Food supplement for use as a cojuvant in the prevention and treatment of cognitive decline associated with Alzheimer's disease.

Abstract

2. A combination comprising a dry extract of Bacopa monnieri, astaxanthin powder, vitamin E, phosphatidylserine and Ashwagandha (Withania somnifera), optionally in combination with an aqueous extract of Sage (Salvia officinalis) and an oily extract of Spanish Sage (Salvia lavandulifolia), for use as a co-adjuvant in the prevention of Alzheimer's disease, which may reduce some of the major modifiable risk factors for cognitive decline associated with Alzheimer's disease, thereby improving memory and cognitive function.

Description

[Technical Field]

[0001] The present invention relates to an oral formulation, particularly an oral formulation for use as a cojuvant to improve memory and cognitive function by reducing major modifiable risk factors for cognitive decline associated with Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease is the most common form of degenerative dementia, which can lead to a gradual and progressive decline in cognitive function (such as memory, thinking, and learning ability). This form of degenerative dementia mainly begins in middle age, but is particularly rare in those under 65 years of age. [1] A decline in cognitive and mental function is an impairment of intellectual abilities that interferes with daily activities. [2] Cognitive decline can be associated with physiological factors (aging) as well as neurodegenerative diseases such as Alzheimer's disease. [3] The World Health Organization's (WHO) 2018 annual report reveals alarming projections of a rise in dementia. According to the report, the 35.6 million cases recorded in 2010 are projected to double by 2030 and triple by 2050, assuming 7.7 million new cases per year, with an estimated annual economic impact of approximately $604 billion on healthcare systems. In Italy, there are over one million people with dementia, approximately 60% of whom have Alzheimer's disease, and an estimated three million people are directly or indirectly involved in the care of such patients. [4] According to a model developed by Dr. Barry Reisberg, clinical director of the Dementia Research Center at New York University School of Medicine, the progression of Alzheimer's disease can generally be divided into seven stages.

[0003] Stage 1: No impairment (normal cognitive function). Alzheimer's patients in Stage 1 show no problems related to memory loss. There is no significant evidence of the manifestation of symptoms attributable to the aforementioned forms of dementia.

[0004] Stage 2: Very mild cognitive decline (early signs of Alzheimer's disease). Patients with Alzheimer's disease in Stage 2 may experience a sense of memory loss. This can be detected by forgetting common words or the location of everyday items. As with Stage 1, no symptoms of dementia can be diagnosed.

[0005] Stage 3: Mild cognitive decline (early Alzheimer's disease). Some individuals may show clear signs of cognitive decline. A decline in memory or concentration can be detected through accurate health checkups.

[0006] Stage 4: Moderate cognitive decline (mild or early-stage Alzheimer's disease). The following symptoms can be detected through accurate health checkups. - Forget recent events; - The ability to perform demanding arithmetic calculations is impaired; - There is significant difficulty in carrying out daily logistical tasks (such as managing personal finances and planning daily activities); - Forget your own career; - Mood and self-control become unstable in socially or mentally challenging situations.

[0007] Stage 5: Moderately severe cognitive decline (moderate or intermediate-stage Alzheimer's disease). Memory and thinking impairments become apparent. Patients with Stage 5 Alzheimer's disease begin to require assistance with daily activities.

[0008] Stage 6: Severe cognitive decline (moderately severe or moderate-stage Alzheimer's disease). Memory gradually deteriorates. Personality changes may also occur. Patients with Stage 6 Alzheimer's disease require considerable assistance to perform daily activities.

[0009] Stage 7: Very severe cognitive decline (severe or advanced Alzheimer's disease). Patients with Stage 7 Alzheimer's disease are unable to respond to daily activities or maintain conversations. In some cases, motor control and motor function are lost. Therefore, such patients require assistance in performing daily activities. They may lose the ability to laugh, sit without support, or lift their heads. Reflexes become abnormal, muscles become rigid, and swallowing ability is impaired. [3] Among the risk factors associated with cognitive impairment, neuroinflammation and oxidative stress are undoubtedly among the most well-studied from a preventative perspective.

[0010] A 2021 study published in the journal Nature Reviews Neurology, titled "Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here?", sheds light on the major role of neuroinflammation in the pathogenesis of Alzheimer's disease. While understanding the mechanisms behind neuroinflammation is relatively complex and controversial, it is known to constitute various inflammatory events in the pathological central nervous system (CNS) and to be associated with the activation of microglia, a major active immune defense in the CNS. Activated microglia play a role in promoting and supporting the neuroinflammatory state by releasing cytokines (including TNF-α, "tumor necrosis factor-alpha," a major marker of inflammatory cytokines produced by various immune cells), reactive oxygen species intermediates, proteases, complement factors, and chemokines. Furthermore, the activation process of microglia is a complex phenomenon characterized by the acquisition of various functional phenotypes related to neurotoxic and neuroprotective functions. This study demonstrates the correlation between activated microglia and their impact on the progression of Alzheimer's disease, depending on disease stage, individual susceptibility, and the activation state of the microglia themselves.

[0011] Activated microglia are the main source of TNF-α in the CNS. The latter (TNF-α) is involved in the activation of various biological processes including apoptosis, differentiation, proliferation and survival. TNF-α plays an important role in brain development under basal conditions; however, particularly under pathological conditions, increased levels of this cytokine excessively activate microglia and cause nerve damage (demyelination and / or neurodegeneration). Excessively activated microglia trigger the release of cytotoxic molecules including TNF-α produced through a positive autocrine activation feedback mechanism. [5]

[0012] Although the basic mechanism by which TNF-α activates microglia has been clarified, specific target molecular mediators that regulate microglial hyperactivation and TNF-α-mediated neuroinflammation have not yet been identified. [6]

[0013] Furthermore, neuroinflammation alters the correct expression of the neurotrophic factor BDNF, and increases the risk of neuronal suffering and death. Indeed, neurotrophic factors belonging to the nerve growth factor (NGF) family, including BDNF, are potent stimulators of neuronal survival under pathological conditions. A study published in the Journal of Neuroscience has shown that through intravitreal administration of BDNF, the response of damaged neurons in the lateral geniculate nucleus (LGN) can be maintained. [7]

[0014] Free radicals (ROS) also play a critical role in the onset and progression of neurodegenerative diseases such as Alzheimer's disease. Indeed, considering that the brain is mostly composed of oxidation-prone lipids; moreover, one of the best-known free radicals is exactly the one containing oxygen (ROS is the abbreviation for reactive oxygen species), and the brain consumes a large amount of oxygen, the risk of free radical generation in this organ is high. [8]

[0015] In addition to the factors mentioned above, cognitive impairment and Alzheimer's disease are also associated with the levels (concentrations) of certain neurotransmitters, such as acetylcholine and dopamine.

[0016] Acetylcholine is responsible for neurotransmission at both the central nervous system (CNS) and peripheral nervous system levels in humans, and therefore plays an essential role in the processes of learning and memory. [9]

[0017] As early as 1999, some scholars had already identified a correlation between cholinergic deficiency and Alzheimer's disease. In fact, Alzheimer's disease is associated with particularly low levels of acetylcholine in the hippocampus and neocortex due to the degeneration of cholinergic neurons.

[10]

[0018] As a result, acetylcholinesterase inhibitors have become one of the most commonly used medications for Alzheimer's disease patients today. These drugs work by inhibiting the enzyme cholinesterase, thereby increasing acetylcholine concentration at the synaptic level and ensuring the continuity of nerve impulses.

[0019] On the other hand, a 2019 meta-analysis revealed that dopamine homeostasis also appears to play a major role in Alzheimer's disease. According to this analysis, a decrease in dopamine neurotransmitter concentration caused by a deficiency of cortical dopamine correlates with the pathophysiology of Alzheimer's disease.

[11] [Overview of the project]

[0020] The applicant has now found that a combination consisting of a dry extract of *Bacopa monnieri*, astaxanthin, vitamin E, phosphatidylserine and *Withania somnifera*, and optionally a combination of an aqueous extract of *Salvia officinalis* and an oily extract of *Salvia lavandulifolia*, can reduce major modifiable risk factors for cognitive decline associated with Alzheimer's disease, and thus can be effectively used for the prevention of Alzheimer's disease. The embodiments of the present invention also include the following: Embodiment 1: A combination for use as an adjuvant to reduce some of the major cognitive decline risk factors associated with Alzheimer's disease, comprising a combination of dried extract of Bacopa monnieri, astaxanthin powder, vitamin E, phosphatidylserine, ashwagandha (Withania somnifera), and optionally an aqueous extract of sage (Salvia officinalis) and an oily extract of Spanish sage (Salvia lavandulifolia). Embodiment 2: The combination for use according to Embodiment 1, wherein the major risk factors are neuroinflammation resulting from oxidative stress, changes in neurotrophic factor expression, and changes in acetylcholine and dopamine-mediated neurotransmission. Embodiment 3 The combination for use according to Embodiment 1 or 2, wherein the combination is contained in an oral formulation in combination with a suitable excipient and / or diluent. Embodiment 4: The combination for use according to Embodiment 3, wherein the oral preparation contains 100 to 300 mg, preferably 150 mg, of Bacopa monnieri, and the minimum bacoside potency is 10%, preferably 20%. Embodiment 5: The combination for use according to Embodiment 3 or 4, wherein the oral formulation contains 1 mg to 2 mg, preferably 1 mg, of astaxanthin. Embodiment 6: The combination for use according to any one of Embodiments 3 to 5, wherein the oral preparation contains vitamin E in an amount of 10 mg to 60 mg, preferably 15 mg, of vitamin E acetate. Embodiment 7: The combination for use according to any one of Embodiments 3 to 6, wherein the oral formulation contains 10 mg to 20 mg, preferably 15 mg, of phosphatidylserine. Embodiment 8: The combination for use according to any one of Embodiments 3 to 7, wherein the oral formulation contains 100 to 300 mg, preferably 200 mg, of ashwagandha (Withania somnifera). Embodiment 9 The combination for use according to any one of Embodiments 1 to 7, wherein the oral preparation optionally contains the combination of a total amount of 200 to 400 mg, preferably 300 mg, of aqueous extract of sage (Salvia officinalis) and oily extract of Spanish sage (Salvia lavandulifolia). Embodiment 10 The combination for use according to any one of Embodiments 3 to 9, wherein the oral formulation is in the form of a single-dose sachet that can be dispersed in water, and is preferably administered twice a day. Embodiment 11: The combination for use according to any one of Embodiments 3 to 10, wherein the oral preparation is a nutritional supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1] Figure 1 (expression assay of PGE2 and 8-iso-PGF2α) shows a histogram comparing the levels (pg / mg) of PGE2 and 8-iso-PGF2α in cortical tissue treated with three different preparations: 60 mM solution of K+; 60 mM solution of K+ + Illumina® preparation; 60 mM solution of K+ + preparation according to the present invention. [Figure 2] Figure 2 (TNF-α level assay) shows a histogram comparing the gene expression (R.Q. value) of TNF-α in cortical tissue treated with three different preparations: 60 mM solution of K+; 60 mM solution of K+ + Illumina® preparation; 60 mM solution of K+ + preparation according to the present invention. [Figure 3]Figure 3 (BDNF level test) shows a histogram comparing the gene expression (RQ value) of BDNF in cortical tissue treated with three different formulations: a 60 mM K+ solution; a 60 mM K+ solution plus Illumina® formulation; and a 60 mM K+ solution plus the formulation according to the present invention. [Figure 4] Figure 4 (Dopamine Protection Test) shows a histogram comparing DOPAC levels in cortical tissue treated with four different formulations: 60 mM K+ solution; 60 mM K+ solution + half volume of Illumina® formulation; 60 mM K+ solution + Illumina® formulation; and 60 mM K+ solution + formulation according to the present invention. [Modes for carrying out the invention]

[0022] In this invention, the definition of “includes” does not exclude the possibility of additional elements existing in addition to those explicitly listed after the definition. In contrast, the definition of “consists of” excludes the possibility of additional elements existing in addition to those explicitly listed after the definition.

[0023] According to a preferred solution, the combination for use that is the subject of the present invention consists of the aforementioned five active ingredients: Bacopa monnieri, astaxanthin, vitamin E, phosphatidylserine, and Withania somnifera, and optionally an aqueous extract of sage (Salvia officinalis) and an oily extract of Spanish sage (Salvia lavandulifolia).

[0024] The combination for use according to the present invention is preferably contained in an oral formulation as the sole active ingredient in combination with a suitable excipient and / or diluent. In a particularly preferred solution, the oral formulation comprises the combination as the sole active ingredient, and in the combination, The dried extract of Bacopa monnieri is contained in an amount of 100-300 mg, preferably 150 mg, with a minimum bacoside potency of 10%, preferably 20%; Astaxanthin is preferably contained in an amount of 1 mg to 2 mg, more preferably 1 mg; Vitamin E is preferably included in the combination as vitamin E acetate, preferably in an amount of 10 mg to 60 mg, preferably 15 mg; Phosphatidylserine is preferably contained in the above combination in an amount of 10 mg to 20 mg, more preferably 15 mg; Ashwagandha (Withania somnifera) is included in the above combination in an amount of 100 to 300 mg, preferably 200 mg; If present, a combination of an aqueous extract of sage (Salvia officinalis) and an oily extract of Spanish sage (Salvia lavandulifolia) is preferably contained in the combination in an amount of 200-400 mg, more preferably 300 mg.

[0025] Astaxanthin is preferably contained in a dry extract of the alga Haematococcus pluvialis Flotow, with a minimum astaxanthin titer of 2%, more preferably 5%.

[0026] More preferably, the oral formulation is in the form of a single-dose sachet that can be dispersed in water. Furthermore, according to a more preferred solution, the oral formulation, in the form of a water-dispersible single-dose sachet containing the combination for use according to the present invention as the sole active ingredient, is administered twice daily.

[0027] More preferably, the oral preparation is a food supplement (nutritional supplement). The composition of a food supplement in the form of a single-dose sachet containing the combination for use according to the present invention as the sole active ingredient is shown in Example 1 below for illustrative purposes, and Example 2 shows a preclinical study demonstrating the efficacy of the combination (whose composition is reported in Example 1) in reducing some of the major modifiable risk factors for cognitive decline associated with Alzheimer's disease. [Examples]

[0028] Example 1 - Formulation of a food supplement in the form of a single-dose sachet

[0029] [Table 1]

[0030] Example 2 - Preclinical Study The purpose of this study is to verify whether the mixture constituting the supplement, which is the objective of the present invention, has a reducing effect on some modifiable risk factors for cognitive decline associated with Alzheimer's disease.

[0031] The effectiveness of the supplement of this invention was evaluated as follows. - DPPH Test (for oxidative stress): A chemical test to evaluate the decay of diphenylpicrylhydrazyl (DPPH) radicals in the absence or presence of antioxidants. Such radicals are used to test the ability of substances to act as free radical scavengers. In solution, DPPH is purple, but when this radical reacts with hydrogen from scavengers, it produces the reduced form DPPH-H, which becomes yellowish-colorless.

[0032] - ABTS test (on oxidative stress): The antioxidant capacity of various biological matrices can be determined by the reaction between the sample being analyzed and radical cations. The latter (radical cation) is generated by oxidizing the diammonium salt of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) with potassium persulfate solution (K2S2O8). Radical cation ABTsy + It has a peak absorption at 734 nm, making it a stable coloring species. A hydrogen atom or electron is radicalized by ABTsy. + Antioxidant compounds that can be transferred to cause discoloration of the solution.

[0033] - Ex vivo studies: Apply experimental models using explanted cortical tissue to evaluate protective effects against induced nerve damage. Such models involve the selection of neurotoxic stimuli induced by β-amyloid peptide, iron(II) sulfate, or hydrogen peroxide, and the evaluation of treatment-related effects against such induced stress. The following items are evaluated in such studies: • Gene expression of neuroprotective factors such as BDNF; • Inflammatory response markers (PGE2 and 8-iso-PGF2 alpha-release doses); • TNF-alpha expression.

[0034] - Enzyme assay: In vitro evaluation of cholinesterase inhibitory effect - Dosage of dopamine metabolite DOPAC result The results obtained from the tests conducted on this formulation were compared with those obtained from a similar Illumina® formulation that does not contain the active ingredient ashwagandha. The results are shown in Tables 2 and 3, and Figures 1, 2, 3, and 4.

[0035] [Table 2]

[0036] Table 2 shows the superior efficacy of the present invention compared to the commercially available formulations Illumina® and ashwagandha in inhibiting both acetylcholinesterase activity and butyrylcholinesterase activity, which are two major enzymes involved in the acetylcholine degradation process.

[0037] [Table 3]

[0038] Table 3 shows the minimum mixture concentration required to inhibit DPPH and ABTS radicals by 50% (IC). 50 The present invention demonstrates higher efficacy of the target formulation compared to the commercially available formulation Illumina®, as evaluated based on numerical values.

[0039] Figure 1 (Expression test of PGE2 and 8-iso-PGF2 alpha): Cortical tissue is treated with a potassium-based solution (left histogram) used as an inflammatory stimulus to increase levels of PGE2 and 8-iso-PGF2 alpha. Treatment with the formulation of the present invention (right histogram, identified as "new") provides greater benefits than the commercially available formulation Illumina® (center histogram, identified as "old" in the figure).

[0040] Figure 2 (TNF-alpha level test): This histogram supports the fact that the target product of the present invention (indicated as "New Illumina") is more effective than the commercially available Illumina® composition (indicated as "Old Illumina") in reducing the expression of TNF-alpha, one of the inflammatory cytokines most involved in neuroinflammation.

[0041] Figure 3 (BDNF level test): This graph shows how treatment with the formulation of the present invention (right histogram identified as "PLUS") provides greater efficacy than the commercially available formulation Illumina® (center histogram indicated as "Old"), as can be seen from the analysis of gene expression of the neurotrophic factor BDNF, which is involved in the development of neurodegeneration and neuroinflammation.

[0042] Figure 4 (Dopamine Protection Test): Cortical tissue is treated with a potassium-based solution (left histogram) as an inflammatory stimulus to increase levels of dopamine metabolites, DOPACs (produced as a result of the breakdown of such neurotransmitters). The graph shows how the target formulation of the present invention (right histogram, identified as Illumina ALZ) further reduces DOPAC expression than the commercially available formulation Illumina® ("Illumina OLD" in the center histogram).

[0043] List of References 1.Brookmeyer R, Gray S, Kawas C. Projections of Alzheimer's disease in the United States and the public health impact of delaying disease onset. Am J Public Health. 1998 Sep;88(9):1337-42. 2. www.issalute.it / index.php / la-salute-dalla-a-alla-z-menu / d / declino-cognitivo-mentale 3. www.alz.org / it / stadi-del-morbo-di-alzheimer.asp 4. www.epicentro.iss.it / alzheimer / iniziative-mondo 5.Leng F, Edison P. Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here? Nat Rev Neurol. 2021 Mar;17(3):157-172.TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity. Cell Death & Disease volume 11, Article number: 415 (2020). 6.Lima Giacobbo B, Doorduin J, Klein HC, Dierckx RAJO, Bromberg E, de Vries EFJ. Brain-Derived Neurotrophic Factor in Brain Disorders: Focus on Neuroinflammation. Mol Neurobiol. 2019 May;56(5):3295-3312.J. Neurosci. 23:287-296, 2003. 7.Huang WJ, Zhang X, Chen WW. Role of oxidative stress in Alzheimer's disease. Biomed Rep. 2016 May;4(5):519-522.The cholinergic hypothesis of Alzheimer's disease:a review of progress. J Neurol Neurosurg Psychiatry1999;66:137-147. 8.Pan X, Kaminga AC, Wen SW, Wu X, Acheampong K, Liu A. Dopamine and Dopamine Receptors in Alzheimer's Disease: A Systematic Review and Network Meta-Analysis. Front Aging Neurosci. 2019;11:175. Published 2019 Jul 11.

Claims

1. An oral formulation for the prevention of Alzheimer's disease, The following active ingredients: Bacopa monnieri dry extract, astaxanthin powder, vitamin E, phosphatidylserine, and ashwagandha (Withania somnifera), where the amount of ashwagandha (Withania somnifera) is in the range of 100-300 mg. An oral preparation comprising an excipient and / or a diluent.

2. The oral formulation according to claim 1, wherein the prevention of Alzheimer's disease is achieved by reducing major risk factors, which are neuroinflammation caused by oxidative stress, changes in neurotrophic factor expression, and changes in acetylcholine and dopamine-mediated neurotransmission.

3. The oral preparation according to claim 1, wherein the oral preparation contains 100 to 300 mg of a dried extract of Bacopa monnieri and has a minimum bacoside potency of 10%.

4. The oral preparation according to claim 1, wherein the oral preparation contains 1 mg to 2 mg of astaxanthin powder.

5. The oral preparation according to claim 1, wherein the oral preparation contains vitamin E in an amount of 10 mg to 60 mg.

6. The oral preparation according to claim 1, wherein the oral preparation contains 10 mg to 20 mg of phosphatidylserine.

7. The oral formulation according to claim 1, in the form of a single-dose sachet that can be dispersed in water.

8. The oral formulation according to claim 1, which is a nutritional supplement.

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