EP2654743A1 - Kombination aus d-asparaginsäure und l-arginin oder salzen davon zur verbesserung der kognitiven aktivitäten und des gedächtnsses bei demenzen wie seniler demenz und morbus alzheimer - Google Patents

Kombination aus d-asparaginsäure und l-arginin oder salzen davon zur verbesserung der kognitiven aktivitäten und des gedächtnsses bei demenzen wie seniler demenz und morbus alzheimer

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Publication number
EP2654743A1
EP2654743A1 EP11820851.1A EP11820851A EP2654743A1 EP 2654743 A1 EP2654743 A1 EP 2654743A1 EP 11820851 A EP11820851 A EP 11820851A EP 2654743 A1 EP2654743 A1 EP 2654743A1
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Prior art keywords
aspartic acid
arginine
salts
memory
vitamin
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English (en)
French (fr)
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Antimo D'ANIELLO
Gemma D'ANIELLO
Enrico D'aniello
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • 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.
  • 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.
  • MCI mild cognitive impairment
  • 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).
  • 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.
  • ⁇ - ⁇ is a protein present on the neuronal plasmatic membrane and is partly located within the neuron membrane and partly outside the neuron (transmembrane protein).
  • ⁇ -secretase and ⁇ - 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).
  • 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).
  • ⁇ - ⁇ ⁇ -Amyloid Precursor Protein
  • the a-secretase and ⁇ -secretase cleave a big size precursor protein (a transmembrane protein) called ⁇ -Amyloid Precursor Protein ( ⁇ - ⁇ ), thus generating a peptide fragment of 42 amino acids which is ⁇ -amyloid.
  • ⁇ - ⁇ ⁇ -Amyloid Precursor Protein
  • the ⁇ -amyloid once generated, becomes insoluble and settles as extracellular aggregates on neurons membranes, thus forming the amyloid plaques.
  • Tau 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.
  • 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 as, for example, physostigmine, galanta- mine, rivastigmine and neostigmine are used in therapy to mitigate the loss of acetylcholine (3).
  • 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 ⁇ -secretase inhibitors. Some synthetic compounds have been proposed belonging to the group of dichlorophenol derivatives (6).
  • dementia senile 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.
  • 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).
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • the invention also concerns a pharmaceutical composition
  • 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
  • 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.
  • 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- acetam
  • 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.
  • a ketoacid such as, e.g., for example, alpha-oxalacetic acid, alpha- ketoglutaric, etc, or a salt thereof.
  • the combination or pharmaceutical composition of the invention may include at least one water-soluble and/or liposoluble vitamin.
  • 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 B9 Vitamin B9 or Vitamin M (folic acid)
  • Vitamin B12 (cobalamin)
  • Vitamin C (ascorbic acid)
  • 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.
  • 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.
  • 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.
  • Figure 1 shows the composition and the stereo-chemical configu- IT2011/000411
  • D-Asp D-Aspartic acid
  • L-Asp L-Aspartic acid
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • synaptic vesicles subcellular particles of neuron endings of 40-60 nm in size
  • concentrations between Alzheimer's brain and control brain.
  • 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 was taken up in 400 ⁇ 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.
  • synaptic vesicles 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
  • 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
  • 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).
  • this fact means that D-aspartic acid which is found in the brain is all concentrated in the synaptic vesicles
  • 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).
  • 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
  • * 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.
  • 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:
  • Alzheimer's brain or control brain was homogenized (1 :5) in 0.02 M phosphate buffer pH 8.0. Then 500 ⁇ of supernatant were mixed with 100 ⁇ I of Na L-aspartate 1 M (sample) or 100 ⁇ of distilled H 2 0 (blank sample) and 10 ⁇ 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 ⁇ of perchloric acid 5 M, mixed and centrifuged at 13,000 rpm for 5 min.
  • 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.
  • 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.).
  • 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.
  • 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.
  • D-Aspartic D-[ 3 H] aspartic acid
  • 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 ⁇ of membrane suspension were mixed with 100 ⁇ I of D-[ 3 H] aspartate (100 nM) and 300 ⁇ 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 ⁇ of 0.1 M NaOH.
  • 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.
  • Ki inhibition constant or affinity constant
  • 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).
  • PSMS postsynaptic membranes
  • 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 ⁇ .
  • 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.
  • 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.
  • 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.
  • Rats which drunk tap water (control)
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • D-aspartate is meant the salt of D-aspartic acid.
  • L-aspartate is meant the salt of L-aspartic acid.
  • Rats were held 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the rats drank a solution of 20 mM D-Aspartic acid + 20 mM L-Aspartic acid for 30 days (see experimental part, section .7).
  • 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.
  • 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.
  • 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 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
  • 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).
  • 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.
  • 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
  • D-Aspartic acid is usually present in the endogenous form in the brain and in synaptic vesicles of rats.
  • concentration of D-aspartic acid is on average 35 ⁇ 5 nmoles / g of brain (4.65 ⁇ 0.66 pg / g).
  • 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).
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.

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