WO2021019580A1

WO2021019580A1 - Composition comprising 5'-ribonucleotides for use in the treatment of alzheimer's disease

Info

Publication number: WO2021019580A1
Application number: PCT/IT2019/000060
Authority: WO
Inventors: Francesco Maione; Maria Daglia; Federica RAUCCI; Anella SAVIANO; Daniele BONVICINI; Alessandro Sgherbini
Original assignee: Prosol S.P.A.
Priority date: 2019-07-26
Filing date: 2019-07-26
Publication date: 2021-02-04

Abstract

The present invention relates to a composition comprising, by weight of the total weight of the composition, at least 40% of 5'-ribonucleotides as active ingredient, for use in treating Alzheimer's disease symptoms; it also relates to a pharmaceutical or food supplement preparation for use in treating Alzheimer's disease symptoms, including the above composition and a carrier acceptable from the pharmaceutical or food standpoint.

Description

Title: Composition comprising 5 -ribonucleotides for use in the treatment of Alzheimer’s disease

DESCRIPTION

Field of application

The present invention relates to the sector of pharmaceutical industry; in particular, the invention relates to a composition comprising 5’-ribonucleotides, as active ingredient, useful in particular for the treatment of Alzheimer’s disease symptoms.

Prior art

Alzheimer’s disease (AD) is one of the most common form of dementia characterized by the deposition of extracellular amyloid- b (Ab) peptides in the brain. The neuronal death, that characterizes this neurodegenerative disorder, mainly involves the hippocampal and the neocortical area causing irreversible cognitive impairment and behavioural alteration.

It is well known that memory problems are typically one of the first signs of cognitive impairment related to Alzheimer’s disease; a decline in non-memoiy aspects of cognition, such as word-finding, vision / spatial issues, and impaired reasoning or judgment, may signal the very early stages of Alzheimer’s disease.

As Alzheimer’s disease is an irreversible, progressive brain disorder, memory and thinking skills, and, eventually, the ability to carry out the simplest tasks are slowly destroyed, till to the most severe condition of the disease in which patients cannot communicate and are completely dependent on others for their care. Current approaches focus on helping people to maintain mental function, manage behavioural symptoms, and slow down certain problems, such as memory loss.

It is also well known that several drugs are used to treat symptoms of this disease, such as, for example, Donepezil (Aricept®), rivastigmine (Exelon®), and galantamine (Razadyne®) for mild and moderate Alzheimer’s; Memantine (Namenda®), the Exelon® patch, and Namzaric® (a combination of memantine and donepezil) for moderate to severe Alzheimer’s.

These drugs work by regulating neurotransmitters, the chemicals that transmit messages between neurons, reducing symptoms and helping with certain behavioural problems.

However, these drugs often show a short-term efficacy, modest benefits and, sometime, they may cause neurotoxicity, especially Memantine; moreover, all of these drugs are veiy expensive (Keith e al., Alternative Medicine Review, vol. 15, n.3, pages 223-244).

Beside synthetic drugs, the use of natural-derived products and/or nutraceuticals could represent a future medical option in industrialized countries.

Nutraceuticals are not proposed as an alternative to drugs, but instead, they can be helpful to complement a pharmacological therapy and prevent the onset of chronic diseases.

Medicinal plants have been reported for possible anti-AD activity in a number of preclinical and clinical trials. Ethnobotany, being popular in China and in the Far East and possibly less emphasized in Europe, plays a substantial role in the discovery of anti- AD agents from botanicals. Chinese Material Medica (CMM) involving Chinese medicinal plants has been used traditionally in China in the treatment of AD. Ayurveda has already provided numerous lead compounds in drug discovery and many of these are also undergoing clinical investigations (Dey et al., Biotechnology Advances, Vol. 35, Issue 2, March-April 2017, Pages 178-216).

It is also known that many compounds, including lignans, flavonoids, tannins, polyphenols, triterpenes, sterols, and alkaloids, have shown various beneficial pharmacological activities, such as anti inflammatory, anti-amyloidogenic, anticholinesterase, and antioxidant activity. It also known that some compounds, such as aged garlic extract, curcumin, melatonin, resveratrol, Ginkgo biloba extract, green tea, and vitamins C and E, have been used in AD patients and yielded positive results (Buy et al., J Basic Clin Physiol Pharmacol 2017; 28(5): 413-423).

A study was carried out in Europe on 225 patients with mild Alzheimer disease, who received 625 mg/ die of UMP, 400mg/die of coline and 1200mg/ die of DHA for 12 weeks orally. At the end of the treatment, behavioral tests showed an improvement in estimated episodic memory. Moreover, blood samples analysis showed a significant increase in EPA and DHA concentrations in the erythrocyte membranes of treated patients. An increase in vitamin E and a decrease in levels of homocysteine was detected in the blood plasma of this group. (Sheltens, P., Kamphius, P.J., Verhey, F.R., Olde Rikkert, M.G., Wurtman, R.J., Wilkinson, D.,Twisk, J.W., Kurz, A. (2010). Efficacy of a medical food in mild Alzheimer’s disease: a randomized, controlled trial. Alzheimer’s Dementia. 6, 1- 10.)

A multicenter clinical study was performed on 527 mild or moderate Alzheimer patients, treated with acetylcholinesterase inhibitors and/or memantine, a non competitive inhibitor of glutamate RNMDA (N- Methyl-D-aspartic acid receptors). Patients received 625 mg/ die of UMP, 400 mg/die of coline and 1200 mg/ die of DHA orally for 24 weeks. In this case, behavioral tests at the end of the treatment didn’t show any significant differences between samples and controls (Shah, R. (201 1). Souvenaid as an add-on intervention in patients with mild to moderate Alzheimer’s disease using Alzheimer’s disease medication: results from a randomized, controlled, double-blind study (S-Connect). (J Nutr Health Aging. 15, S30.)

A clinical study on 250 mild Alzheimer patients, treated with acetylcholinesterase inhibitors and/or memantine has been recently carried out. Patients received 625 mg/die of UMP, 400 mg/die of coline and 1200 mg/die of DHA orally for 24 weeks. Due to the impossibility of quantifying the number of dendritic spines in patients, an estimate of this value was determined measuring the electrical activity of the brain by electroencephalogram (EEG). Moreover, the Neuropsychological Test Battery (TBS) was used to evaluate memory. EEG and TBS results were significantly better for the samples compared to the controls. Higher levels of DHA, EPA and E vitamin and lower levels of homocysteine in the blood were confirmed (Sheltens, P., Twisk, J.W., Blesa, R., Scarpini, E., Von Arnim, C.A., Bongers, A., Harrison, J., Swinkels, S.H., Stam, C.J., De Waal, H., Wurtman, R.J., Wieggers, R.L., Vellas, B., Kamphius, P.J. (2014). Efficacy of Souvenaid in mild Alzheimer’s disease: results from a randomized, controlled trial. J Alzheimers Disease. 31, 225-236.

A double-blind clinical study was carried out on 331 people of different European nationalities with prodromal Alzheimer's, characterized by sporadic memory disorders. Patients received 625 mg/die of UMP, 400 mg/die of coline and 1200 mg/die of DHA orally for 24 months. The primary endpoint, the NTB results and the psychometric tests showed no statistically significant differences between the treated group and the control group after 24 months. However, the secondary endpoint, given by the evaluation of cerebral atrophy and measure of cognitive function, revealed a significant decrease (45% less) in the worsening of cognitive function in the treated group, compared to the control. The assessment of cerebral atrophy also revealed a significant improvement in the treated group compared to the control, in terms of hippocampal volume and ventricular volume, which indicates that treatment with Souvenaid has a positive effect in patients suffering from prodromal Alzheimer's disease (Soininen, H., Solomon, A., Visser, P.J., Hendrix, S.B., Blennow, K., Kivipelto, M., Hartmann, T. (2017). 24-month intervention with a specific multinutrient in people with prodromal Alzheimer’s disease (LipiDiDiet) : a randomized, double-blind, controlled trial. Lancet Neurology. 16, 965-975.

The problem underlying the present invention was to provide an alternative composition of natural origin, which is able to treat cognitive and behavioural synthoms of senile dementia, in particular, Alzheimer’s disease, for example, slowing down mnemonic and spatial working memory decline.

Summary of the invention

The problem has been solved by providing a composition comprising, by weight of the total weight of the composition, at least 40% of 5'-ribonucleotides as active ingredient, for use in treating Alzheimer’s disease symptoms.

Preferably, the above composition comprises 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of adenosine 5'-monophosphate, 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of uridine 5'-monophosphate, 9.1% to 18.2%, preferably 1 1.7% to 15.6% and advantageously 13%, of disodium salt heptahydrate of cytidine 5'-monophosphate, and 11.7% to 19.5%, preferably 14.3% to 16.9% and advantageously 15.6%, of disodium salt heptahydrate of guanosine 5'-monophosphate by weight of the total weight of the composition.

Preferably, the composition further comprises 15% to 22%, more preferably 17% to 20% and advantageously 18%, of compounds selected from nucleosides and nucleotides different from 5'- ribonucleotides by weight of the total weight of the composition.

Preferably, the composition comprises 1% to 5%, more preferably 2% to 3%, of a mixture of amino acids by weight of the total weight of the composition.

Preferably, the mixture of amino acids comprises methionine, cysteine, threonine, phenylalanine, tryptophan and lysine.

Preferably, the composition is obtained from an extract of fungal microorganism.

Preferably, the fungal microorganism is a yeast belonging to a genus selected from the group comprising Saccharomyces, Kluyveromyces or Candida.

Preferably, the composition has a shelf life of at least 12 months, more preferably 18 months.

The present invention also relates to a pharmaceutical or food supplement preparation for use in treating Alzheimer’s disease symptoms, including a composition as described above, and a carrier acceptable from the pharmaceutical or food standpoint.

Preferably, said 5 '-ribonucleotides are contained in an amount ranging from 0.01 to 2.0 g, more preferably from 0.3 to 1.0 g.

Preferably, the above preparation is suitable for oral administration .

Preferably, the preparation is in the form of tablets, syrups, capsules, film coated tablets or sachets of powder or granules.

In another aspect thereof, the present invention also discloses a method of treating Alzheimer’s disease symptoms, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising, by weight of the total weight of the composition, at least 40% of 5 '-ribonucleotides as active ingredient.

Preferably, said composition comprises 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of adenosine 5'-monophosphate, 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of uridine 5'-monophosphate, 9.1% to 18.2%, preferably 11.7% to 15.6% and advantageously 13%, of disodium salt heptahydrate of cytidine 5'-monophosphate, and 1 1.7% to 19.5%, preferably 14.3% to 16.9% and advantageously 15.6%, of disodium salt heptahydrate of guanosine 5'-monophosphate by weight of the total weight of the composition. .

Preferably, the composition is obtained from an extract of fungal microorganism belonging to a genus selected from the group comprising Saccharomyces, Kluyveromyces or Candida.

Preferably, the composition is included in a pharmaceutical or food supplement preparation together with a carrier acceptable from the pharmaceutical or food standpoint.

Preferably, the pharmaceutical or food supplement preparation contains said 5'-ribonucleotides in an amount ranging from 0.1 to 2.0 g, more preferably from 0.3 to 1.0 g.

Preferably, the pharmaceutical or food supplement preparation is administered orally.

Preferably, the pharmaceutical or food supplement preparation is in the form of tablets, syrups, capsules, film coated tablets or sachets of powder or granules.

In a further aspect thereof, the present invention also discloses a use of a composition comprising, by weight of the total weight of the composition, at least 40% of 5'-ribonucleotides as active ingredient, for the manufacture of a medicament for treating Alzheimer’s disease symptoms.

Preferably, the composition comprises 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of adenosine 5'-monophosphate, 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of uridine 5'-monophosphate, 9.1% to 18.2%, preferably 1 1.7% to 15.6% and advantageously 13%, of disodium salt heptahydrate of cytidine 5'-monophosphate, and 1 1.7% to 19.5%, preferably 14.3% to 16.9% and advantageously 15.6%, of disodium salt heptahydrate of guanosine 5'-monophosphate by weight of the total weight of the composition.

Preferably, the composition further comprises 15% to 22%, more preferably 17% to 20% and advantageously 18%, of compounds selected from nucleosides and nucleotides different from 5'- ribonucleotides by weight of the total weight of the composition. Preferably, the composition further comprises 1% to 5%, more preferably 2% to 3%, of a mixture of amino acids by weight of the total weight of the composition.

Preferably, said mixture of amino acids comprises methionine, cysteine, threonine, phenylalanine, tryptophan and lysine.

The compositions according to the invention may be administered with any available and efficient delivery system, comprising, but not limited to, oral, buccal, parenteral, inhalatoiy routes, topical application, by injection, by transdermic or rectal route (for ex. by means of suppositories) in dosage unit formulations containing conventional, pharmaceutically acceptable and non-toxic carriers, adjuvants and vehicles. The administration by parenteral route comprises subcutaneous, intravenous, intramuscular, intrasternal injection or infusion techniques.

The solid dosage forms for the administration by oral route comprise, for example, capsules, tablets, powders, granules and gels. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as, for example, sucrose, lactose or starch. These dosage forms normally also comprise additional substances different from the inert diluents, such as, for example, lubricating agents like magnesium stearate.

The pharmaceutical or food supplement preparations for the use according to the present invention may be produced by using conventional pharmaceutical techniques, as described in the various pharmacopoeias or handbooks of the field such as, for example, “Remington’s Pharmaceutical Sciences Handbook”, Mack Publishing, New York, 18th Ed., 1990.

The average daily dosage of the 5’-ribonucleotides contained in the composition or preparation according to the present invention depends on many factors, such as, for example, the seriousness of the disease and the conditions of the patient (age, weight, sex): The dose may generally vary from 10 mg to 2000 mg per day, preferably 300 mg to 1000 mg, of 5’-ribonucleotides, optionally divided into more administrations.

The Applicant has surprisingly found out the unexpected ability of 5’-ribonucleotides to slow down and favour the reversion of mnemonic and spatial working memory decline in a mouse model of Alzheimer’s disease (AD).

Due to its antioxidant properties, the composition of the invention helps to reduce the concentration of reactive oxygen species (ROS) in brain tissue and cells, preventing oxidative damage associated with the abnormal marked accumulation of Ab and the consequent or concomitant deposition of neurofibrillary tangles typical in AD subjects.

The present composition also helps to maintain intact the membrane lipid bilayer, showing a potential protective effect against lipid oxidation of brain cell membranes.

A further potential advantage represented by the use of the composition according to the invention consists in favouring the inhibition of the formation of breakpoints in the DNA, caused by a high intracellular concentration of ROS and likely to determine genomic instability and inclination to generation of site-specific genetic mutations. Brief description of figures

Figure 1 shows bar graphs regarding the effect of RIBODIET® at the dosage of 0.1, mg/mouse, 1.0 mg/ mouse and 10.0 mg/mouse in the object recognition test (A) and olfactory discrimination test (B). Data were expressed as media ± S.E.M, and were analysed by one-way ANOVA, followed by Bonferroni test for multiple comparisons. ^#P<0,05 vs Ctrl; ^##P<0,01 vs Ctrl; *P<0,05 vs bi-42; **P<0,01 vs bi-42.

Figure 2 shows bar graphs regarding the effect of RIBODIET® at the dosage of 0.1, mg/mouse, 0.3 mg/mice and 10.0 mg/mouse in the Y-maze test (A) and locomotor activity test (B). Data were expressed as media ± S.E.M, and were analysed by one-way ANOVA followed by Bonferroni test for multiple comparisons. #P<0,05 vs Ctrl or b_1-42.

Figure 3 (A-C) shows a qualitative analysis of ~40 cytokines by ELISA Spot assay in the following experimental conditions: (A) Control [(b_1-42 + RIBODIET® vehicle saline solution]; (B) b_1-42 + RIBODIET® 0.1 mg/ mouse; (C) b_1-42 + RIBODIET® 1 mg/ mouse; (D) b_1-42 + RIBODIET® 10 mg/ mouse).

Figure 3 (E-G) shows levels (expressed as INT/mm²) of C5a, ICAM, MCSF, SDF (E) and CXCL13, CXCL1, IL-1a, IL-Req, IL-16, KC, MIP1a, MIP2, TIMP1, and TREMl(F-G) in animals administered with b_1- ₄₂ + RIBODIET® vehicle saline solution, b_1-42 + RIBODIET® 0, 1 mg/mouse, b_1-42 + RIBODIET® 1 mg/mouse and b_1-42 + RIBODIET® 10 mg/ mouse. Data were expressed as media ± S.E.M, and were analysed by one-way ANOVA followed by Bonferroni test for multiple comparisons. ^#P<0,05 vs b_1-42; ***P<0,001 vs b_1-42.

Figure 4 (A)-(D) shows a qualitative analysis of 28 cytokines by ELISA Spot in the following experimental conditions: (A) Control (b_1-42 + RIBODIET® vehicle saline solution); (B) b_1-42 + RIBODIET® 0.1 mg/mouse; (C) b_1-42 + RIBODIET® 1 mg/mouse; (D) b_1-42 + RIBODIET® 10 mg/ mouse).

Figure 4 (E)-(F) shows levels (expressed in INT/mm²) of C5a and SDF (E) and BLC, IP- 10, KC, MIP1a, MIP-2, and HSP60 (F) in animals administered with b_1-42 + RIBODIET® vehicle saline solution, b_1-42 + RIBODIET® 0.1 mg/mouse, b_1-42 + RIBODIET® 1 mg/mouse and b_1-42 + RIBODIET® 10 mg/mouse. Data were expressed as media ± S.E.M and were analysed by one-way ANOVA followed by Bonferroni test for multiple comparisons. #P<0,05 vs b_1-42; ***P<0,001 vs b_1-42.

Figure 5 shows a table reporting pro-inflammatoiy, chemotactic and tissue/organ homeostatic activities of a set of chemokines and cytokines, in different experimental conditions, related to their activation of neutrophils, macrophages and lymphocytes.

Detailed description of the invention

For several years, the Applicant has been producing a composition based on 5'-ribonucleotides, marketed under the name RIBODIET® as a food supplement, having an anti-inflammatory and immuno stimulatory functions in promoting the health of the intestinal tract.

In view of the beneficial effects found with the use as a dietary supplement of the aforementioned composition due to its anti inflammatory and immunizing properties, and the ease of industrial production of the RIBODIET® product, the Applicant decided to verify whether this product had a positive effect also in the treatment of Alzheimer’s disease symptoms.

A b(1-42) peptide has been recognized as a key factor in the neurodegeneration in AD patients and it often mediates its harmful effect via inducing oxidative stress in brain cells. In fact, enhanced apoptosis accompanied by a poor antioxidant status is one of the mechanisms for AD pathogenesis; superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide (ROS) in the oxidative stress mediate neurodegeneration in AD.

In particular, the Applicant intended to test the effect of the antioxidant properties of RIBODIET® in brain tissues characterized by high contents of Ab -peptide, which are rich in harmful oxidants.

To this end, several in vivo tests were carried out in a mouse model.

The novel object recognition (NOR) test is a commonly used behavioural method for the investigation of various aspects of learning and memory in mice; it is a relatively fast and efficient means for testing different phases of learning and memory in mice.

This test assesses animal’s behaviour when it is exposed to a novel and a familiar object; in particular, it relies primarily on a rodent’s innate exploratory behaviour in the absence of externally applied rules or reinforcement.

The task procedure normally consists of three phases: habituation, familiarization, and test phase (An tunes et al., Cogn Process. 2012 May; 13(2): 93-110). In the habituation phase, each animal is allowed freely exploring the open-field arena in the absence of objects. The animal is then removed from the arena and placed in its holding cage.

During the familiarization phase, a single animal is placed in the open-field arena containing two identical sample objects (A + A), for a few minutes. After a retention interval, during the test phase, the animal is returned to the open-field arena with two objects, one is identical to the sample and the other is novel (A + B).

Normal (i.e. wild-type) mice spend more time exploring the novel object during the first few minutes of the test phase. In fact, the strongest novel object preference scores tend to occur early in the test phase; while the novel object is still relatively novel, since in the course of time, the novel object became familiar.

The NOR task is particularly attractive because it requires no external motivation, reward, or punishment but a little training or habituation is required, and it can be completed in a relatively short time.

As previously mentioned, when animals are exposed to a familiar and a novel object, they approach frequently and spend more time exploring the novel than the familiar one; in fact, the recognition of novelty requires more cognitive skills from the subject, relative to tasks measuring exploration of novel environments or a single novel object.

Another test carried out by the Applicant relates to olfactory discrimination ability, because early olfactory dysfunction has been consistently reported in AD patients.

The herein reported olfactory discrimination test (OD test) takes advantage of the fact that rodents prefer places impregnated with their own odor (familiar compartments) instead of places with non-familiar odors.

To this purpose, each mouse was placed for few minutes in a cage divided in two equal areas separated by an open door, where it could choose between one compartment with fresh sawdust (non-familiar compartment) and another with unchanged sawdust (familiar compartment).

Finally, the Applicant carried out a test related to spatial working memory.

In fact, spatial disorientation is one of the early manifestations of Alzheimer’s disease (AD), besides the well-known memory impairment. The research in spatial deficits in this neurodegenerative disease has grown rapidly in last years and decline in spatial navigation abilities may become another diagnostic mark for AD in the near future.

To this end, spontaneous alternation using a Y-shaped maze was used as a test for habituation and spatial working memory.

This test is based on the innate preference of mice to alternate arms when exploring a new environment. Various modifications are available with different levels of difficulty and different demands on specific types of cognition. One version that is particularly popular for the study of cognitive changes in AD transgenic models is the spontaneous alternation version of the Y-maze.

In this instance, test animals are placed in a Y-shaped maze for few minutes and the number of arms entered, as well as the sequence of entries, is recorded and a score is calculated to determine alternation rate (degree of arm entries without repetitions). A high alternation rate is indicative of sustained cognition as the animals must remember which arm was entered last to not renter it.

Surprisingly, in NOR test, AD mice administered with RIBODIET® showed an improved spontaneous tendency to spend more time exploring the novel object than the familiar one than AD mice, thus indicating an improvement in learning and recognition memory.

In the ODR test, AD mice administered with RIBODIET® were able to discriminate and prefer the familiar compartment containing sawdust impregnated with their own odor than the other compartment containing fresh sawdust. Such a behaviour was completely different from that of AD mice, which instead did not distinguish familiar and unfamiliar compartments, spending most time in the unfamiliar one.

In the Y-maze test, AD mice administered with RIBODIET^® preferred to investigate a new arm of the maze rather than returning to one that was previously visited, scoring a higher alternation rate and correct arm entries than AD mice.

In all of the above-mentioned tests, AD mice administered with RIBODIET^® showed a behaviour very similar to the wild-type mice, suggesting that this product, based on 5'-ribonucleotides, is able to significantly reduce memory and spatial impairments typical of AD subjects.

The method for the production of RIBODIET®, sold by the Applicant PROSOL S.p.a., is disclosed in Italian patent application No. 1020160001 12436, and it is briefly summarized below.

The starting raw material is the liquid obtained by DNA extraction process from yeasts (for example, Kluyveromyces or Saccharomyces ) .

Such a liquid has been filtered by microfiltration (membranes with a pore size of 0.45 mm) to separate suspended particles from the process liquid.

This process liquid has 10% of dry substance, which has a hydrolysable RNA content of between 60% and 90% (Schmidt & Tannhauser method).

A first dilution is carried out by adding osmotic water, and the pH, if necessary, is corrected up to a value of 5.5.

The mass thus obtained is subjected to a first heat treatment step, at a temperature between 90°C and 100°C for 20-30 minutes; then, it is cooled by adding water in a quantity sufficient to lower the temperature to 70°C.

As the heating step normally causes a drop in pH of 0.2 to 0.5 points, a second pH correction is made by adding 30% NaOH, reporting the value in a range between 5.3 and 5.5.

The conditions thus obtained (70 ° C and pH between 5.3 and 5.5) are those considered optimal for the activity of the enzyme necessary in order to hydrolyse RNA (ribonuclease).

The enzyme is weighed (0.33% on the dry substance) and added into the reactor, after being dissolved in a separated container in 10-15 L osmotized water.

Hydrolysis is carried out for 10 h at a temperature of 70 ° C; in this step, pH decreases due to the enzymatic activity (decrease of 0.4-0.7 points) .

After hydrolysis, the pH is brought to 6.30 by adding 30% NaOH. Then, the mass is subjected to a centrifugation step in a clarifying centrifuge and a subsequent concentration step in a concentrator under vacuum.

Thus, 1400-1500 L of concentrated liquid having a dry substance of 32-37% is obtained. The concentrated liquid is then cooled and, finally, pumped to the spray drying plant.

The so-obtained composition in powder form presents a 5’- ribonucleotide content from 40% up to 65% (52%-84.5%, considering 5’- ribonucleotides in the disodium salt heptahydrate form), generally between 50-65%.

The composition also contains nucleosides and other nucleotides different from 5’-ribonucleotides (about 20% w/w) and a mixture of amino acids (about 5% w/w) including methionine, cysteine, threonine, phenylalanine, tryptophan and lysine.

Further features and advantages of the present invention will become apparent from the following examples, given for purposes of illustration and not of limitation.

EXAMPLE 1

The purpose of the following tests was to evaluate the potential therapeutic efficacy of the commercial product RIBODIET®, produced by the Applicant, in the treatment of Alzheimer’s disease symptoms by in vitro and in vivo test.

Materials

Animals for in vivo experiments were CD-I mice (20-25g in weight) purchased by Charles River (Italy).

The reagents, solvents, kits and all the necessary for in vitro and ex vivo experiments were sold by Sigma Aldrich (Milan, Italy) and R&D System (Milan, Italy). Laboratory plastic consumables were sold by BioCell company (Naples, Italy). Methods

The above-mentioned animals were housed in plexiglass cages equipped with enrichment material (“mouse house tecniplast”) with free access to food and water, in a room with controlled temperature (22 ± 2 ° C) and humidity (45-65%), with 12h: 12h light:dark cycles.

Before the experimental procedures, the animals were housed in the enclosure for a period of 7 days (adaptation period). In the experiments, CD1 mice were subdivided into different experimental groups (n = 7-10).

All experimental groups underwent the behavioural tests described below.

Induction of the experimental model in vivo.

The surgical procedure to perform the intracerebroventricular administration (i.c.v.) of peptide Ab_1-42 (single administration at 3mg/3ml dose) was performed according to the method described by Haley and McCormick (1957) and subsequently modified by Porreca (Porreca et al., 1984).

The surgical procedure started with the anaesthesia of the animals with a solution of ketamine (100 mg / kg) and xylazine (10 mg / kg) or alternatively enfluorane. The weighed and anesthetized animals were shaved on the head and positioned on a pre-heated pad to avoid the onset of post-anesthetic hypothermia. After disinfecting the surgical area with betadine, the animals were gently placed on the stereotaxic device and a longitudinal incision from the area between the eyes (previously kept hydrated with 2 drops of eye drops) to the back of the ears was made by a disposable scalpel.

The connective tissue was removed, and the surface of the skull was kept hydrated and disinfected with H2O2 to visualize and distinguish the lines of bregma and lambda.

Then, the area was washed and, by means of the stereotaxic device, a small mark was made on the point of the skull where a 2mm perforation was subsequently carried out (by means of a millimetric chisel) according to the coordinates described by Paxinos and Franklin, 2001 (namely, -0.2 mm in antero-posterior direction and -2 mm in the middle-lateral direction) and subsequently modified by Maione and collaborators (Maione et al, 2008).

The i.c.v. administration was performed with a 2 mm needle in order to allow a direct administration of the Ab_1-42 peptide in the left ventricle (3 mΐ) with an infusion rate of 1m1/60 seconds.

Finally, after the procedure and the i.c.v. administration, the animals received a subcutaneous single administration of analgesic (meloxicam 1 mg/ kg) and were monitored daily for the duration of experiments.

RIBODIET® administration

Some of the above Alzheimer's induced mice (AD mice) were administered with RIBODIET® at the dosages of 0.1 mg/mice, 1 mg/mice and 10 mg/ mice by oral gavage 3 times weekly for 21 days. RIBODIET® was dissolved in a saline solution (0.9% NaCl) and the resulting solution was administered by gavage.

Such AD mice administered with RIBODIET® (hereinafter, “RIBODIET® mice”) were the subjects to test the effect of RIBODIET® on impaired mnemonic memory and spatial working memory, which are typical symptoms in Alzheimer’s subjects.

Tested mice

In the Novel Object Recognition (NOR) test, Olfactory Discrimination (OD) test and Y-shaped maze test described in detail below, the following experimental mice groups were tested:

- RIBODIET® mice: AD mice administered with RIBODIET®, obtained as described above;

- Control mice (CTRL): mice subjected to i.c.v. surgical procedure, but not administered with Ab_1-42 peptide)

- AD mice: mice administered with Ab_1-42 peptide and “RIBODIET® vehicle saline solution”, namely a 0.90% w/v NaCl solution.

NOR (Novel Object Recognition) Test

The NOR test was carried out in two sessions in the same context, divided by an intersession interval (ISI) equal to 24 hours. During the first session (familiarization session), the animal is free to explore two similar objects, and during the second session (test session), one of the objects is replaced by a novel, unfamiliar object.

In particular, during the first session, the animals were allowed to freely explore for 2 days the area where test took place.

Then, two equal objects (A and B) were placed in the test area. Each animal could explore both objects for 10 minutes. The animals explored the object when their head was turned to the object or when mice interacted with the object sniffing it or touching it. The time spent on exploration was recorded by an operator and expressed as a percentage of exploration time.

In the test session, one of the two objects was replaced by a new object (C). The animal could explore the novel object for 5 minutes and the time spent on exploration was appropriately recorded. The time of exploration was normalized as a percentage of the total exploration time.

The preference for a novel object meant that presentation of the familiar object existed in animals’ memory.

OD (Olfactory Discrimination) Test

The OD test was an olfactory test consisting of two different sessions: a training session followed by a testing session carried out 24 h later.

During the training session, two compartments were placed (A and B) in the test area, one containing sawdust from the original cage (A) and the other containing new sawdust (B) .

The animals were allowed to freely explore both compartments for 5 minutes and the time spent in compartment A and B was registered. The preference towards compartment A (evaluated after 24h) was therefore considered as a maintenance of memory towards the family environment.

Y-maze test

The tested Y-Maze consisted of a maze composed of 3 arms (40 cm in length) at a 120° angle from each other.

After introduction to the centre of the maze, the animal was allowed to freely explore the three arms, by visually registering the sequence and number of entries in the various arms (named A, B and C). Over the course of multiple arm entries, the subject should show a tendency to enter a less recently visited arm.

During the test, after the above habituation phase, the animal was tested by evaluating the alternation of its entrances in arms A, B and C, dividing a hypothetical sequence ABCB in ABC-BCB, where the ABC sequence was considered as spontaneous and expressed alternation episode evaluated as "% of correct alternations".

At the same time, locomotor activity was (indirectly) recorded by assessing the total number of entries (expressed as "arm entries”) of mice within the arms of the Y-maze.

ELISA Spot

In order to evaluate possible differences in the modulation of neuro-inflammatory response related to the different RIBODIET^® dosages (0.1 mg/mouse, 1.0 mg/mouse and 10.0 mg / mouse), cytokine and chemokine profile, in the various experimental conditions, was assessed by ELISA Spot.

The following 40-cytokines panel was tested: CXCL-13, C5a, G- CSF, GM-CSF, CCL1, Eotaxin, ICAM, IFN-y, IL-la, IL- 1, IL -IReq, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-12p70, IL-16, IL-17, IL-23, IL-27, IP- 10, I-TAC, KC, MCSF, JE, MCP-5, MIG, MIP- la, MIP-lp, MIP-2, RANTES, SDF-1, TARC, TIMP-1, TNF-a, TREM-1).

Moreover, a 28 chemokines panel was also tested by ELISA Spot: 6Ckine, BLC, CIO, C5a, CCL28, Chemerin, CTACK, CXCL16, Eotaxin, Fractalkine, IL-16, IP- 10, I-TAC, JE, KC, LIX, MCP-2 , MCP-5, MDC, MIG, MIR-1a/b, MIP-ly, MIP-2, RANTES, SDF, Complement factor D, gpl30 and HSP60.

The assay was carried out as follows.

Equal volumes (- 1.5 ml) of supernatant obtained from the lysis of single brains of the above CD1 mice, in the different experimental conditions, were incubated with the Precoated Proteome Profiler array kit (R&D Systems, Milan, Italy).

The signal of the (semi-quantitative) expression of different cytokines and chemokines was detected with the ECL (Enhanced Chemiluminescence) system and captured by Image Quant (Biorad, Italy).

Finally, the densitometric analysis of the relative spots was carried out using a specific software (GS 800 imaging densitometer software, Biorad, Italy).

Statistical analysis

The results were expressed as mean + standard error of the mean (S.E.M.) and were analysed by means ANOVA one-way or ANOVA two-way, followed by Bonferroni test for multiple comparisons. Statistical analysis was performed by GraphPad software version 6.0. Values lower than P <0.05 were considered significant.

Results

NOR (Novel Object Recognition) test and OD (Olfactory Discrimination) test showed that control mice (Ctrl, subjected to i.c.v. surgical procedure, but not administered with Ab_1-42 peptide) showed a normal behaviour in the novel object recognition (TF: time familiar; TNF: time non-familiar) compared to mice administered with Abi-4₂ peptide (Beta) - see Fig. 1.

Notably, the administration of RIBODIET^® reverts the mnemonic decline observed in AD mice.

In fact, the AD mice administered with RIBODIET^® show the preference for the novel object more than all. The recognition of novelty requires more cognitive skills from the subject, relative to tasks measuring exploration of novel environments or a single novel object.

Similarly, in the Y-maze test, RIBODIET^® mice showed a reversion of the mnemonic decline of AD mice. In fact, they reported a higher number of correct alternations compared to AD mice (Beta), maintaining a normal locomotor activity (Fig. 2).

Regarding the modulation of chemokines and cytokines depending on RIBODIET^® dosages, ELISA Spot assay showed that there was a significant (P <0.05) decrease in C5a, ICAM, MCSF and SDF cytokines in RIBODIET® mice compared to the AD mice (Fig. 3E). Furthermore, a total reversion of the cytokine content of CXCL13, CXCL1, IL-la, IL-1R, IL-16, KC, MIPla, MIP2, TIMP1 and TREMI (Figures 3F and 3G) was observed.

Instead, a significant (P <0.05) decrease in C5a and SDF chemokines (Figure 4E) and a total reversion of BLC, IP- 10, KC, MIP-2 and HSP60 (Figure 4F) was observed.

The ex vivo results seem to indicate a modulation, at the CNS level, of the main cytokines and chemokines (such as BCL, C5a, 1-309, ICAM, IL- 1a, IL-16, IP- 10, KC, MIPs, SDF-1, TIMP-1, TREM-1 and HSP60) involved in the neuro-inflammation and neuro-degeneration process. The results of this study suggest the potential role of RIBODIET® as nutraceutical product useful for neurodegenerative disease such as AD, thanks to its anti-inflammatory and neuroprotective effect.

The results of these semi-quantitative tests have been summarized in Figure 5, where the modulated activity of the detected cytokines and chemokines and the cellular systems involved in this modulation process was reported.

The above reported results clearly indicated that RIBODIET® is a product that, at low dosages, is able to improve and revert the mnemonic decline and cognitive impairment typical in an AD subjects.

The ex vivo results also indicated a modulation a level of the CNS and the main cytokines and chemokines involved in the process of neuroinflammation and neuro-degeneration.

Claims

1. A composition comprising, by weight of the total weight of the composition, at least 40% of 5'-ribonucleotides as active ingredient, for use in treating Alzheimer’s disease symptoms.

2. The composition for the use according to claim 1, comprising 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3% , of disodium salt heptahydrate of adenosine 5'-monophosphate, 10.4% to 18.2% , preferably 13% to 15.6% and advantageously 14.3% , of disodium salt heptahydrate of uridine 5'-monophosphate, 9.1% to 18.2% , preferably 11.7% to 15.6% and advantageously 13% , of disodium salt heptahydrate of cytidine 5'-monophosphate, and 1 1.7% to 19.5% , preferably 14.3% to 16.9% and advantageously 15.6% , of disodium salt heptahydrate of guanosine 5'- monophosphate by weight of the total weight of the composition.

3. The composition for the use according to claim 1 or 2, further comprising 15% to 22%, preferably 17% to 20% and advantageously 18%, of compounds selected from nucleosides and nucleotides different from 5'-ribonucleotides by weight of the total weight of the composition.

4. The composition for the use according to any one of claims 1-3, comprising 1% to 5%, preferably 2% to 3%, of a mixture of amino acids by weight of the total weight of the composition.

5. The composition for the use according to claim 4, wherein said mixture of amino acids comprises methionine, cysteine, threonine, phenylalanine, tryptophan and lysine.

6. The composition for the use according to any one of claims 1-5, wherein said composition is obtained from an extract of fungal microorganism .

7. The composition for the use according to claim 6, wherein said fungal microorganism is a yeast belonging to a genus selected from the group comprising Saccharomyces, Kluyveromyces or Candida.

8. The composition for the use according to any one of claims 1-7, wherein said composition has a shelf life of at least 12 months, preferably 18 months.

9. A pharmaceutical or food supplement preparation for use in treating Alzheimer’s disease symptoms, including a composition according to any one of claims 1 to 8 and a carrier acceptable from the pharmaceutical or food standpoint.

10. A pharmaceutical or food supplement preparation for the use according to claim 9, wherein said 5'-ribonucleotides are contained in an amount ranging from 0.01 to 2.0 g, preferably from 0.3 to 1.0 g·

1 1. A preparation for the use according to claim 9 or 10, characterized in that it is suitable for oral administration.

12. A preparation for the use according to claim 1 1, characterized in that it is in the form of tablets, syrups, capsules, film coated tablets or sachets of powder or granules.

13. A method of treating Alzheimer’s disease symptoms, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising, by weight of the total weight of the composition, at least 40% of 5 '-ribonucleotides as active ingredient.

14. The method according to claim 13, wherein said composition comprises 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of adenosine 5'-monophosphate, 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of uridine 5'- monophosphate, 9.1% to 18.2%, preferably 11.7% to 15.6% and advantageously 13%, of disodium salt heptahydrate of cytidine 5'- monophosphate, and 1 1.7% to 19.5%, preferably 14.3% to 16.9% and advantageously 15.6%, of disodium salt heptahydrate of guanosine 5 '-monophosphate by weight of the total weight of the composition.

15. The method according to claim 13 or 14, wherein said composition further comprises 15% to 22%, preferably 17% to 20% and advantageously 18%, of compounds selected from nucleosides and nucleotides different from 5' -ribonucleotides by weight of the total weight of the composition.

16. The method according to any one of claims 13-15, wherein said composition comprises 1% to 5%, preferably 2% to 3%, of a mixture of amino acids by weight of the total weight of the composition.

17. The method according to claim 16, wherein said mixture of amino acids comprises methionine, cysteine, threonine, phenylalanine, tryptophan and lysine.

18. The method according to any one of claims 13-17, wherein said composition is obtained from an extract of fungal microorganism belonging to a genus selected from the group comprising Saccharomyces, Kluyveromyces or Candida.

19. The method according to any one of claims 13-18, wherein said composition is included in a pharmaceutical or food supplement preparation together with a carrier acceptable from the pharmaceutical or food standpoint.

20. The method according to claim 19, wherein said pharmaceutical or food supplement preparation contains said 5 '-ribonucleotides in an amount ranging from 0.1 to 2.0 g, preferably from 0.3 to 1.0 g.

21. The method according to claim 19 or 20, wherein said pharmaceutical or food supplement preparation is administered orally.

22. The method according to claim 21, wherein said pharmaceutical or food supplement preparation is in the form of tablets, syrups, capsules, film coated tablets or sachets of powder or granules.

23. Use of a composition comprising, by weight of the total weight of the composition, at least 40% of 5'-ribonucleotides as active ingredient, for the manufacture of a medicament for treating Alzheimer’s disease symptoms.

24. The use according to claim 23, wherein said composition comprises 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of adenosine 5'- monophosphate, 10.4% to 18.2%, preferably 13% to 15.6% and advantageously 14.3%, of disodium salt heptahydrate of uridine 5'- monophosphate, 9.1% to 18.2%, preferably 11.7% to 15.6% and advantageously 13%, of disodium salt heptahydrate of cytidine 5'- monophosphate, and 11.7% to 19.5%, preferably 14.3% to 16.9% and advantageously 15.6%, of disodium salt heptahydrate of guanosine 5'-monophosphate by weight of the total weight of the composition.

25. The use according to claim 23 or 24, wherein said composition further comprises 15% to 22%, preferably 17% to 20% and advantageously 18%, of compounds selected from nucleosides and nucleotides different from 5'-ribonucleotides by weight of the total weight of the composition.

26. The use according to any one of claims 23 to 25, wherein said composition further comprises 1% to 5%, preferably 2% to 3%, of a mixture of amino acids by weight of the total weight of the composition.

27. The use according to claim 26, wherein said mixture of amino acids comprises methionine, cysteine, threonine, phenylalanine, tiyptophan and lysine.

28. The use according to any one of claims 23-26, wherein said composition is obtained from an extract of fungal microorganism.

29. The use according to claim 28, wherein said fungal microorganism is a yeast belonging to a genus selected from the group comprising Saccharomyces, Kluyveromyces or Candida.

30. The use according to any one of claims 23-28, wherein said composition is included in a pharmaceutical or food supplement preparation together with a carrier acceptable from the pharmaceutical or food standpoint.

31. The use according to claim 30, wherein said pharmaceutical or food supplement preparation contains said 5 '-ribonucleotides in an amount ranging from 0.1 to 2.0 g, preferably from 0.3 to 1.0 g.

32. The use according to claim 30 or 31, wherein said pharmaceutical or food supplement preparation is administered orally.

33. The use according to claim 32, wherein said pharmaceutical or food supplement preparation is in the form of tablets, syrups, capsules, film coated tablets or sachets of powder or granules.