75-80 60-70 60-70 55-65 45-55 40-50 40-50 65-75 45-55

Each patient took a daily dose consisting of 2.5 g of D-Aspartic acid and 1.5 g of L-Arginine and fed for 30 days. The assessment of recovery of cognitive activities and memory has been expressed in terms of arbitrary evaluating before and after treatment, giving a value of 100% and cognitive activities and memory of people without dementia and of the same age group.

GENERAL CONCLUSIONS

In this study has been evaluated the possibility of using a mixture of D-Aspartic acid and L-Arginine acid by appropriate molecular ratios for the improvement of cognitive activities and memory in patients with dementia senile and Alzheimer's. The first study was carried out on different experimental groups of rats and then on a group of patients with dementia.

Rats were subjected to oral treatment of some amino acids alone and in association with each other. After a period of 30 days of treatment, the rats were tested by the Water-Maze Learning System, universally rec- ognized as a method suitable for this purpose. This study showed that, among the various combinations of amino acids tested, the mixture consisting of 20 mM D-aspartate acid and 10 mM L-arginine was found to be the most effective for the improvement of memory in rodents. In addition, the mixture consisting of 20 mM D-Aspartic acid and 20 mM L-arginine has efficiently responded for increasing memory.

It is also important to report that treatment with D-Aspartic acid alone or L-arginine alone, or a combination of D-Aspartic acid and L- Aspartic acid also improve learning and memory in rats, but their activity is only 1/ 5 of that induced by the association of D-Aspartic acid and L- arginine.

The human study consisted in giving a drinking dose per day including 2.5 g of D-Aspartic acid and 1.5 g of L-arginine dissolved in 1/2 glass of water for 30 consecutive days to a group of elder persons suffering from dementia senile with tendency to the Alzheimer's disease. The result showed that in all patients there was a recovery of 60-70% of memory. Bibliography

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