WO2025083658A1

WO2025083658A1 - A combination for use in a method for the prevention and treatment of cognitive decline and/or forms of dementia

Info

Publication number: WO2025083658A1
Application number: PCT/IB2024/060321
Authority: WO
Inventors: Rita Paola PETRELLI; Valeria CURTI
Original assignee: Kolinpharma SpA
Current assignee: Kolinpharma SpA
Priority date: 2023-10-19
Filing date: 2024-10-21
Publication date: 2025-04-24
Anticipated expiration: 2026-04-19

Abstract

An object of the present invention is a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade. Further, the present invention relates to a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade for use in a method for the prevention and/or treatment of cognitive decline and/or forms of dementia. The present inventions also has as its object a composition, and a formulation containing said composition, for use in a method for the prevention and treatment of cognitive decline and/or forms of dementia, preferably has as its object a composition, and a formulation containing said composition, to counteract inflammation, oxidative stress, and cerebrovascular damage at the level of the central nervous system as causes of neurodegeneration leading to the onset of cognitive decline and development of dementia. A further object of the present invention is an association or combination of said composition and/or formulation and at least one drug for the treatment of forms of dementia and/or treatment of cognitive decline. An object of the present invention is, in addition, an association or combination of said composition and/or formulation and said at least one drug for use in a method for the prevention and/or treatment of cognitive decline and/or forms of dementia.

Description

A COMBINATION FOR USE IN A METHOD FOR THE PREVENTION AND TREATMENT OF COGNITIVE DECLINE AND/OR FORMS OF DEMENTIA

An object of the present invention is a composition and a formulation containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade. Further, the present invention relates to a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade for use in a method for the prevention and/or treatment of cognitive decline and/or forms of dementia. An object of the present invention is, in addition, a composition, and a formulation containing said composition, for use in a method for the prevention and treatment of cognitive decline and/or forms of dementia, preferably an object is a composition and a formulation containing said composition, to counteract inflammation, oxidative stress, and cerebrovascular damage at the level of the central nervous system as causes of neurodegeneration leading to the onset of cognitive decline and development of dementia. A further object of the present invention is an association or combination of said composition and/or formulation and at least one drug for the treatment of forms of dementia and/or the treatment of cognitive decline. An object of the present invention is, in addition, an association or combination of said composition and/or formulation and said at least one drug for the prevention and/or treatment of cognitive decline and/or forms of dementia.

Technical Background

The generic term neurodegenerative diseases (NDs) refers to a number of conditions that affect the central nervous system and, specifically, cells called neurons. These cells lack regenerative capacity, so any damage to the nervous system is permanent.

Neurodegenerative diseases are debilitating and untreatable conditions; they are caused by the progressive functional and structural degeneration and/or death of nerve cells. Depending on where the degeneration is localized, disorders in movement (ataxias) or cognitive disorders (dementias) result. For example, if the involvement is at the encephalic level, Alzheimer's disease or Parkinson's disease may develop; if, on the other hand, the degeneration is at the motor neuron level, amyotrophic lateral sclerosis will be found; if the degeneration affects the myelin sheath of the axon, manifestations such as multiple sclerosis will occur. All neurodegenerative diseases, indiscriminately, have a significant public health impact in terms of incidence, disability, complexity of disease management, and need for ongoing treatment and care.

The etiopathogenesis of these diseases is still being studied, but to date some shared cellular mechanisms underlying neurodegeneration have been identified, such as: excessive oxidative stress, oligomerization of proteins to create toxic aggregates, a deficit in axonal transport, deregulation of calcium concentration at the intersynaptic and cellular levels, mitochondrial damage in terms of both function and structure, excessive inflammation/neuroinflammation condition, abnormal neuro-glial interaction, incorrect RNA processing or DNA damage.

Factors such as age, sex, conditions associated with metabolic syndrome (e.g., hypertension, obesity, and diabetes), and the presence of comorbidities that already alter the inflammatory profile may increase the risk of neurodegeneration onset and thus constitute risk factors to be monitored. According to WHO, there were 35.6 million people with dementia worldwide in 2010, a number that is expected to triple by 2050. In fact, an average of 7.7 million new cases of dementia are diagnosed each year, with the cost attributable to medical expenses increasing. In Italy, it is estimated that the number of people diagnosed with dementia exceeds 1 million, while there are about 3 million people involved in caring for loved ones with the disease. This finding takes on even greater significance if one considers that dementia can be difficult to clinically diagnose, due to its multifactorial nature and the large number of overlapping symptoms, which mean that the disease can present with very varied clinical manifestations. This heterogeneity makes it possible to identify different types of dementia, which are classified into four main categories: Alzheimer's disease (AD), Lewy body dementia (LBD), frontotemporal dementia (FTD), and vascular dementia (VaD). In general, all dementia states are caused by neurodegenerative processes that result in the onset of cognitive decline that becomes increasingly severe as time passes. At the pathophysiological level, neurodegeneration is always associated with oxidative stress and neuroinflammation, which are then accompanied by other factors that are characteristic of each individual type of dementia.

Alzheimer's disease (AD)-related dementia is the most common form of dementia, and is characterized by distinctive neuropathological features such as senile plaque formation at the extracellular level and intraneuronal accumulation of neurofibrillar clusters. The formation of senile plaques is due to the accumulation of p-amyloid peptide (Ap), especially its isoform Ap42, which originates from amyloid precursor protein (APP), a transmembrane protein that can be processed, at the physiological level, by a-, p- and y-secretases through two pathways: amyloidogenic and non-amyloidogenic. During the amyloidogenic pathway, p-secretase processes APP near the N-terminal region of its amyloid domain (Ap), causing the formation of a C-terminal fragment, called p-CTF, which is released into the extracellular space. p-CTF, in turn, is processed by y-secretase at the C-terminal region of the Ap domain, causing the formation of the peptide Ap42 and its extracellular accumulation. In the non-amyloidogenic pathway, on the other hand, the processing of APP by o-secretase, which occurs in the central region of the Ap domain of APP, precludes the possibility of generating the amyloid peptide. In general, all characteristic markers of Alzheimer's disease are associated with neurodegeneration, neuroinflammation, microglia activation, blood-brain barrier (BBB) malfunction, leading to cognitive decline and memory impairment.

One of the most common forms of dementia in the elderly is Lewy body dementia (LBD), characterized by the abnormal aggregation of the synaptic protein o-synuclein within neurons and typically associated with the course of Parkinson's disease.

The term frontotemporal dementia (FTD), on the other hand, denotes a large and heterogeneous group of neurodegenerative dementias that can have different clinical and pathological profiles and are characterized by atrophy of the frontal and temporal lobes, accompanied by neuronal loss, gliosis, and spongiosis, resulting in aphasia, severe personality disorders, memory loss, and language difficulties.

Finally, many cognitive disorders share a vascular origin and are grouped under the classification of vascular dementias (VaDs). Specifically, VaDs are characterized by reduced cerebral blood flow, which can cause hypoxic states and altered blood-brain barrier (BBB) permeability, with neurotoxic effects that promote neurodegeneration and amyloid protein accumulation. The cognitive decline experienced by patients with dementia is often accompanied by secondary disorders, which can negatively affect the patient's quality of life. Among these, states of anxiety and depression are very common. In particular, in patients with Alzheimer's disease, depression is considered a risk factor for the development and progression of the disease and is observed in nearly 50 percent of cases. In dementia, depression and anxiety have an endogenous component, related to the physiological changes in the brain caused by neurodegeneration, and a reactive component, in response to the awareness of the pathology and the gradual loss of independence. In addition to anxiety and depression, sleep disorders are also very common. Neurodegenerative diseases, in fact, can alter sleep architecture, affecting patients' state of fatigue and apathy, even to the point of causing acute confusional states {delirium). Moreover, sleep deprivation is itself one of the risk factors related to dementia, especially Alzheimer's disease, as it can affect cognitive functions. The aforementioned disorders related to anxiety, depression, and sleep deprivation can, in very severe cases, result in true neuropsychiatric disorders, which manifest themselves in patients in the form of illusions, especially of a paranoid nature, and hallucinations, often visual. In addition, patients with dementia may suffer from agitation and aggression, mainly due to confusion resulting from the inability to fully comprehend what is happening to them. This condition can lead to behaviors that are dangerous for caregivers and the patient himself.

Treatments currently employed in the field of dementia focus on slowing the progression of cognitive decline and restoring function by attempting to remedy the damage already suffered by the brain. In this direction, several therapeutic strategies have been developed, both pharmacological and nonpharmacological. The first approach used to slow the progression of the disease is to have the patient follow an appropriate diet, such as MIND (Mediterranean-DASH Intervention for Neurodegenerative Delay), accompanied, if possible, by the performance of exercise and the implementation of strategies to intervene on cardiovascular risk factors. As for symptom relief, there are drugs presently available (acetylcholinesterase inhibitors) which can improve the symptoms of the disease and temporarily slow its progression. Acetylcholinesterase inhibitors, which are presently prescribed by specialized centers to patients with Alzheimer's disease of mild-to-moderate severity, are donepezil (Aricept or Memac), rivastigmine (Exelon or Prometx) and galantamine (Reminyl). The effectiveness of these drugs is similar, what changes is the method of administration (donepezil is to be taken once a day in a single dose, rivastigmine and galantamine several times a day in increasing doses) and the profile of side effects, such as nausea, vomiting and diarrhea (some of these drugs are better tolerated than others). Donepezil and rivastigmine are also available in the form of slow-release patches. Memantine (Ebixa) has been approved for the treatment of moderately severe- to-severe Alzheimer's disease.

As mentioned above, the main drugs used to treat dementia are cholinesterase inhibitors, such as donepezil (CAS number 120014-06-4), which have proven useful because they can increase levels of acetylcholine, which plays a key role in memory and attention processes. In addition to these, also widely used are NMDA receptor antagonists, such as memantine (CAS number 19982-08-02), which prevent pathological overactivation of NMDA receptors. However, the use of both classes of drugs mentioned above can be accompanied, as already mentioned above, by side effects, including nausea, vomiting, loss of appetite and diarrhea, headache, and confusion. Cognitive interventions, which can help mitigate some of the cognitive-related symptoms of dementia, are often added to drug therapy. Among these, the main ones are environmental enrichment, defined as creating a stimulating environment for the patient, and cognitive neurorehabilitation, conducted by speech therapists and neuropsychologists.

Pharmacological therapies are also often used to counter symptoms related to secondary disorders associated with the disease, such as anxiety, depression, sleep disorders, agitation, and aggression. Among the most commonly used drugs are selective serotonin reuptake inhibitors (SSRIs), which are considered useful both for states of anxiety and depression and to mitigate agitation and aggression. However, SSRIs are often accompanied by major side effects, and in the case of depression associated with dementia, some studies find their use to be ineffective. With regard to problems related to agitation and aggression, antipsychotics (which, however, can cause very serious side effects), mood stabilizers (although there is no evidence regarding their effectiveness in dementia- related cases), or benzodiazepines (to be administered only in cases where the patient actually becomes dangerous to himself or herself and others) can also be used in very severe cases. As for sleep disorders, when necessary, patients can be treated with antidepressants, such as trazodone and mirtazapine, or low-dose nonbenzodiazepines.

In general, the therapies available to date, in addition to having several side effects of some significance, are not sufficient to effectively counteract the progression of the disease and its clinical manifestations.

For this reason, it is of paramount importance to intervene on patients from the early stages of the neurodegenerative process, before a diagnosable stage of dementia is reached. The intermediate state between a normal cognitive condition and the diagnosis of dementia is termed mild cognitive impairment (MCI) and groups together heterogeneous cases, which may result in different cognitive-related pathologies. Specifically, MCI cases can be classified as amnestic or nonamnestic, depending on whether the memory sphere is involved or not. Subsequently, both classes of MCI can be further divided into single-domain or multiple-domain cases, depending on the number of cognitive areas involved. Accordingly, single-domain amnestic MCI refers to a condition, often a precursor to Alzheimer's disease, in which subjects have memory impairment but have not yet reached the state of dementia. Multiple-domain amnestic MCI, also a precursor to Alzheimer's disease, includes those cases in which, in addition to memory-related problems, other alterations in other cognitive domains also occur. As for non-amnestic MCI cases, on the other hand, single-domain MCI cases are characterized by disorders affecting a single cognitive domain other than memory, while multiple-domain MCI cases manifest alterations in multiple cognitive domains, such as those related to language, visual-spatial abilities, and executive functions.

Mild cognitive decline, in general, is a common disorder in the elderly population with the following current prevalence estimates in relation to age groups: 6.7% between 60 and 64 years, 8.4% between 65 and 69 years, 10.1 % between 70 and 74 years, 14.8% between 75 and 79 years, and 25.2% between 80 and 84 years. Risk factors associated with MCI are comparable to those for dementia and include, in addition to age, poor education, vascular risk factors, hypertension, diabetes, obesity, history of heart attack or heart disease, and genetic factors. It has also been estimated that the risk of a patient with MCI worsening to a state of dementia is 10% each year in individuals over 65. Those diagnosed with MCI are three times more likely to develop dementia in the 5 years after diagnosis than healthy individuals.

In order to take timely action on the reversible factors that can lead to the onset of cognitive decline, it is crucial to be able to correctly diagnose a situation of MCI. Diagnosis, which is often difficult because of the heterogeneity and complexity of MCI cases, usually, is based on the patient's own or close people's report of a disorder and objective evidence of alterations in one or more cognitive domains, which can be assessed through clinical interviews, validated scales, such as MoCA {Montreal Cognitive Assessment) and MMSE (Mini-Mental State Examination), and neuropsychological tests. For proper diagnosis, it is important to be able to distinguish problems related to abnormal neurodegeneration from age-related physiological problems. Also common in patients with MCI are mood-related alterations, including depression, with a prevalence of 25-40%. The relationship between MCI and depression is complicated: on the one hand, cognitive impairment could be a consequence of depression; on the other hand, depression could be an early manifestation of cognitive decline. Other widespread symptoms are anxiety, irritability, aggression, and apathy. Overall, the neuropsychiatric symptoms observed in cases of MCI are similar to those associated with Alzheimer's disease, but with less frequency and severity. The main challenge for the treatment of patients with MCI is to be able to act early on risk factors to slow the progression of neurodegenerative processes and avoid or delay the onset of dementia. This is complemented, where necessary, by interventions to counter depression and sleep disorders, taking special care to ensure that the therapies used do not have side effects that worsen patients' quality of life.

In general, of great importance seems to be the management of cardiovascular risk factors. The main strategies that can be undertaken to counter the progression of a mild state of cognitive decline involve dietary control, exercise, and the implementation of cognitive interventions for memory stimulation. In this case, cholinesterase inhibitors, normally used in dementia cases, fail to prevent or slow the progression of cognitive decline and the transition from MCI to dementia.

Purposes of the invention

One purpose of the present invention is to make available a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade.

Another purpose of the present invention is to make available a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade, that counteracts the onset and/or slows the progression of dementia and/or cognitive decline states.

A further purpose of the present invention is to make available a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade, that improves the quality of life of patients with cognitive decline and/or dementia.

Still another purpose of the present invention is to make available a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade, which can be employed, without causing side effects, as a support or complement to conventional therapy for neurodegenerative diseases, particularly cognitive decline and/or dementia, that acts on the underlying causes of the neurodegenerative disease, and is thus capable of preventing and/or slowing down the disease itself.

A further purpose of the present invention is to make available an association or combination of said composition and/or formulation and at least one drug intended for treatment of forms of dementia and/or treatment of cognitive decline.

Another purpose of the present invention is to make available an association or combination of said composition and/or formulation and at least one drug intended for treatment of forms of dementia and/or treatment of cognitive decline that counteracts the onset and/or slows the progression of dementia and/or cognitive decline states.

Still another purpose of the present invention is to make available an association or combination of said composition and/or formulation and at least one drug intended for treatment of forms of dementia and/or treatment of cognitive decline that improves the quality of life of patients with cognitive decline and/or dementia.

These and still other purposes, which will be clear from the detailed description that follows, are achieved by the object of the present invention, which provides: (I) a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade; (ii) a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade, for use in a method for the prevention and treatment of cognitive decline and/or forms of dementia; an object is preferably a composition and a formulation, containing said composition and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade, to counteract inflammation, oxidative stress and cerebrovascular damage at the level of the central nervous system; (ill) a combination of said composition and/or formulation and at least one drug intended for treatment of forms of dementia and/or treatment of cognitive decline; (iv) a combination of said composition and/or formulation and at least one drug intended for treatment of forms of dementia and/or treatment of cognitive decline for use in a method for the prevention and treatment of cognitive decline and/or forms of dementia.

Description of the invention

An object of the present invention is a composition comprising or alternatively consisting of:

(I) an epigallocatechin gallate (EGCG), preferably an epigallocatechin-3-gallate (EGCG), and/or an extract of Camellia sinensis (L.) titrated in epigallocatechin gallate (EGCG) or, preferably, titrated in epigallocatechin-3-gallate (EGCG), and

(ii) an extract of Crocus sativus (L), and/or tryptophan.

Preferably, said (I) in said composition may be a green tea extract, e.g., of the type 90%P (total polyphenols) 60%C (total catechins) 40E (EGCG - epigallocatechin gallate) or similar, e.g., an extract from green tea leaves made with an extraction solvent, e.g., water/ethyl acetate.

Preferably, said (I) in said composition may be a green tea extract that, for example, may comprise or, alternatively, consist of polyphenols, catechins, EGCG and/or caffeine, e.g., said (I) may be a green tea extract that, for example, may comprise or, alternatively, consist of from 90% to 98% of total polyphenols, from 60% to 90% of total catechins, from 40% to 55% of EGCG and from 5% to 15% of caffeine, as measured with the HPLC method. Preferably, said (i) in said composition can also be a green tea extract with a reduced caffeine content or without caffeine (decaffeinated green tea), e.g., it can be a green tea extract that comprises or alternatively consists of from 90% to 98% of total polyphenols, from 60% to 90% of total catechins and from 40% to 55% of EGCG as measured with the HPLC method.

For example, a dry extract of green tea leaves with added caffeine may be of the type:

CAS: 490-46-0 EINECS: 207-710-1 ; 2 - (3, 4 - dihydroxyphenyl) - 2, 3, 4 - trihydro - 3, 5, 7 - trihydroxychromene 10-20%;

CAS: 1257-08-5 (-)-Epicatechin 3-gallate 10-20%;

CAS: 58-08-2 EINECS: 200-362-1 caffeine 2.5-10%;

CAS: 4233-96-9 (-)-Gallocatechin 3-gallate 2.5-10%;

CAS: 154-23-4 EINECS: 205-825-1 cyanidanol 1-3%;

CAS: 3371-27-5 2H-1-benzopyran-3, 5, 7- triol,3,4-dihydro-2- (3,4,5-trihydroxyphenyl), (2S,3R) 1-3%.

Preferably, said (ii) in said composition may be, for example, a dry extract of Crocus sativus L, Iridaceae, e.g., a dry saffron extract or saffron powder capable of containing safranal in an amount from 0.1% to 50% by weight (UV- visible ISO 3632 analytical method; UHPLC), preferably from 0.2% to 30% by weight, even more preferably from 0.3% to 15%, e.g., from 0.35% to 5%. Saffron is made from the stigmas of the crocus. The actives are selected, for example, from safranal, crocin and/or picrocrocin. For example, harvested saffron stigmas are dried and then extracted in water. The aqueous phase is filtered. Then an extraction of the aqueous phase is done in 30%-50% ethanol . The ethanol is removed by vacuum separation. Preferably, said (ii) in said composition may be an extract of Crocus sativus (L), and/or tryptophan.

Preferably, said an epigallocatechin gallate (EGCG), preferably an epigallocatechin-3-gallate (EGCG), present in said (i), as well as the actives safranal, crocin and/or picrocrocin present in said (ii), may also be present as extracts, for example extracts of natural origin or extracts originating from natural sources, or they may be present as "natureidentical” molecules (e.g., Reg. (EC) N 1334/2008) prepared by synthesis or fermentation.

Preferably, said composition may also comprise (iii) a trans-resveratrol, or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera, to give a (i)+(ii)+(iii) composition.

Preferably, said component (iii) may be, for example, a trans-resveratrol or resveratrol, e.g., it may have formula C14H12O3 and/or molecular weight of about 228.24 g/mol and/or CAS No. 501 -36-0, preferably it may be from Polygonum cuspidatum or Vitis vinifera,' said component (iii) may give a (i)+(ii)+(iii) composition, where said (ii) can be an extract of Crocus sativus (L), and/or tryptophan.

Preferably, said trans-resveratrol and/or resveratrol, present in said (iii), may be present as extracts, for example extracts of natural origin or extracts originating from natural sources, or they may be present as "nature-identical” molecules (e.g., Reg. (EC) N 1334/2008) prepared by synthesis or fermentation. Preferably, said composition may also comprise (iv) citicoline or other sources of choline, to give a (i)+(ii)+(iv) composition, or (i)+(ii)+(iii)+(iv) composition, where said (ii) may be an extract of Crocus sativus (L), and/or tryptophan. Preferably, said tryptophan, present in said (iv), may be present as an extract, for example an extract of natural origin or extract originating from natural sources, or it may be present as a "nature-identical” molecule (e.g., Reg. (EC) N 1334/2008) prepared by synthesis or fermentation.

Said component (iv) may, for example, currently be marketed under the trade name Cognizin ®.

Preferably, said composition may, in addition, comprise (v) at least one compound selected from the group comprising or, alternatively, consisting of selenium, zinc, vitamin C, vitamin D, vitamin E, or vitamin B12, to give a

(i)+(ii)+(v), or (i)+(ii)+(iii)+(v), or (i)+(ii)+(iv)+(v), or (i)+(ii)+(iii)+(iv)+(v) composition.

Preferably, said composition may, in addition, comprise (vi) an oleuropein, preferably obtained from a dry extract of the olive tree Olea europaea (L), to give a (i)+(ii)+(vi), or (i)+(ii)+(iii)+(vi), or (i)+(ii)+(iv)+(vi), or

(i)+(ii)+(iii)+(iv)+(vi), or (i)+(ii)+(v)+(vi), or (i)₊(ii)₊(iii)₊(v)₊(vi), or (i)₊(ii)₊(iv)₊(v)₊(vi), or (i)₊(ii)₊(iii)₊(iv)₊(v)₊(vi) composition.

Preferably, said composition may, in addition, comprise (vii) at least one substance selected from the group comprising or, alternatively, consisting of a dry extract of Bacopa monnieri, a dry extract of Curcuma longa (L), omega-3 fatty acids, preferably DHA or docosahexaenoic acid, or phosphatidylserine, to give a (i)+(ii)+(vii), or (i)+(ii)+(vi)+(vii), or (i)+(ii)+(iii)+(vi)+(vii), or (i)+(ii)+(iv)+(vi)+(vii), or (i)+(ii)+(iii)+(iv)+(vi)+(vii), or (i)+(ii)+(v)+(vi)+(vii), or (i)+(ii)+(iii)+(v)+(vi)+(vii), or (i)+(ii)+(iv)+(v)+(vi)+(vii), or (i)+(ii)+(iii)+(iv)+(v)+(vi)+(vii) composition.

A further object of the present invention is an association or combination of a composition comprising or, alternatively, consisting of:

(i) an epigallocatechin gallate (EGCG), preferably an epigallocatechin-3-gallate (EGCG), and/or an extract of Camellia sinensis (L.) titrated in epigallocatechin gallate (EGCG),

(ii) an extract of Crocus sativus (L.) and/or tryptophan,

(iii) a trans-resveratrol or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera, and

- at least one drug selected from the group comprising or, alternatively, consisting of drugs having, as an active ingredient, at least one active ingredient that inhibits acetylcholinesterase, said at least one active ingredient being selected from donepezil, preferably Aricept® or Memac®, rivastigmine, preferably Exelon® or Prometax®, galantamine, preferably Reminyl®, and memantine, preferably Ebixa®.

(ii) an extract of Crocus sativus (L.) and/or tryptophan

(iii) a trans-resveratrol, or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera and at least one cholinesterase inhibitor drug, such as donepezil, and/or at least one NMDA receptor antagonist drug, such as memantine.

A further object of the present invention is an association or combination of a composition comprising or, alternatively, consisting of: (i) an epigallocatechin gallate (EGCG), preferably an epigallocatechin-3-gallate (EGCG), and/or an extract of Camellia sinensis (L.) titrated in epigallocatechin gallate (EGCG),

(II) an extract of Crocus sativus (L.) and/or tryptophan,

(ill) a trans-resveratrol or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera,

(iv) citicoline or other sources of choline, and

Another object of the present invention is an association or combination of a composition comprising or, alternatively, consisting of:

(ii) an extract of Crocus sativus (L.) and/or tryptophan,

(iii) a trans-resveratrol, or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera,

(iv) citicoline or other sources of choline and at least one cholinesterase inhibitor drug, such as donepezil, and/or at least one NMDA receptor antagonist drug, such as memantine.

In the context of the present invention said at least one drug, preferably donepezil and/or memantine, may be administered in association or combination with said compositions of the present invention (i)+(ii)+(vii), or (i)+(ii)+(vi)+(vii), or (i)+(ii)+(iii)+(vi)+(vii), or (i)+(ii)+(iv)+(vi)+(vii), or (i)+(ii)+(iii)+(iv)+(vi)+(vii), or (i)+(ii)+(v)+(vi)+(vii), or (i)+(ii)+(iii)+(v)+(vi)+(vii), or (i)+(ii)+(iv)+(v)+(vi)+(vii), or (i)+(ii)+(iii)+(iv)+(v)+(vi)+(vii).

The combination envisages that said at least one drug is administered together with at least one of said compositions; in practical terms, said drug and said composition are administered at the same time (a same administration).

The association, by contrast, envisages that said at least one drug is administered separately from said at least one of said compositions; in practical terms, said drug and said composition are administered without a precise sequence and at different times; for example, said drug and said composition may be administered in a time interval comprised from 30 seconds to 60 minutes, preferably from 1 minute to 30 minutes (two separate administrations staggered over time).

The doses and amounts of the drugs used are in line with current medical practices and medical protocols, known to persons skilled in the art, such as physicians and pharmacologists.

An object of the present invention is also a composition, among those mentioned above, for use as a medicament; preferably, said composition is for use in the prevention and/or treatment of neurodegenerative diseases; preferably, said neurodegenerative diseases are selected from cognitive decline and/or dementia.

An object of the present invention is also a formulation comprising a composition, among those mentioned above, for use as a medicament; preferably, said formulation is for use in the prevention and/or treatment of neurodegenerative diseases; preferably, said neurodegenerative diseases are selected from cognitive decline and/or dementia.

An object of the present invention is also a combination, among those mentioned above, for use as a medicament; preferably, said combination is for use in the prevention and/or treatment of neurodegenerative diseases; preferably, said neurodegenerative diseases are selected from cognitive decline and/or dementia.

The composition object of the present invention comprises or alternatively consists of (I) an epigallocatechin gallate (EGCG), preferably an epigallocatechin-3-gallate, preferably derived or obtained from dry extract of green tea Camellia sinensis (L.) titrated in epigallocatechin gallate (EGCG), preferably containing epigallocatechin-3-gallate, and/or (ii) a saffron extract (Crocus sativus), preferably a saffron dry extract or a saffron powder (Crocus sativus), and/or tryptophan.

Preferably, said (I) is a green tea extract of the type 90%P (total polyphenols) 60%C (total catechins) 40E (EGCG -epigallocatechin gallate) or similar, e.g., extracted from green tea leaves with an extraction solvent, e.g., water/ethyl acetate.

Preferably, said (I) is a green tea extract that may comprise or, alternatively, consist of polyphenols, catechins, EGCG and/or caffeine, e.g., said (I) may be a green tea extract that, for example, may comprise or, alternatively, consist of from 90% to 98% of total polyphenols, from 60% to 90% of total catechins, from 40% to 55% of EGCG and from 5% to 15% of caffeine, as measured with the HPLC method.

Preferably, said (I) can also be a green tea extract with reduced caffeine content or without caffeine (decaffeinated green tea) e.g., it can be a green tea extract that comprises or alternatively consists of from 90% to 98% of total polyphenols, from 60% to 90% of total catechins and from 40% to 55% of EGCG as measured with the HPLC method.

Preferably, said (II) may be a dry extract, or powder, of saffron (Crocus sativus) titrated in safranal, for example, at 0.2%; or 0.3%; or 0.4%; 0.5%; or 1 %; or 1 .5%; or 2%; or 3%; or 4%; or 5%, by weight. For example, it is an extract having the name Satiereal WS, marketed by the company Inoreal.

For example, from the stigmas of the plant (Crocus sativus) a solid/liquid extraction can be made with an extraction solvent containing from 30% to 50% of ethanol and from 70% to 50% of water; the extract is supported on acacia gum, for example.

Preferably, the composition may comprise, in addition to (I) and (II), also (ill) a trans-resveratrol, or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera, where said (II) may be an extract of Crocus sativus (L), and/or tryptophan.

Preferably, the composition may comprise, in addition to (I), (II) and (ill), also (iv) a citicoline or other source of choline such as phosphatidylcholine, where said (II) may be an extract of Crocus sativus (L), and/or tryptophan. Preferably, the composition may comprise, in addition to (I), (II), (ill), and (iv), also (v) a source of selenium such as, for example, an L-selenomethionine obtained from Saccharomyces cerevisiae strains, and/or a source of zinc such as, for example, a zinc bis-glycinate, and/or one or more vitamins such as, for example, a vitamin C (CAS No. 50- 81-7), a vitamin D, preferably a vitamin D3 (CAS No. 67-97-0), a vitamin E (CAS No. 7695-91-2) or a vitamin B, preferably a vitamin B12 (CAS No. 68-19-9). Preferably, the composition may comprise, in addition to (i), (ii), (iii), (iv) and (v), also (vi) an oleuropein, preferably extracted from a dry extract of olive tree titrated 40% in oleuropein (CAS No. 32619-42-4).

Also according to the present invention, the composition may further comprise:

Dry extract of Bacopa monnieif,

Dry extract of Curcuma longa L. ;

Omega-3 fatty acids;

Phosphatidylserine.

Specifically, again according to the present invention, said epigallocatechin-3-gallate (EGCG) is preferably derived from dry extract of green tea {Camellia sinensis).

Epigallocatechin-3-gallate (EGCG) is a molecule with antioxidant and anti-inflammatory properties, as well as being able to cross the blood-brain barrier.

Said dry extract of saffron {Crocus sativus) is used, according to the present invention, to take advantage of its characteristic of significantly improving sleep quality and mood, as often treated subjects tend to suffer from anxiety and depression.

According to the present invention, said trans-resveratrol or resveratrol is preferably derived from Polygonum cuspidatum or Vitis vinifera.

The brain requires constant blood flow through the network of cerebral veins and arteries to replenish itself with oxygen, glucose, and other essential nutrients, but also to remove waste products of metabolism.

Preferably, according to the invention, a source of choline is, for example, citicoline.

Citicoline is used as a precursor to choline, an essential nutrient involved in numerous processes at the neuronal level. Choline, in cholinergic neurons, is acetylated to give acetylcholine, a key neurotransmitter involved in numerous areas, including memory. As an example, citicoline may be the one currently commercially available as Cognizin ®, in the form of a white crystalline powder.

Vitamin E, another preferred component of the composition according to the present invention, is a fat-soluble antioxidant par excellence, protecting the cell from free radical-induced damage at the level of cell membranes. Vitamin C, on the other hand, contributes to the normal functioning of the nervous system and normal psychological function.

Vitamin B12 contributes to the normal functioning of the nervous system and contributes to normal psychological function.

Vitamin D, preferably vitamin D2 and/or D3, could be useful in the composition of the present invention.

Zinc contributes to normal cognitive function and the protection of cells from oxidative stress.

Selenium contributes to the protection of cells from oxidative stress.

Again, according to the present invention, said oleuropein is preferably derived from a dry extract of olive tree {Olea eurepaea L).

Oleuropein is one of the main phenolic components of the olive tree {Olea europaea L). Specifically, the chemical structure of oleuropein contains an ortho-diphenolic group that can act as a scavenger of ROS, stabilizing free radicals via an intramolecular hydrogen bond. Tryptophan is an essential amino acid: the body is unable to synthesize it ex novo. Therefore, it must be taken in through the diet. Being the precursor of serotonin (5-HT), supplementation with tryptophan during the day can increase the levels of this neurotransmitter in the brain.

Bacopa monnieri extract is useful for memory and cognitive function, as well as promoting mental well-being. The extract derived from Bacopa monnieri, due to its bioactive components, including Bacoside A, could play a role in protecting the brain from oxidative damage and cognitive impairment.

Said omega-3 fatty acids are, for example, according to the present invention, preferably DHA or docosahexaenoic acid. DHA contributes to the maintenance of normal brain function.

Phosphatidylserine is one of the main phospholipids that make up cell membranes and has been found, in particular, at the level of neuronal cells, where it plays an essential role related to membrane viscosity.

The effectiveness of the composition comes from the surprising synergistic effect of its essential components in association with each other, further enhanced by the presence of its optional components, also in association with each other. This synergistic effect is evident from the fact that although the individual components are known as such for example to show efficacy on inflammation, oxidative stress, to have generic antioxidant activity, protective activity on the cerebrovascular system and cognitive function, and to have mood-regulating activities, their combination does not provide individual benefits on each of these aspects, but rather has an overall action in the prevention and/or treatment of neurodegenerative diseases, particularly cognitive decline and dementia.

In addition, the composition, particularly the composition comprising or, alternatively, consisting of components (I), (ii) and (ill), also defined hereinafter as Mix 4, or the composition comprising or, alternatively, consisting of components (I), (ii), (ill) and (iv), also defined hereinafter as Mix 3, has revealed to be particularly effective if in combination with at least one cholinesterase inhibitor drug, such as donepezil, and/or at least one NMDA receptor antagonist drug, such as memantine. In this case, the compositions Mix 3 and Mix 4 have revealed to be capable of amplifying the effect of the known drugs donepezil and memantine when administered in combination with the latter.

With reference to the attached Figures, which have the purpose of illustrating and not limiting the invention:

Figure 1 shows the results of the tests conducted for dose-response assessment with regard to cell viability for components (I), (ill), (ii);

Figure 2 shows the results of tests conducted to evaluate the best combination of components (I), (ii), (ill) and (iv) according to the invention, excluding a cytotoxic effect;

Figure 3 shows the results of tests conducted to evaluate the passage through the BBB (blood-brain barrier); Figure 4 shows the results of tests designed to determine the integrity of the BBB (Marveld and Claudin 5);

Figure 5 shows the results of tests designed to evaluate the protective effect of compounds and compositions thereof according to the invention on mitochondrial metabolism in the presence of oxidative stress;

Figure 6 shows the results of tests designed to evaluate the antioxidant effect of the compounds and compositions of the invention, in the presence of oxidative stress;

Figure 7 shows the results of tests designed to assess nitric oxide (NO) production in the presence of oxidative stress; Figure 8 shows the results of tests designed to assess inflammatory markers (IL-2 and TNF-a) in the presence of oxidative stress;

Figure 9 shows the results of tests designed to assess the production of BDNF (brain-derived neurotrophic factor) in the presence of oxidative stress;

Figure 10 shows the results of tests designed to assess the activation of intracellular apoptotic mechanisms (p53 and cytochrome C) activated in the presence of oxidative stress;

Figure 11 shows the results of tests designed to evaluate the activation of intracellular energy metabolism mechanisms (SIRT1 and NRF2) activated in the presence of oxidative stress;

Figure 12 shows the results of tests designed to assess the activation of intracellular mechanisms activated in the presence of oxidative stress, such as APP and pTau expression;

Figure 13 shows the results of tests designed to evaluate the protective effect of compounds and combinations thereof in compositions according to the invention in the presence of iron accumulation;

Figure 14A shows the results of tests designed to assess neurodegenerative damage caused by iron accumulation;

Figure 14B shows the results of tests designed to verify the absence of Fe³⁺ accumulation in astrocytes;

Figure 15 shows the results of tests designed to evaluate lipid peroxidation in the presence of iron accumulation; Figure 16 shows the results of tests to evaluate inflammatory markers (IL-ip and TNF-a) expressed in the presence of iron accumulation;

Figure 17 shows the results of tests for evaluating the activation of intracellular mechanisms activated in the presence of iron accumulation (evaluated as BDNF brain-derived neurotrophic factor-panel A; cytochrome C activity-panel B; p53 activity - panel C);

Figure 18 shows the results of tests for evaluating the activation of intracellular mechanisms activated in the presence of iron accumulation (evaluated as SIRT1 - panel A; NRF2 - panel B; APP - panel C; pTAU - panel D). Figure 19 shows the results of tests for evaluating mitochondrial metabolism through an assessment of cell viability;

Figure 20 shows the results of tests for evaluating the level of oxidative stress through an assay for the quantification of ROS production;

Figure 21 shows the results of tests for the quantification of homocysteic acid production;

Figure 22 shows the results of tests for evaluating the trend in the main markers involved in neurodegeneration (APP, pTAU, NRF2 and SIRT1).

According to a preferred, but not limiting, embodiment of the present invention, the composition which is the object of the invention is as follows:

1. Composition C1 -Main components:

- Epigallocatechin-3-gallate (EGCG), preferably derived from green tea {Camellia sinensis) dry extract, from 50 mg to 400 mg, preferably from 100 mg to 300 mg, such as 200 mg, per daily dose; and

- Saffron {Crocus sativus) dry extract from 5 mg to 50 mg, preferably from 10 mg to 40 mg, e.g., 30 mg, per daily dose.

2. Composition C2 -Main components: - Epigallocatechin-3-gallate (EGCG), preferably derived from green tea (Camellia sinensis) dry extract, from 50 mg to 400 mg, preferably from 100 mg to 300 mg, e.g., 200 mg, per daily dose; and

-tryptophan from 50 mg to 400 mg, preferably from 100 mg to 350 mg, e.g., 300 mg, per daily dose.

3. C3 composition -Main components: - Epigallocatechin-3-gal late (EGCG), preferably derived from green tea (Camellia sinensis), dry extract from 50 mg to 400 mg, preferably from 100 mg to 300 mg, e.g., 200 mg, per daily dose; and

- Saffron (Crocus sativus) dry extract from 5 mg to 50 mg, preferably from 10 mg to 40 mg, e.g., 30 mg, per daily dose; and

-tryptophan from 50 mg to 400 mg, preferably from 100 mg to 350 mg, e.g., 300 mg, per daily dose. 4. Composition C4 -Main components:

A trans-resveratrol or resveratrol preferably from Polygonum cuspidatum or Vitis vinifera from 50 mg to 300 mg, preferably from 100 mg to 200 mg, e.g., 150 mg, per daily dose, and/or an Oleuropein preferably extracted from dry extract of olive tree (Olea eurepaea L.) from 10 mg to 150 mg, preferably from 30 mg to 100 mg, e.g., 70 mg, is added to the C1, or C2, or C3 compositions.

Table 1 Additional components:

Table 2

Preferably, in the composition according to the invention, said epigallocatechin-3-gallate is provided in an amount from 2 mg to 500 mg, preferably from 10 mg to 300 mg, even more preferably from 25 mg to 200 mg, and is preferably derived from dry extract of green tea {Camellia sinensis). The amount of green tea extract will depend on the concentration of the extract.

Said saffron extract is provided in an amount from 1 mg to 50 mg, preferably from 5 mg 40 mg, even more preferably from 10 mg to 30 mg, of saffron dry extract.

Said trans-resveratrol or resveratrol preferably from polygonum cuspidatum is provided in an amount from 5 mg to 500 mg, preferably from 10 mg to 300 mg, even more preferably from 40 mg to 200 mg.

Said citicoline is present in an amount from 10 mg to 5 g, preferably from 50 mg to 4 g, even more preferably from 100 mg to 3 g.

Said vitamin E is present in an amount from 2 mg to 60 mg, preferably from 5 mg to 50 mg, even more preferably from 6 mg to 40 mg.

Said vitamin C is present in an amount from 10 mg to 500 mg, preferably from 40 mg to 400 mg, even more preferably from 60 mg to 300 mg.

Said vitamin B12 is present in an amount from 0.1 ug to 1 mg, preferably from 1 ug and 0.5 mg.

Said vitamin D, preferably vitamin D2 and/or D3, is present in an amount from 1 ug to 50 ug, preferably from 2 ug to 30 ug.

Said zinc is present in an amount from 1 mg to 15 mg, preferably from 3 mg to 12 mg, even more preferably from 5 mg to 10 mg.

Said selenium is present in an amount from 5 ug to 100 ug, preferably from 10 ug to 70 ug, even more preferably from 25 ug to 50 ug.

Said oleuropein and/or extract from leaves of Olea europaea L. preferably titrated 20%, or 30% or 40% or 50% in oleuropein, is present in an amount from 5 mg to 1 g, preferably from 10 mg to 0.75 g, even more preferably from 50 mg to 0.5 g.

Said tryptophan is present in an amount from 5 mg to 500 mg, preferably from 40 mg to 400 mg, even more preferably from 80 mg to 300 mg. Said Bacopa monnieri extract is present in an amount from 50 mg to 1 g, preferably from 100 mg to 600 mg, even more preferably from 200 mg to 400 mg.

Said curcuma is present in an amount from 10 g to 500 g of extract, preferably from 25 mg to 300 mg, even more preferably from 50 mg to 200 mg.

As for said omega-3s, they are present in an amount from 50 mg to 5 g, preferably DHA is present in an amount from 50 mg to 1 g, preferably from 100 mg to 0.5 g, of DHA. The composition according to the invention will provide an amount of omega-3 such that an intake of 250 mg of DHA per day is provided.

Said phosphatidylserine is present in an amount from 50 mg to 1 g, preferably from 100 mg to 0.75 g, even more preferably from 200 mg to 0.5 g.

All quantities expressed by weight mentioned above refer to the amount of the individual components with respect to the weight of the composition, according to the present invention.

The composition according to the present invention may be administered according to a dosage of one or two daily administrations, such as one or two tablets per day.

For example, a tablet may be of the type:

Active components: Amount/compound:

Epigallocatechin-3-gallate from green tea dry extract (250 mg): 100 mg

Trans-resveratrol: 75 mg

Citicoline: 250 mg

Saffron: 15 mg

Selenium: 30 ug

Vitamin E: 6 mg

Vitamin C: 60 mg

Zinc: 5 mg

Vitamin B12: 1.25 ug

Vitamin D3: 2.5 ug

An object of the present invention is also formulations that comprise the composition of the invention, as well as acceptable, commonly used additives and formulation agents or carriers of food or pharmaceutical grade. Said formulations may be in tablet, pouch, orosoluble stick, stick or gel form. The composition and formulation of the invention are advantageously formulated for oral (or sublingual) administration such as in sachet or tablet form. The dosage form of the formulation of the invention may be a solid form, such as a tablet, chewable tablet, effervescent tablet, multi-layered tablet (e.g., time-release), capsule, lozenge, granules or powder (granules or powder to be dissolved in water or orosoluble granules or powder), or a semisolid form, such as a soft gel, or a liquid form, such as a solution, suspension, dispersion, emulsion, or syrup; preferably, the formulation of the invention is in a solid form for oral use, more preferably in a tablet or powder/granule form to be dispensed in sachets to be dissolved in water.

Said composition of the invention can be a pharmaceutical composition, a composition for medical devices (Medical Device Regulation (EU) 2017/745 (MDR)), a food supplement and/or a food for special medical purposes (FSMP). Promising results are obtained by applying the following in-vitro study protocol, which employs a human astrocyte cell model.

The protocol generally provides for, among other things:

• Analysis of Passage through the blood-brain barrier (BBB) through a model of co-culture between astrocytes and HUVECs;

• Solubility of raw materials to be tested;

• Cell viability assays with treatment with individual actives (i)-(iv) - at least 4 assays for each sample without toxic stimulus:

- (I) an epigallocatechin gallate (EGCG), or

- (ii) an extract of Crocus sativus (L.), or

- (iii) a trans-resveratrol, or

- (iv) citicoline or other sources of choline.

• Cell viability assays with treatment with (i)+(ii) - at least 4 assays for each sample without toxic stimulus:

- (I) an epigallocatechin gallate (EGCG), and

- (II) an extract of Crocus sativus (L.);

• TOXIC STIMULATION: Fe³⁺ and H₂O₂ ;

• Validation of the AD model with Fe³⁺ and H₂O₂ for 24 h for 1050;

• Analysis of neuroprotective effects exerted by individual actives (I), (II), (iii) and (iv), and (i)+(ii) by analyzing two different aspects of neurodegeneration (oxidative stress and iron accumulation);

• NO analysis;

• BDNF (brain-derived neurotrophic factor) Quantification

• Apoptosis markers

• ROS analysis

• Lipid Peroxidation/MDA (malondialdehyde) analysis.

• Nrf2/intracellular mechanisms.

The substances used are as follows:

1 . Green tea, from 25piM to 200piM

2. Saffron, from 25piM to 500piM

3. Resveratrol, from 1 piM to 10piM

4. Citicoline (e.g., the product currently commercially available as Cognizin®) 10OpiM

Phase 1 of the experimental protocol involves:

• Cell viability assays with treatment with the active compounds taken individually and in combination using a neuronal cell model known in the literature for studies on cognitive decline (CCF-STTG1, human astrocytic-like cell line).

Phase 2 involves: • Analysis of the passage through the blood-brain barrier (BBB) through a model of co-culture between astrocytes and HUVECs

• Evaluation of barrier integrity (TJ): claudin 5 and MARVELD

Phase 3 involves:

• Validation of cognitive decline model with Fe³⁺ and H2O2 for 24h, through cell viability assays

• Study of neuroprotective mechanisms exerted by active compounds taken individually and combinations thereof.

• Analysis of mitochondrial metabolism

• Analysis of oxidative stress by analysis of ROS, NO and lipid peroxidation

• Analysis of the inflammatory picture by quantifying IL-1 b, IL-2, TNF-a

• BDNF production

• Analysis of iron accumulation by cytochemical staining and quantification

• Analysis of apoptotic mechanisms

• Analysis of key markers involved in neurodegeneration.

Figure 1 (dose-response evaluation) shows the results of tests conducted to evaluate the best concentration to be used for the components shown as (I), (II) and (ill) according to the present invention.

As shown in Figure 1 , all tested substances demonstrate an increase in cell viability for all tested concentrations (p<0.05), The data obtained show that the ranges tested are effective and the best concentration for Green Tea (compound (I) according to the present invention) was lOOpiM, for Resveratrol (compound (ill) according to the present invention) 1 piM, and for Saffron (compound (ii) according to the present invention) 25piM; therefore these concentrations will be maintained for subsequent experiments and will be combined with Cognizin® (compound (iv) according to the present invention) lOOpiM, a concentration already confirmed by a previous study [ref: Francesca Uberti., et al. "Effect of Mixed Lipoic Acid, Vitamin D, Phosphatidylserine and Homotaurine to Obtain a New Formulation for Brain Ageing Prevention." EC Neurology 11.5 (2019)].

Figure 2 shows the results of tests performed to evaluate the best combination of individual components (I), (ii), (ill) and (iv) according to the present invention, excluding a cytotoxic effect. The tested combinations increased cell viability compared to the control (p < 0.05), but also compared to the individual substances (p < 0.05). Thus, the analysis showed that the tested substances are able to stimulate cell viability, excluding any cytotoxic effect and confirming the safety of all tested compositions. In particular, the data obtained show a synergistic effect of the tested substances, suggesting that their combination is able to improve neuronal homeostasis. Among the proposed mixtures, Mix 4 seems to satisfy mitochondrial metabolism better, as it increases cell viability in a statistically significant manner compared to the other combinations (p < 0.05).

Figure 3 shows the results of tests performed to evaluate passage through the blood-brain barrier (BBB). Specifically, tests on the BBB identified that the compositions according to the present invention are able to pass through the blood-brain barrier with a maximum peak at 12h, maintaining their effect even at 24h. In particular, the combined effect of the substances is able to increase permeability (p < 0.05), suggesting a possible antioxidant and neuroprotective role.

Composition Mix4, according to the invention, achieves a better value at 12h than the other compositions (p < 0.05). In addition, no composition gave rise to barrier exchange problems.

Next, tight junctions were analyzed in order to confirm the integrity of the BBB reproduced in vitro and the absence of any alteration due to treatment. In particular, claudin 5, which is involved in membrane pore selectivity and is linked to its stability to the passage of molecules, and MARVELD, which enables the maintenance of the barrier to the passage of macromolecules and allows the release of effectors downstream, were evaluated. As shown in Figure 4, data from the tight junction evaluation tests on the BBB showed an increase in the activity of the two TJs analyzed, confirming the maintenance of structural integrity to the passage of substances, both alone and in combination (p<0.05 vs control). In particular, both TJs were found to be more active following treatment with the combinations (p < 0.05), especially following treatment with Mix 1 and Mix 4, compared with Mix 2 and Mix 3 (p < 0.05), for both parameters analyzed.

Figure 5 shows the results of tests designed to evaluate the protective effect of compounds and compositions thereof according to the invention in the presence of oxidative stress. Analysis of mitochondrial metabolism shows that brain damage induced by H2O2 reduces cell viability compared with the untreated control (p<0.05). All test substances are able to maintain and increase normal cellular physiological conditions. This effect is amplified for the combinations according to the present invention, stimulating mitochondrial well-being (p<0.05). These data demonstrate that the compositions according to the present invention are able to activate survival mechanisms more effectively than the components taken individually (p<0.05 vs individual substances). Among the analyzed compositions, the Mix4 composition according to the invention increases the antioxidant effect the most (p<0.05). Since the main theory behind brain aging concerns the oxidative condition, further experiments on ROS production were conducted. Individual substances and combinations were able to maintain ROS production below the physiological level (p < 0.05 vs control), supporting the hypothesis of their safety during use (Figure 6). In contrast, exposure of astrocytes to H2O2 significantly increased intracellular production compared with the control (p < 0.05). The data obtained from these analyses demonstrate that ROS production is inhibited by the test substances (p<0.05), which, especially when combined, are able to exert a significantly greater protective effect than when the actives are administered individually (p < 0.05), positively influencing the antioxidant capacities of neuronal cells by attempting the restoration of homeostasis, suggesting that the combined actives exert a neuroprotective effect.

In addition, NO production was observed by means of the Griess assay. As shown in Figure 7, treatment with H2O2 increased NO production compared with the control (p < 0.05), supporting the hypothesis of cell loss previously observed through the viability and ROS production assay. The harmful action of H2O2 was counteracted by stimulation with the single substances and combinations (p < 0.05). In particular, the beneficial effect of combinations was significantly greater in reducing NO production (p < 0.05) than treatment with single substances. The analysis on NO production confirms its central role in neurodegenerative processes.

Figure 8 shows the results of tests designed to assess inflammatory markers during oxidative stress. It is reported that the oxidative stress condition correlates with an increase in IL-2 and TNFa. The analysis of inflammatory processes confirms the data obtained previously: the compositions examined decrease inflammatory markers, decreasing the inflammation that is generated following the induction of oxidative damage. In particular, all the compositions examined were seen to decrease the production of IL-2 and TNFa compared to cells treated with H2O2 (p<0.05). Furthermore, the compositions according to the present invention were found to be more effective and provided with a synergistic effect, causing a decrease in the inflammatory picture related to oxidative stress compared to the control. Again, the Mix4 composition according to the invention is confirmed to have a greater effect than that attributable to the sum of the effects of the individual active ingredients composing it (p<0.05).

Figure 9 shows the results of tests aimed at assessing the production of BDNF (brain-derived neurotrophic factor), a neurotrophin required for the survival of neurons. Its presence was assessed following damage induced by H2O2 and stimulation with individual components and combinations thereof according to the compositions of the present invention. These data were also compared with respect to treatment with exogenous BDNF, which was employed as a positive control to assess whether the substances/combinations were able to act on the endogenous mechanism of neurotrophin production.

Again, all substances considered were able to stimulate BDNF production compared with the untreated control (X- axis) and compared with damage induced with H2O2 (p < 0.05). With regard to the mixtures, in all cases considered, there is better stimulation of BDNF than with the individual compounds (p < 0.05). In particular, the Mix 4 combination results in a significant increase in BDNF production compared with the other mixtures (p < 0.05).

Figure 10 shows the results of tests designed to assess the activation of intracellular mechanisms triggered in the presence of oxidative stress. Loss of mitochondrial potential under conditions of oxidative stress triggers a cascade of events that activate apoptosis. In this context, p53 activity, as a key factor involved in aging, oxidative stress and neurodegeneration, and cytochrome C activity, as a key regulator of cellular energy metabolism and apoptosis, were studied. The results obtained show an increase in p53 activity following induction of oxidative stress. There is a reduction in p53 activity after stimulation with the compounds taken individually and in combination in compositions according to the present invention (p < 0.05 compared with control). Regarding cytochrome C activity, cells treated with H2O2 showed an increase in its activity (p < 0.05) compared with the control. Stimulation with compounds (I), (ii), (ill) and (iv) reduced cytochrome C activity back to basal levels. Treatment with the compositions of the invention results in a statistically significant reduction in cytochrome C activity compared to the individual actives (p<0.05).

Figure 11 shows the results of tests designed to assess the activation of intracellular mechanisms activated in the presence of oxidative stress. SIRT1 (an enzyme that deacetylates proteins that contribute to cellular regulation in response to stressors and in longevity) controls energy metabolism through gluconeogenic/glycolytic pathways via the PGC-1a (peroxisome proliferator gamma coactivator 1)/NRF2 (nuclear transcription factor erythroid-2) pathway, leading to increased mitochondrial function. Low levels of SIRT1 in some brain regions are correlated with a slowdown in mitochondrial activity. The transcriptional co-activator NRF2, originally described as a metabolic regulator in peripheral tissues, plays a key role in transcriptional dysregulation and mitochondrial dysfunction in the brain when it decreases its production, negatively affecting BDNF (brain neurotrophic factor) production. As can be seen from Figure 11 , the stimulus given by hydrogen peroxide decreases the activity of both SIRT 1 and NRF2. Treatment with the single actives is able to increase the activity of these two markers. Treatment with the mixtures is also found to statistically significantly increase the activity of SIRT1 and Nrf2 compared with the single actives (p<0.05). In particular, among the mixtures, the most active appears to be Mix 4.

In addition, APP and pTAU activity were also analyzed to investigate the mechanisms activated in the neurodegeneration process (Figure 12). With regard to APP, the previously observed data about the beneficial effect on brain trophism are confirmed; in fact, combinations of the substances under investigation amplify the beneficial effect compared to single substances (p<0.05), but only Mix 1 , Mix 2 and Mix 4 induce a statistically significant effect regarding this parameter (p<0.05). Similarly, data obtained from the analysis of pTAU support the hypothesis of prevention of cognitive decline, and again, combinations were able to induce significant improvement with respect to F Ch-induced damage (p<0.05).

To study the potential action of the test substances, alone and in combination, in preventing cell damage under conditions of cellular accumulation, mitochondrial metabolism, iron accumulation, and lipid peroxidation were assessed by pretreating neuronal cells with 300 pM Fe³⁺.

Figure 13 shows the results of tests designed to evaluate the protective effect of compounds and combinations thereof in compositions according to the invention in the presence of iron accumulation. As can be seen in Figure 13, exposure to Fe³⁺ significantly reduced mitochondrial metabolism compared with the control (p<0.05); in contrast, all substances under investigation are able to maintain and increase normal cellular physiological conditions. In particular, this effect is amplified when the substances are combined in that they stimulate mitochondrial well-being more than the control (p<0.05) and compared with Fe³⁺ (p<0.05). These data demonstrate that the compositions according to the invention exert a noncytotoxic, neuroprotective effect.

Figures 14 A and B show the results of tests designed to assess neurodegenerative damage caused by iron accumulation. Iron can be accumulated progressively in the brain during normal aging, and its accumulation is an important cause of brain damage. In particular, in neurodegenerative disorders, it may be stored abnormally, altering its transport mechanisms. An analysis was conducted to assess the amount of intracellular iron, and it was observed that all the compositions of the present invention are able to decrease its levels significantly (p<0.05) compared with the control. In particular, it is evident how the synergistic action of the individual agents in the compositions according to the invention is able to further improve this condition, especially with regard to the efficacy of Mix 4.

To confirm these data, a cytochemical investigation was carried out by exploiting the Peris staining method (which detects ferric iron Fe³⁺) and Turnbull staining (which detects ferrous iron Fe²⁺) (Figure 14B).

Astrocytes possess two distinct systems that exploit iron, one linked to the DMT1 receptor that internalizes Fe²⁺ and one linked to transferrin that involves Fe³⁺. Normally these systems are aimed at the balancing of the oxidized state to Fe²⁺ by the astrocyte, also converting the internalized Fe³⁺. If this mechanism is dysregulated, Fe³⁺ remains accumulated within the membrane of astrocytes, causing cognitive decline. In this context, as shown in Figure 14A, it is important to note that single actives are able to decrease iron accumulation only marginally. In contrast, treatment with the combined actives was able to modulate this accumulation. In fact, the number of positive cells decreased significantly compared with treatment with the individual agents alone (Figure 14B).

Figure 15 shows the results of tests designed to assess lipid peroxidation as measured by MDA levels in the presence of iron accumulation. Exposure to Fe³⁺ increased MDA levels significantly compared to the control (p<0.05). Treatment with test substances (I), (II), (ill) and (iv) was found to significantly reduce MDA levels (p<0.05), and all combinations thereof were found to significantly increase the neuroprotective effect (p<0.05), suggesting an active role of these new formulations in counteracting lipid peroxidation. These results confirm the ability of the combinations to counteract the oxidative condition caused by iron accumulation and indicate the ability of the combinations to counteract iron-dependent damage by preventing iron accumulation. In particular, the Mix 4 composition better supports protection.

Since iron accumulation is considered an important factor in the development of an inflammatory condition, cytokines required to activate the innate immune response, specifically IL-1 p and TNF-o, were analyzed. In Figure 16 A, it can be seen that Fe³⁺ increases IL-1 p production compared to the control (p<0.05); in contrast, treatment with the substances, alone and in combination, was able to decrease its levels. Specifically, although all substances were able to reduce IL-1 p levels compared with Fe³⁺ (p<0.05), only Green Tea (compound (I) according to the invention) reduced IL-1 p levels compared with the control (p<0.05). The same result was obtained for TNF-o analysis (Figure 16 B). As for the mixtures, all combinations were able to reduce values compared with the control treated with Fe³⁺ (p<0.05), but only Mix 1 , 2, and 4 were able to decrease inflammation levels (expressed as IL- i and TNF-a) to a greater and statistically significant extent compared with the control and treatment with the individual actives (p<0.05).

Figure 17 A shows the test results for the evaluation of BDNF (brain-derived neurotrophic factor) in the presence of damage generated by iron accumulation. All compounds considered showed the ability to induce BDNF production compared with the untreated control (X-axis) and especially compared with damage induced by Fe³⁺ accumulation (p < 0.05).

Recovery from damage was amplified by using the substances in combination, confirming the positive role of the mixtures in counteracting brain damage (p<0.05). Specifically, Mix 1 and Mix 4 were the most effective. These data suggest that combinations are able to enhance BDNF production during damage induction.

To examine the involvement of apoptotic pathways, the activity of p53 and cytochrome C was analyzed. As can be seen from the data shown in Figure 17 (panels B and C) pretreatment with Fe³⁺ induces an increase in the activity of both markers (p<0.05) compared with the control (X-axis). In contrast, following treatment with the test substances, alone and in combination, a reduction in p53 and cytochrome C activity was observed, which was amplified by the combinations (p<0.05).

In particular, Mix 1 and Mix 4 had the most appreciable effects, with a statistically significant reduction in P53 and cytochrome C activity compared with Fe³⁺-treated control cells and the individual actives (p<0.05).

Finally, the activation of intracellular mechanisms involved in the presence of iron accumulation was analyzed. Specifically, the activities of SIRT1, known to play a role in neuroprotection against the progression of neurodegeneration, NRF-2, a protein implicated in neurogenesis, APP, a precursor of p-amy I old protein, and pTAU, an important marker of aging and neurodegeneration during oxidative stress and age-related disorders, were analyzed.

As shown in Figure 18, panels A and B, pretreatment with Fe³⁺ induced a decrease in the activity of SIRT1 and NRF-2 (p<0.05) compared with the control (X-axis); in both cases, the test substances alone and in combination enabled recovery from Fe³⁺-induced damage (p<0.05). Mix 1 and 4 had a greater beneficial effect in inducing an increase in SIRT1 and NRF2 activity (p<0.05).

With regard to APP, Figure 18 panel C shows how combinations of the test substances amplify the effect of the individual actives: APP activity decreases statistically significantly (p<0.05) with respect to both iron-treated cells and individual actives for all mixtures considered.

Finally, the data from the analysis of pTAU (Figure 18 D) support the hypothesis of prevention of cognitive decline in that the substances under consideration, alone and in combination, were able to induce significant improvement over Fe³⁺-induced damage (p < 0.05). In particular, the synergistic effect of the substances is also visible here as the combinations considered further reduced pTAU activity compared to Fe³⁺ (p < 0.05).

Thus, it is possible to infer that the combinations obtained by combining the substances under consideration are able to amplify the beneficial effect, confirming a key role of these formulations in counteracting neurodegenerative processes.

The data obtained, as illustrated in the attached Figures and commented on above, showed that: saffron - compound (ii), green tea - compound (I), citicoline - compound (iv), currently commercially available, for example, as Cognizin®, and resveratrol - compound (ill), are used as novel nutraceuticals against cognitive decline. These substances are able to cross the BBB.

When combined in compositions according to the present invention, their synergistic effect amplifies their activity. The compositions of the invention are able to maintain proper mitochondrial metabolism and are able to modulate antioxidant and anti-inflammatory effects that, during cognitive decline, are impaired.

The compositions according to the invention inhibit key molecular pathways involved during cognitive decline, maintaining proper homeostasis of neuronal cells.

The compositions of the invention can be freely used, having shown no inherent toxicity. The compositions according to the invention have shown different synergies of efficacy.

Associations/combinations (i), (ii) and (iii) - Mix 4 and (i), (ii), (iii) and (iv) - Mix 3 with drugs for the treatment of dementia and cognitive decline

An experimental protocol was also developed in order to evaluate the efficacy of combining the compositions according to the invention, in particular the composition of components (I), (ii) and (iii) - Mix 4 and the composition of components (I), (ii), (iii) and (iv) - Mix 3, and at least one known drug used in the treatment of dementia and cognitive decline, in particular at least one drug selected from cholinesterase inhibitors, such as donepezil, and/or at least one drug selected from NMDA receptor antagonists, such as memantine. The doses and amounts of the drugs used are in line with current medical practices and medical protocols. According to this protocol, in PHASE 1 absorption and bioavailability were evaluated in a validated intestinal model and the product of digestion was collected and administered to the brain part of the subsequent phase. In PHASE 2 the creation of the brain-gut axis was achieved by forming a cell monolayer in the basolateral compartment of the Transwell® system using CCF-STTG1 astrocytic-like cells to evaluate the effects at the cerebral level by pretreating the astrocytic-like cells for 30' with H2O2 200piM to recreate a condition of neurodegeneration. The following were then analyzed:

• mitochondrial metabolism through an assessment of cell viability

• the level of oxidative stress by means of an assay for the quantification of ROS production

• the quantification of homocysteic acid production

• main markers involved in neurodegeneration (APP, pTAU, NRF2 and SIRT1)

The combinations of the Mix 3 or Mix 4 compositions with donepezil or memantine showed a significant enhancement of the effect of the drugs donepezil or memantine taken individually.

The data deriving from Figures 19 and 20 confirm the ability of the Mix 3 and Mix 4 compositions to counteract the damage of neurodegeneration by preventing cell loss, as observed through cell viability. At the same time, the action of the drugs donepezil and memantine is not altered by the presence of the Mix 3 and Mix 4 compositions, demonstrating that the aforesaid drugs donepezil and memantine can be advantageously administered in association or combination with the Mix 3 and Mix 4 compositions.

ROS production is inhibited by the substances under examination (p<0.05), which, if combined, are capable of having a protective effect by acting on the antioxidant capacities of neuronal cells by attempting the restoration of homeostasis.

In particular, the Mix 4 combination shows a statistically greater protective power in terms of both cell viability and the production of oxidative stress if administered in combination with the drugs donepezil or memantine, as compared with the administration of the drugs alone.

It has been reported that high levels of homocysteic acid HCA may induce memory disorders in mice with AD. As may be noted from Figure 21 , it is possible to observe a reduction in HCA production, demonstrating the potential activity of the examined substances and compositions in protecting the brain against cognitive disorders.

The Mix 3 and Mix 4 compositions both show, advantageously, a statistically greater power in reducing the levels of homocysteic acid if administered in association with the drugs donepezil and memantine, as compared with the administration of the pharmacological treatment with donepezil and memantine alone.

The expression of pTau, a marker of cognitive decline, and of SIRT1, for mitochondrial function, was analyzed. The data which emerged, shown in Figure 22, reveal that the Mix 3 and Mix 4 compositions amplify the effect of the drugs donepezil and memantine. In particular, as regards the Mix 3 composition, the administration thereof in association with memantine shows a statistically higher efficacy on the activity of these two markers. With regard to the Mix 4 composition, by contrast, its association or combination with both drugs, donepezil and memantine, proves, advantageously, to be statistically more effective than the pharmacological treatment alone.

In order to further assess the action of the Mix 3 and Mix 4 compositions on mitochondrial function, the activity of NRF2 was also investigated. In this case, both compositions, Mix 3 and Mix 4, advantageously demonstrated to be statistically more effective if administered in association or combination with the drugs donepezil and memantine, as compared with the pharmacological treatment alone. As regards the expression of APP, p-amyloid precursor, only the combination of memantine + Mix 4 shows a statistically higher efficacy in reducing APP levels compared to the treatment with memantine alone.

Claims

1. An association or combination comprising or, alternatively, consisting of:

- a composition comprising or, alternatively, consisting of:

(II) an extract of Crocus sativus (L.) and/or tryptophan.

(ill) a trans-resveratrol, or resveratrol, preferably from Polygonum cuspidatum or Vitis vinifera.

- at least one cholinesterase inhibitor drug, and/or at least one NMDA receptor antagonist drug.

2. The association or combination according to claim 1 , wherein said composition may in addition comprise: (iv) citicoline or other sources of choline.

3. The association or combination as per claim 1 or 2, wherein said cholinesterase inhibitor drug is donepezil.

4. The association or combination as per claim 1 or 2, wherein said NMDA receptor antagonist drug is memantine.

5. The association or combination according to any one of claims 1 -4, wherein said combination is for use as a medicament.

6. The association or combination according to claim 5, wherein said composition is for use in the prevention and/or treatment of neurodegenerative diseases.

7. The association or combination according to claim 6, wherein said neurodegenerative diseases are selected from cognitive decline and/or dementia.

8. A formulation comprising the association or the combination according to any one of claims 1-7 and, optionally, additives or excipients or carriers of acceptable food or pharmaceutical grade.

9. The formulation according to claim 8, wherein said formulation is for use as a medicament; preferably, said formulation is for use in the prevention and/or treatment of neurodegenerative diseases.

10. The formulation according to claim 9, wherein said neurodegenerative diseases are selected from cognitive decline and/or dementia.