Composition and methods for enhancing or promoting healthy metabolic aging

Abstract

To provide compositions for use in the treatment and / or prevention of disorders responsive to positive modulation of a ghrelin receptor, such as diabetes, obesity-related disorders, and most preferably for the treatment or prevention of age-related conditions or diseases, for example, by stimulating appetite, inhibiting insulin secretion and lowering insulin resistance, increasing growth hormone release, enhancing muscle vitality or fragility, and the like.SOLUTION: Provided is a composition, pharmaceutical composition, or nutraceutical or food composition, or dietary supplement comprising D-pinitol, D-chiro-inositol and / or myo-inositol, or any salt thereof, for use in preventing or slowing the onset of the clinical manifestations of mild cognitive impairment or in preventing or slowing the onset of the clinical manifestations of a tauopathy in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the delivery of compositions, preferably pharmaceutical compositions, comprising D-pinitol, D-chiro-inositol, or myo-inositol, or any pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disorder, disease, or condition responsive to stimulation of the ghrelin receptor in a subject in need thereof. [Background technology]

[0002] The aging process in humans is associated with physical decline and impaired metabolic homeostasis (1). Physical decline is associated with increased frailty and the development of sarcopenia, a decline in whole-body muscle mass and muscle cell performance, while dysregulation of metabolic networks leads to age-related increases in obesity, insulin resistance, diabetes, and hypertension (2). Aging impairs the activity of key metabolic signaling pathways, and the subsequent metabolic dysregulation accelerates aging. This contributes to the frailty of the elderly, associated with frailty, impaired metabolic homeostasis, and hypertension. Sexuality is increasing, limiting quality of life and indeed lifespan.

[0003] A major signaling system impaired in aging is insulin signaling. Impaired activity of the insulin signaling pathway with aging leads to insulin resistance (1). Subsequent hyperglycemia as a result of dysregulated glucose clearance promotes the formation of advanced glycation end products (AGEs), which cause tissue damage, further exacerbate metabolic dysregulation, and accelerate the aging process. The key hepato-muscular loop is profoundly affected by the development of obesity and insulin resistance, and the link between hepatic glucose production and glucose consumption by muscle is dysregulated as a result of impaired insulin signaling, leading to hepatic glucose overproduction and inhibiting its utilization by muscle, which is already affected by aging-related frailty (1).

[0004] Among the various metabolic signaling pathways controlling energy homeostasis and muscle vitality, ghrelin, a peptide hormone produced by specialized cells in the gastrointestinal tract, has emerged as a potential target for age-related metabolic dysregulation and frailty (3-5). Ghrelin can stimulate appetite, inhibit insulin secretion, increase growth hormone release, improve muscle vitality through increased net muscle mass, and improve cognition (6-9). Given that aging leads to anorexia, insulin hypersecretion, insulin resistance, muscle frailty, and cognitive impairment, the physiological effects of ghrelin are positioned to counteract all of these age-related pathologies. Therefore, because reduced ghrelin levels are positively associated with age-related hypertension (10), enhancing ghrelin release or administering ghrelin receptor agonists has been proposed as a strategy for combating these age-related pathologies, including hypertension (5). Summary of the Invention

[0005] The present invention relates to a composition, preferably a pharmaceutical composition, or a nutraceutical or food composition, comprising D-pinitol, D-chiro-inositol and / or myo-inositol, or any of their pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, for use in the treatment or prevention of a disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin, preferably plasma levels, that are lower than normal preprandial circulating levels of active ghrelin, in a subject in need thereof, wherein the disorder, disease or condition is characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin, and the disorder, disease or condition can be detected by standard conventional assays such as ELISA or radioimmunoassay. This can be determined by determining whether the patient is characterized by a change in the concentration of circulating active ghrelin as measured by the method.

[0006] In the context of the present invention, "normal pre-prandial baseline level" is understood as the pre-prandial baseline level in a healthy subject. Plasma levels of ghrelin are discussed in particular in European Endocrinology, 2015;11(2):90-5 DOI: 10.17925 / EE.2015.11.02.90.

[0007] In the context of the present invention, "active ghrelin" is understood as an acylated (usually n-octanoylated) form of ghrelin. Active ghrelin is generated upon post-translational esterification of a fatty acid (n-octanoic acid, or to a lesser extent, n-decanoic acid) on the serine residue at position 3 of secreted ghrelin. This acylation is necessary for ghrelin activity.

[0008] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating basal levels of active ghrelin is an age-related condition.

[0009] In the context of the present invention, "age-related pathology" is understood to mean any physiological change or pathological disorder that is most commonly associated with the progression of aging. Normal physiological changes associated with aging include a decrease in muscle mass and an increase in muscle frailty or mild cognitive impairment. Pathological disorders whose prevalence increases exponentially with age include atherosclerosis, cardiovascular disease, type 2 diabetes, osteoporosis, hypertension, Alzheimer's disease, arthritis, cataracts, and cancer.

[0010] Preferably, the disorder, disease or condition responsive to modulation of the ghrelin receptor is an age-related condition, and the composition is preferably used to improve muscle vitality and reduce frailty by increasing net muscle mass.

[0011] Preferably, the disorder, disease or condition responsive to modulation of the ghrelin receptor is an age-related condition, and the composition is preferably used to treat or prevent sarcopenia.

[0012] In the context of the present invention, "sarcopenia" is defined as the loss of skeletal muscle mass and strength as a result of aging.

[0013] Preferably, the disorder, disease or condition responsive to modulation of the ghrelin receptor is hypertension and the composition is used to treat or prevent hypertension, preferably age-related hypertension.

[0014] In the context of the present invention, "age-related hypertension" is understood as the increase in systolic blood pressure with age, which is caused by the age-related increase in total (and renal vascular) resistance as a result of the progressive loss of viscoelastic properties of the conduit vessels, the increase in atherosclerotic arterial disease, and the hypertrophy and stiffening of muscular arteries and arterioles.

[0015] Preferably, the disorder, disease, or condition characterized by impaired ghrelin secretion, resulting in circulating levels of active ghrelin lower than normal preprandial basal levels, is an age-related condition, and the composition is preferably used to inhibit insulin secretion and reduce insulin resistance to avoid the progression of insulin resistance syndrome, a condition characterized by a progressive increase in insulin levels and subsequent loss of cellular response to insulin, leading to endocrine pancreatic fatigue. Diseases that benefit from reducing excess insulin may include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, nonalcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease.

[0016] Preferably, the disorder, disease, or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial basal levels of active ghrelin is an age-related condition, and the composition is used to increase circulating growth hormone release to compensate for the physiological decrease in growth hormone release associated with aging, and is used in conditions where increased athletic performance, muscle mass, and bone density are required due to decreased growth hormone secretion, such as in human immunodeficiency virus (HIV) infection.

[0017] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin is an age-related condition, and the composition is preferably used for the treatment or prevention of cognitive impairment or for improving cognition.

[0018] In the context of the present invention, "cognitive impairment" is defined as a deficit in cognitive abilities (including learning, memory, perception, and problem-solving) that is acquired (as opposed to "cognitive decline" associated with normal developmental aging) and may be caused by a brain lesion. "Cognitive improvement" is understood as the amplification or broadening of core mental abilities through improvements in information processing systems. Cognitive improvement can be achieved by interventions that include the administration of compounds covered by this patent.

[0019] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin is selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease and Huntington's disease.

[0020] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin is selected from the group consisting of diabetes, preferably type 2 diabetes.

[0021] Preferably, the composition further comprises a pharmaceutically acceptable carrier.

[0022] The composition is preferably administered orally or intragastrically to a subject in need thereof.

[0023] Additionally, the present invention further relates to: 1. Non-therapeutic use of a composition comprising D-pinitol, D-chiro-inositol or myo-inositol, or any acceptable salt thereof, to stimulate appetite. Non-therapeutic use of a composition comprising D-pinitol, D-chiro-inositol or myo-inositol, or any acceptable salt thereof, to increase net muscle mass. Non-therapeutic use of a composition comprising D-pinitol, D-chiro-inositol or myo-inositol, or any acceptable salt thereof, to improve cognition. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the structures and relationships of D-pinitol ((1S,2S,4S,5R)-6-methoxycyclohexane-1,2,3,4,5-pentol), D-chiro-inositol (1R,2R,3S,4S,5S,6S)-cyclohexane-1,2,3,4,5,6-hexol, and D-myo-inositol ((1R,2S,3r,4R,5S,6s)-cyclohexane-1,2,3,4,5,6-hexol). Inositol is a polyalcohol with insulin-mimetic properties. D-pinitol, derived from natural sources (i.e., carob fruit), can be demethylated and converted to D-chiro-inositol in the acidic medium of the stomach. Additionally, other inositols found in food are isomers of D-chiro-inositol and can be converted to D-chiro-inositol by the enzymatic action of epimerases. [Figure 2] FIG. 1 shows that oral administration of pinitol (100 mg / kg) dissolved in sterile water to adult male Wistar rats a) results in an increase in circulating plasma ghrelin concentrations, which is associated with B) a decrease in insulin release into the circulation, C) a decrease in insulin resistance as measured by the HOMA index, and D) an inhibition of the expression of pyruvate kinase, a key enzyme in the conversion of phosphoenolpyruvate to glucose production. [Figure 3]FIG. 1 shows that oral administration of pinitol (500 mg / kg) to adult male Wistar rats A) results in increased circulating plasma ghrelin concentrations, which is associated with B) decreased insulin release into the circulation, C) maintained plasma glucose levels, D) decreased insulin resistance as measured by the HOMA index, E) increased glucagon secretion, and F) activation of hypothalamic mTOR signaling in the hypothalamus by phosphorylation. [Figure 4] FIG. 1 shows that oral administration of D-chiro-inositol (500 mg / kg) enhances ghrelin secretion as measured by monitoring circulating plasma ghrelin concentrations. [Figure 5] Figure 1 shows a proposed model of inositol's role in metabolic aging. Both D-pinitol and D-chiro-inositol promote ghrelin secretion, a metabolic profile characterized by reduced insulin demand from the endocrine pancreas, hepatic gluconeogenesis, increased muscle glucose utilization with muscle growth, and increased mTOR signaling in the hypothalamus, leading to increased appetite. The overall results of this unique pharmacological profile may include protection from pancreatic fatigue due to age-related insulin resistance and obesity, increased muscle vitality (preventing sarcopenia and frailty characteristic of the elderly), and better directing glucose disposal by the body. [Figure 6](A) Kinases and phosphatases analyzed by Western blotting in the present invention to assess activity and / or expression. (C) Schematic diagram of tau phosphorylation due to dysfunction of the Akt-GSK3 pathway or activation of additional kinases, such as protein kinase A (PKA) or cyclin-dependent kinase (CDK5). Under physiological conditions, Akt is activated by phosphorylation. In pathological conditions, such as brain insulin resistance, Akt activates the kinase GSK-3β, which phosphorylates tau. This can also occur with overactivation of other kinases (PKA, CDK5). When hyperphosphorylated, tau protein dissociates from microtubules and forms insoluble aggregates called neurofibrillary tangles (NFTs) in neurons and glial cells. Both microtubule destabilization and NFT aggregation lead to cell apoptosis. (B) Chemical structure of D-pinitol (DPIN). DPIN is the 3-O-methyl form of D-chiro-inositol (DCI), found as a cyclitol, a cyclic polyol. DPIN is a known antidiabetic agent isolated from the pulp of carob fruit (Ceratonia siliqua). Experimental design: Chronic oral administration of DPIN and DCI to 20 male Wistar rats was performed for 10 consecutive days. Plasma and brain samples were collected from both experimental groups. Abbreviations: DPIN, D-pinitol; DCI, D-chiro-inositol. [Figure 7]Figure 1 shows the effect of oral administration of DPIN or DCI for 10 days on tau dephosphorylation in the hippocampus of Wistar rats. A) Bar graphs represent the ratio between phosphorylated tau (AT8: Ser202, Thr205) and total tau, and the amount of total tau compared to α-adaptin for Wistar rats. Histograms represent the mean ± SEM (n = 8 Wistar rats). B) Western blot membranes after each antibody incubation (target proteins identified to the right of the bands). Molecular weights (MW) are indicated in kilodaltons (kD). Blots shown are from four out of eight independent samples from a group of Wistar rats. Corresponding α-adaptin expression is shown as a loading control for each lane. One-way ANOVA and Tukey's test were performed: (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 vs. vehicle group. [Figure 8] Figure 1 shows the effect of oral administration of DPIN or DCI for 10 days on the activity of the tau kinase cyclin-dependent kinase 5 (CDK5) in the hippocampus of Wistar rats. A) Bar graphs represent the ratios between the amounts of p25, p35, and total CDK5 tau kinase compared to α-adaptin for Wistar rats. Histograms represent the mean ± SEM (n = 8 Wistar rats). B) Western blot membranes after each antibody incubation (target proteins identified to the right of the bands). Molecular weights (MW) are indicated in kilodaltons (kD). Blots shown are from three out of eight independent samples from Wistar rat groups. Corresponding α-adaptin expression is shown as a loading control for each lane. An unpaired t-test was performed for the Wistar rat analysis. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 vs. vehicle group. [Figure 9]Figure 1 shows the effect of oral administration of DPIN or DCI for 10 days on the phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the hippocampus of Wistar rats. A) Bar graphs represent the ratio between GSK-3β phosphorylated (Ser9 / Tyr216) and total GSK-3β, and the amount of total GSK-3β compared to α-adaptin for Wistar rats. B) Western blot membranes after each antibody incubation (target proteins identified to the right of the bands). Molecular weights (MW) are indicated in kilodaltons (kD). Blots shown are from three out of eight independent samples from Wistar rat groups. Corresponding α-adaptin expression is shown as a loading control for each lane. An unpaired t-test was performed for the analysis of Wistar rats. (*) P<0.05, (**) P<0.01, and (***) P<0.001 vs. the vehicle group. [Figure 10] Figure 1 shows the effect of oral administration of DPIN or DCI for 10 days on other hippocampal tau kinases: mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), and protein kinase A (PKA) in the hippocampus of Wistar rats. A) Bar graphs represent the ratio between the amount of total tau kinase compared to α-adaptin for Wistar rats. Histograms represent the mean ± SEM (n = 8 Wistar rats). B) Western blot membranes after each antibody incubation (target proteins identified to the right of the bands). Molecular weights (MW) are indicated in kilodaltons (kD). Blots shown are from three out of eight independent samples from Wistar rat groups. Corresponding α-adaptin expression is shown as a loading control for each lane. An unpaired t-test was performed for the analysis of Wistar rats. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 vs. vehicle group. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a method for increasing ghrelin levels by oral administration of D-pinitol, a natural cyclic polyol derived from plants, including the pods of the carob tree, or related inositols, such as D-chiro-inositol or its epimer, myo-inositol (Figure 1). Other stereoisomers of inositol, such as cis-inositol, epi-inositol, alolo-inositol, muco-inositol, neo-inositol, L-chiro-inositol, scyllo-inositol, or any of their pharmaceutically acceptable salts, esters, tautomers, solvates, and hydrates, or any combination thereof, may also be used. As shown in the Examples contained herein, oral administration of D-pinitol (100 mg / kg) (Figure 2) or 500 mg / kg D-chiro-inositol (Figure 3) increased ghrelin secretion, reduced circulating insulin, and activated the phosphorylation of mTOR in the hypothalamus, a metabolic sensor required for appetite stimulation (Figure 3). The net effect was a reduction in insulin demand, reflected by a decrease in HOMA (insulin resistance index) (Figures 2 and 3), without leading to hypoglycemia or hyperglycemia. This may be due to a combination of the direct effects of D-pinitol and D-chiro-inositol on glucose uptake by muscle cells and net hepatic glucose production (gluconeogenesis) through the inhibition of pyruvate kinase, which converts glycolysis to glucose production in the liver (Figure 2). Furthermore, administration of D-chiro-inositol increased ghrelin secretion (Figure 4).

[0026] Additionally, as shown in Figure 5, the use of D-pinitol and D-chiro-inositol to stimulate ghrelin secretion coordinates insulin and glucagon secretion, reduces insulin demand, and redirects hepatic glucose production toward muscle utilization. Additionally, given the known effects of ghrelin, D-pinitol or related inositols, such as D-chiro-inositol or its epimer, myo-inositol, are predicted to increase muscle mass and energy and reduce muscle frailty. Because ghrelin levels decline with age (16), these compounds may be useful in promoting healthy metabolic aging.

[0027] Meanwhile, the present authors have surprisingly discovered a role for D-pinitol in preventing cognitive impairment in aging and tauopathy. In this sense, D-pinitol's insulin-sparing properties and its ability to enhance ghrelin secretion have cognitive-promoting effects that can be used as a preventive strategy for cognitive impairment associated with neurodegeneration. Brain insulin resistance is associated with cognitive impairment, particularly by altering hippocampal function. Meanwhile, ghrelin is a unique hormone that can promote cognition through its ability to cross the blood-brain barrier and its interaction with growth hormone secretagogue receptors. Therefore, the novel effects described in this invention may provide a rationale for the use of D-pinitol in aging or neurodegenerative disorders, particularly Alzheimer's disease, to alleviate cognitive impairment in the early stages of disease when symptoms can be diagnosed.

[0028] However, recent studies have emphasized the need to focus on the molecular target most associated with the development of mild cognitive impairment and major dementia: tau protein. Abnormal phosphorylation or acetylation of tau leads to tau protein deposition, resulting in the appearance of neurofibrillary tangles (NFTs), a histopathological biomarker of a group of diseases collectively known as tauopathies. NFT formation correlates more strongly with cognitive decline than the distribution of senile plaques formed by polymorphic β-amyloid (Ax) protein deposits, a pathological hallmark of Alzheimer's disease, one of the major dementias. Therefore, tau deposition is a crucial factor in the cognitive impairment observed in Alzheimer's disease (when β-amyloid deposition is insufficient to cause dementia, as well as in the progression to mild cognitive impairment and dementia observed in chronic traumatic encephalopathy).

[0029] Therefore, tauopathy is considered a key feature of neurodegeneration and normal brain aging, and its prevention before the onset of clinical symptoms is a clear medical need. Taking the above points into consideration, we tested the effect of D-pinitol administration on the phosphorylation state of tau, a complex process regulated by multiple proteins (Figure 6). Surprisingly, we found that oral administration of D-pinitol significantly reduced tau phosphorylation (Figure 7) through a mechanism dependent on the reduction of the activity of cyclin-dependent kinase 5, one of the major tau phosphorylation enzymes (Figure 8). The action of D-pinitol is specific, as D-pinitol did not affect other tau-regulating proteins (Figure 9), demonstrating the unique pharmacological profile of this natural inositol.

[0030] Based on the above findings, the present invention further proposes that administration of D-pinitol results in: a) Prevention or delay of the onset of mild cognitive impairment and subsequent progression to dementia, where the condition promotes tau hyperphosphorylation and the intent is to prevent or delay its clinical manifestation. These conditions include Pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathies including argyrophilic grain dementia, globular glial tauopathy, neurofibrillary tangle dementia, chronic traumatic encephalopathy (CTE), and age-related tauastrogliopathy. b) Primary age-related tauopathies, including Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain dementia, glioglobular tauopathy, and neurofibrillary tangle dementia, chronic exocytic leukemia, Treatment of diagnosed tauopathies, including chronic traumatic encephalopathy (CTE) and age-related tauastrogliopathy.

[0031] Thus, the present invention relates to inositols, such as D-pinitol, D-chiro-inositol, and its epimer, myo-inositol (hereinafter, "compounds of the present invention"), for increasing ghrelin levels. The compounds of the present invention are represented by the structural formula identified in Figure 1 or any pharmaceutically acceptable salts derived therefrom. Furthermore, the present invention further relates to compounds of the present invention for preventing or delaying the progression of clinical symptoms of a tauopathy or mild cognitive impairment in a subject, preferably a subject before the onset of such clinical symptoms, more preferably a human subject, by reducing tau phosphorylation.

[0032] In the present invention, the term "asymptomatic subject" is understood as a subject that does not show clinical symptoms of a disease, in particular a tauopathy.

[0033] In the present invention, the term "onset of clinical symptoms of a tauopathy" is understood as the appearance of cognitive, neuropsychological and / or neurological symptoms and signs, including objective diagnostic procedures (i.e., magnetic resonance imaging, positron emission tomography and cerebrospinal fluid biomarkers), that are diagnostic indicators of a tauopathy.

[0034] In the present invention, the term "onset of clinical symptoms of mild cognitive impairment (MCI)" is understood as a stage between the expected cognitive decline of normal aging and the more serious deterioration of dementia, which is characterized by problems with memory, language, thinking, or judgment.

[0035] In the present invention, the term "prevention" means avoiding the occurrence of a disease or pathological condition in an individual, particularly when the individual has a predisposition to the pathological condition but has not yet been diagnosed. In the present invention, the disease or pathological condition is preferably a "tauopathy".

[0036] In the present invention, the term "tauopathy" refers to a group of neurodegenerative diseases associated with the aggregation of tau protein into neurofibrillary tangles (NFTs) or gliofibrillary tangles in the human brain. The tangles are formed by hyperphosphorylation of the microtubule protein known as tau, causing the protein to dissociate from microtubules and form insoluble aggregates (these aggregates are also called paired helical filaments). Examples of tauopathies are selected from the group consisting of Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain dementia, globular glial tauopathy, primary age-related tauopathies including neurofibrillary tangle dementia, chronic traumatic encephalopathy (CTE), and age-related tauastrogliopathy.

[0037] As already indicated, the compounds of the present invention are effective in elevating ghrelin levels. Therefore, they are useful for the treatment and / or prevention of disorders characterized by impaired ghrelin secretion, resulting in circulating levels of active ghrelin lower than normal preprandial basal levels, such as age-related conditions. Normal physiological changes associated with aging include a loss of muscle mass and an increase in muscle frailty or mild cognitive impairment. Further pathological disorders whose prevalence increases exponentially with age include atherosclerosis, cardiovascular disease, type 2 diabetes, osteoporosis, hypertension, Alzheimer's disease, arthritis, cataracts, and cancer. Preferably, the disorder, disease, or condition characterized by impaired ghrelin secretion, resulting in circulating levels of active ghrelin lower than normal preprandial basal levels, is hypertension, preferably age-related hypertension.

[0038] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin lower than normal preprandial circulating levels of active ghrelin is an age-related condition, and the composition of the present invention preferably prevents the progressive rise in insulin levels and It is used to inhibit insulin secretion and reduce insulin resistance to avoid the progression of insulin resistance syndrome, a condition characterized by the progressive loss of cellular response to insulin, which subsequently leads to endocrine pancreatic fatigue.Diseases that benefit from the reduction of excess insulin may include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, non-alcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease.

[0039] Preferably, the disorder, disease, or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial basal levels of active ghrelin is an age-related condition, and the compositions of the present invention are used to increase circulating growth hormone release to compensate for the physiological decrease in growth hormone release associated with aging, and are used in conditions where increased athletic performance, muscle mass, and bone density are required due to decreased growth hormone secretion, such as in human immunodeficiency virus (HIV) infection.

[0040] Preferably, the disorder, disease or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin is an age-related condition, and the compositions of the present invention are preferably used for treating or preventing cognitive impairment or improving cognition.

[0041] Preferably, the disorder, disease, or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial basal levels of active ghrelin is selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, and Huntington's disease.

[0042] Thus, one aspect of the present invention provides a method of treating or preventing any of the above-mentioned diseases, disorders, or conditions in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of D-pinitol, D-chiro-inositol, and / or myo-inositol, or any pharmaceutically acceptable salt thereof. The present invention also provides a method of treating or preventing any of the above-mentioned diseases, disorders, or conditions in a subject in need thereof by administering D-pinitol, D-chiro-inositol, and / or myo-inositol, or any pharmaceutically acceptable salt thereof, in combination with a therapeutically or prophylactically effective amount of another agent known to be useful in treating or preventing any of the above-mentioned conditions or diseases. Another aspect of the present invention provides a pharmaceutical composition comprising a compound having the structural formula of D-pinitol, D-chiro-inositol, and / or myo-inositol and a pharmaceutically acceptable carrier.

[0043] Yet another aspect of the present invention relates to the use of D-pinitol, D-chiro-inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for the treatment, prevention, or suppression of diseases characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin in a subject in need thereof, in particular the treatment, prevention, or suppression of any of the diseases, disorders, or conditions mentioned above.

[0044] Yet another aspect of the present invention relates to the non-therapeutic use of an amount of D-pinitol, D-chiro-inositol and / or myo-inositol, or any salt thereof, in a subject in need thereof for stimulating appetite, inhibiting insulin secretion and reducing insulin resistance, increasing growth hormone release, improving muscle strength or weakness by increasing net muscle mass, improving cognition, and preventing age-related hypertension.

[0045] In addition, as already shown, the compounds of the present invention significantly reduce tau phosphorylation, thereby preventing the onset or progression of clinical symptoms of tauopathy in subjects, preferably those who have not yet developed such clinical symptoms. These compounds are useful for preventing or delaying the onset or progression of clinical symptoms of a tauopathy in a subject before or after its onset. Accordingly, a further aspect of the present invention provides a method for preventing or delaying the onset or progression of clinical symptoms of a tauopathy in a subject. More preferably, the present invention provides a method for preventing or delaying the onset of clinical symptoms of a tauopathy in a subject, i.e., a method for preventing or delaying the onset of such clinical symptoms in said subject, preferably a healthy subject. Furthermore, a further aspect of the present invention provides a method for preventing or delaying the onset or progression of clinical symptoms of mild cognitive impairment in a subject. More preferably, the present invention provides a method for preventing or delaying the onset of clinical symptoms of mild cognitive impairment in a subject, i.e., a method for preventing or delaying the onset of such clinical symptoms in said subject, preferably a healthy subject.

[0046] Yet another aspect of the present invention relates to the use of D-pinitol, D-chiro-inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for preventing or delaying the progression of clinical symptoms of a tauopathy in a subject, i.e., for preventing or delaying the onset of such clinical symptoms in said subject, preferably a healthy subject. Furthermore, a further aspect of the present invention relates to the use of D-pinitol, D-chiro-inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for preventing or delaying the progression of clinical symptoms of mild cognitive impairment in a subject. More preferably, the present invention relates to the use of D-pinitol, D-chiro-inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for preventing or delaying the onset of clinical symptoms of mild cognitive impairment in a subject, i.e., for preventing or delaying the onset of such clinical symptoms in said subject, preferably a healthy subject.

[0047] In addition to the above, the present invention also encompasses the possibility that the composition of the present invention is in the form of a dietary supplement or nutritional composition comprising D-pinitol, D-chiro-inositol and / or myo-inositol, or any salt thereof.In this sense, when the composition of the present invention is formulated as a nutritional composition, the nutritional composition may be a food product, or may be incorporated into food or food products intended for both human and animal consumption.Therefore, in certain embodiments, the nutritional composition is selected between a food product (which may be a food product for specific nutritional purposes or a medicinal food product) and a nutritional supplement.

[0048] In the present invention, the term "nutritional composition" refers to a food product that provides nutrients to a subject consuming it, thereby beneficially affecting one or more functions of the body, resulting in better health and wellness.

[0049] "dietary supplement," "nutritional supplement," "food supplement," or "dietary supplement" The term "supplement," which is synonymous with either the term "alimentary supplement" or "alimentary complement," refers to a product or preparation intended to supplement the normal diet, consisting of a source of concentrated nutrients or other substances that have a nutritional or physiological effect. In the present invention, the "substance" that has a nutritional or physiological effect on an individual when the nutritional complement is ingested is D-pinitol, D-chiro-inositol, or myo-inositol, or any salt thereof, which is part of any of the compositions of the present invention. Dietary supplements can be in single or combined form and can be sold in dosage forms, i.e., capsules, pills, tablets, and other similar forms, powder sachets, liquid ampoules and dropper bottles, and other similar forms of liquids and powders designed to be taken as a single dose. Dietary supplements can also be any plant-derived supplement containing D-pinitol, including syrup derived from carob fruit.

[0050] Preferably, the nutritional composition or dietary supplement is effective in preventing the progression of clinical symptoms of a tauopathy in a subject, preferably before or prior to the onset of such clinical symptoms, more preferably Preferably, it is intended for use in preventing or delaying in healthy subjects. In addition, the nutritional composition or dietary supplement may further be intended for use in preventing or delaying the progression of clinical symptoms of mild cognitive impairment in a subject, more preferably for preventing or delaying the onset of clinical symptoms of mild cognitive impairment in a subject, i.e., in said subject before the onset of such clinical symptoms, preferably in healthy subjects.

[0051] A wide range of nutrients and other elements may be present in nutritional supplements, including in particular vitamins, minerals, amino acids, essential fatty acids, fiber, enzymes, plants and plant extracts. Their role is to supplement the supply of nutrients in the diet, so they should not be used as a substitute for a balanced diet, and intakes should not exceed the daily amounts explicitly recommended by a doctor or nutritionist.

[0052] Examples of foods that may contain the compositions of the present invention include, but are not limited to, feed, dairy products, vegetable products, meat products, snacks, chocolate, beverages, baby food, cereals, fried foods, industrial bakery products, and biscuits. Examples of dairy products include, but are not limited to, fermented milk-derived products (e.g., yogurt or cheese) or non-fermented milk-derived products (e.g., ice cream, butter, margarine, or whey). Vegetable products include, but are not limited to, cereals and snacks, whether fermented (e.g., soy yogurt, oat yogurt, etc.) or non-fermented. Beverages may be non-fermented milk, but are not limited to these. In certain embodiments, the food product or food product is selected from the group consisting of fruit or vegetable juice, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, freeze-dried or air-dried products (suitable for reconstitution with a liquid vehicle), cereals, baked goods, milk-based products, meat products, and beverages.

[0053] D-pinitol, D-chiro-inositol, and / or myo-inositol, or any pharmaceutically acceptable salt or ester compound thereof, can be provided in a kit. Such a kit typically contains the active compound D-pinitol, D-chiro-inositol, or myo-inositol in a dosage form for administration. The dosage form contains a sufficient amount of the active compound to provide a beneficial effect when administered to a subject at regular intervals, such as once, twice, three times, four times, five times, or six times per day, for one or more days. The kit preferably includes instructions for use of the dosage form and the amount of dosage form to be taken over a specified period of time.

[0054] The term "subject" means a mammal. One embodiment of the term "mammal" is a "human," and the human is either male or female. The compounds are also useful for treating or preventing age-related conditions or diseases in cats and dogs, such as treating or preventing sarcopenia by promoting appetite, inhibiting insulin secretion and reducing insulin resistance, increasing growth hormone release, improving muscle strength or weakness, and increasing net muscle mass, improving cognition, and treating or preventing age-related hypertension. Thus, the term "mammal" includes pets such as cats and dogs. The term "mammal in need thereof" refers to a mammal in need of treatment or prevention as determined by a researcher, veterinarian, physician, or other clinician.

[0055] The term "composition," such as pharmaceutical composition, is intended to encompass a product comprising an active ingredient(s) and an inactive ingredient(s) constituting a carrier, as well as any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more components, or the dissociation of one or more components, or any other type of reaction or interaction of one or more components. Thus, pharmaceutical compositions of the present invention encompass any composition made by mixing a compound of the present invention with a pharmaceutically acceptable carrier. The term "composition" is also intended to encompass nutraceutical or food compositions. Nutraceuticals are foods or food products that provide health and medical benefits, including the prevention and treatment of disease in humans.

[0056] It is understood that the compounds of the present invention include hydrates, solvates, polymorphs, crystalline, hydrous crystalline, and amorphous forms of the compounds of the present invention, as well as pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Ammonium, calcium, lithium, magnesium, potassium, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compound of the present invention is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, malonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid, etc. Citric acid, fumaric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid are particularly preferred.

[0057] Preferably, as previously indicated herein, it should be understood that, as used herein, reference to the compounds D-pinitol, D-chiro-inositol, and myo-inositol is also intended to include pharmaceutically acceptable salts, such as hydrochlorides. The compounds of the present invention are useful for treating, controlling, or preventing diseases, disorders, or conditions characterized by impaired ghrelin secretion, resulting in circulating levels of active ghrelin that are lower than normal preprandial circulating levels of active ghrelin. In particular, the compounds of the present invention are useful for promoting healthy metabolic function during aging. Such promotion is achieved by treating or preventing diseases, disorders, or conditions whose prevalence increases exponentially with age, including, but not limited to, atherosclerosis, cardiovascular disease, type 2 diabetes, osteoporosis, hypertension, Alzheimer's disease, arthritis, cataracts, and cancer. Preferably, the disorder, disease, or condition responsive to modulation of the ghrelin receptor is hypertension, preferably age-related hypertension. Preferably, the disorder, disease, or condition responsive to modulation of the ghrelin receptor is an age-related condition, and the compounds of the present invention are preferably used to inhibit insulin secretion and reduce insulin resistance in order to avoid the progression of insulin resistance syndrome, a condition characterized by a progressive increase in insulin levels and subsequent progressive loss of cellular response to insulin, leading to endocrine pancreatic fatigue. Diseases that benefit from the reduction of excess insulin may include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, nonalcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease. Preferably, the disorder, disease, or condition responsive to modulation of the ghrelin receptor is an age-related condition, and the compounds of the present invention are used to increase circulating growth hormone release to compensate for the physiological decrease in growth hormone release associated with aging, and in conditions where reduced growth hormone secretion, such as human immunodeficiency virus (HIV) infection, necessitates increased athletic performance, muscle mass, and bone density. Preferably, the disorder, disease, or condition responsive to modulation of the ghrelin receptor is an age-related condition, and the compounds of the present invention are preferably used to increase circulating growth hormone release to compensate for the physiological decrease in growth hormone release associated with aging, and in conditions where increased athletic performance, muscle mass, and bone density are required due to decreased growth hormone secretion. Preferably, the disorder, disease, or condition responsive to ghrelin modulation is selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, and Huntington's disease.

[0058] Therefore, the compositions of the present invention are particularly effective in treating type 2 diabetes. The compounds or combinations of the present invention are also useful for treating and / or preventing gestational diabetes. Treatment of diabetes refers to the administration of the compounds or combinations of the present invention to treat diabetes. One outcome of treatment may be increasing insulin levels and enhancing insulin sensitivity. Another outcome of treatment may be reducing insulin resistance in a subject with elevated insulin resistance. Prevention of diabetes refers to the administration of the compounds or combinations of the present invention to prevent the onset of diabetes in a subject at risk of diabetes.

[0059] The terms "administration" of a compound and / or "administering" a compound should be understood to mean providing a compound of the present invention or a prodrug of a compound of the present invention to a subject in need of treatment. Administration of a compound of the present invention to practice the present methods of treatment is carried out by administering a therapeutically effective amount of the compound to a subject in need of such treatment or prevention. The need for prophylactic administration according to the methods of the present invention is determined via the use of known risk factors.

[0060] The term "therapeutically effective amount," as used herein, refers to an amount of active compound that elicits in a tissue, system, subject, mammal, or human the biological or medical response desired by a researcher, veterinarian, physician, or other clinician, including alleviation of symptoms of the disorder being treated. The novel treatment methods of the present invention are for disorders known to those of skill in the art. The term "prophylactically effective amount," as used herein, refers to an amount of active compound that elicits in a tissue, system, subject, mammal, or human the biological or medical response desired by a researcher, veterinarian, physician, or other clinician to prevent the onset of the disorder in obesity or a subject at risk for the disorder. The therapeutically or prophylactically effective amount or dosage of a particular compound will be determined by the attending physician in the final analysis and will depend on factors such as the precise disorder being treated, the severity of the disorder and other diseases or conditions the patient may suffer from, the selected route of administration, other drugs and treatments the patient may require concomitantly, and other factors within the physician's judgment.

[0061] Administration and Dose Ranges. Any suitable route of administration can be used to provide a subject or mammal, particularly a human, with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and other routes can be used. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. The compounds of the present invention are preferably administered orally. The effective dosage of the active ingredient employed can vary depending on the particular compound employed, the mode of administration, the condition being treated, and the severity of the condition being treated. Such dosages can be readily ascertained by one skilled in the art.

[0062] The magnitude of a prophylactic or therapeutic dose of a compound of the invention will, of course, vary depending on the particular compound employed, the mode of administration, the condition being treated, and the severity of the condition being treated. The size of the dose will also vary according to the age, weight, and response of the individual patient. Such dosages can be readily ascertained by one skilled in the art.

[0063] As already mentioned, the compounds of the present invention can be used in combination with other drugs used in the treatment / prevention / suppression or amelioration of diseases or conditions for which the compounds of the present invention are useful. Such other drugs can be administered simultaneously with or sequentially to the compounds of the present invention by a route and in an amount commonly used therefor. When the compounds of the present invention are used simultaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compounds of the present invention is preferred.

[0064] Compositions of the present invention include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), intraocular (ophthalmic), pulmonary (nasal or buccal inhalation), or intranasal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods known in the art of pharmacy. In practical use, the compounds of the present invention may be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous) administration. In preparing compositions for oral dosage forms, any of the usual pharmaceutical media may be used, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. in the case of oral liquid preparations, such as suspensions, elixirs, and solutions, or carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. in the case of oral solid preparations, such as powders, hard capsules, soft capsules, and tablets, with oral solid preparations being preferred over liquid preparations.

[0065] Because of their ease of administration, tablets and capsules are typical oral dosage unit forms, in which case solid pharmaceutical carriers are commonly employed. Tablets may be coated, if desired, by standard wet or dry coating techniques. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of active compound in these compositions may, of course, vary and may conveniently be about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compound may also be administered intranasally, for example, as drops or a spray. Tablets, pills, capsules, and the like may contain binders such as gum tragacanth, gum acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor.

[0066] The compounds of the present invention can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water, suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0067] Finally, the compounds of the present invention can also be administered in nutraceutical or food compositions, such as beverages.

[0068] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily syringable. The form must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0069] The following examples serve to illustrate the invention but not to limit it. [Example]

[0070] Example 1 Methodology of Example 1 Animals and ethical guidelines Experimental procedures using animals were performed in strict accordance with the recommendations of European Commission Directive 2010 / 63 / EU and Spanish legislation regulating the care and use of laboratory animals (Real Decreto 53 / 2013, BOE 34 / 11370-11421, 2013). The protocol was approved by the Ethics Committee for Animal Experiments at the University of Malaga. All studies using animals were conducted in accordance with the ARRIVE guidelines for reporting experiments with animals (Kilkenny, C., Browne, W.). J., Cuthill, IC, Emerson, M. & Altman, DG Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PLoS Biol 8, e1000412, https: / / doi.org / 10.1371 / journal.pbio.1000412 (2010)) All efforts were made to minimize animal suffering and reduce the number of animals used. Experiments were performed on 4- to 5-week-old male Wistar rats (Crl:WI; Charles River Laboratories, Barcelona, ​​Spain). Animals were kept under a standard 12-h light-dark cycle in a temperature- and humidity-controlled room. All rats had free access to water and a commercially available standard pelleted rat diet (STD) (3.02 kcal / g, consisting of 30 kcal% protein, 55 kcal% carbohydrate, and 15 kcal% fat).

[0071] Drug Preparation and Dosage D-Pinitol (3-O-methyl-D-chiroinositol) was kindly provided in the form of a fine crystalline powder by EURONUTRA SL (https: / / www.euronutra.com / , Malaga, Spain). For acute treatment, D-pinitol was dissolved in water and administered by gavage (orally) at different concentrations (100 mg / kg and 500 mg / kg), the drug being administered in a volume of 1 ml / kg.

[0072] Treatment guidelines After 18 hours of fasting, D-pinitol dissolved in water was acutely administered by gavage (100 mg / kg in the first study, 500 mg / kg in subsequent studies). After administration, animals were divided into groups and sacrificed at different time points (D-pinitol administration was time point 0). For the first study (100 mg / kg), groups of animals were sacrificed at 10, 20, 30, 60, 120, 240, and 360 minutes after administration; for the second study (500 mg / kg), animals were sacrificed at 60, 120, and 240 minutes after administration. As a control group, one group received water alone (by gavage).

[0073] Sample collection Animals were anesthetized with sodium pentobarbital (50 mg / kg, i.p.), and blood, brain, and liver samples were collected. Blood was centrifuged (2100 g for 8 min at 4°C), and plasma was saved for further analysis. Liver and brain samples were flash-frozen in liquid N2 and then stored at -80°C until further analysis.

[0074] Measurement of metabolites in plasma Plasma levels of insulin and ghrelin were measured using commercially available kits, catalog number EZRMI-13K and catalog number EZRMI-13K, from EMD Millipore Corporation (Billerica, MA, USA). Plasma glucagon levels were determined by enzyme-linked immunosorbent assay (ELISA) using RGRT-91K and RGRT-91K. Plasma glucagon levels were determined using Sigma-Aldrich (Saint Louis, MO, USA) catalog number R The Glucagon Enzyme Immunoassay (EIA) Kit from AB0202 was used to determine the levels of steroid hormones. All serum samples were assayed in duplicate in a single assay, and the results were expressed relative to the specific standard hormone.

[0075] RNA isolation and cDNA synthesis The Trizol™ method was used according to the manufacturer's instructions (GIBCO BRL Life Technologies). Total RNA was extracted from liver tissue portions (100 mg to 300 mg) according to the manufacturer's instructions. To ensure the purity of the mRNA sequences, RNA samples were isolated using the RNeasy Minelute Cleanup Kit (Qiagen), including digestion with a DNase I column (RNase-free DNase set, Qiagen), according to the manufacturer's instructions. Total RNA was quantified using a spectrophotometer (Nanodrop 1000 Spectrophotometer, Thermo Scientific) with an A260 / A280 ratio of 1.8 to 2.0. The Transcriptor Reverse Transcriptase kit and random hexamer primers (Transcriptor RT, Roche Diagnostics) were used. Reverse transcription was performed from 1 μg of RNA using a PCR kit (Promega Biosciences GmbH). A negative control was performed by omitting the reverse transcriptase. The reaction involved reverse transcription.

[0076] Real-time quantitative polymerase chain reaction (qPCR) and gene expression analysis Real-time qPCR was performed according to the MIQE guidelines (Bustin, SA et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55, 611-622 (2009)). Polymerase chain reaction was performed for each cDNA template using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). 9 μl of cDNA (100-fold diluted) was added. The target rat gene was amplified in a 20 μl reaction volume containing 11 μl of a master mix (TaqMan, Life Technologies) containing the primers. The primers were derived from the TaqMan™ Gene Expression Assay and FAM™ dye-labeled format (Life Technologies). Each reaction was performed in duplicate. The parameters were 50°C for 2 minutes to inactivate dUTP-containing single- and double-stranded DNA, 95°C for 10 minutes to activate Taq DNA polymerase, followed by 40 cycles of 95°C for 15 seconds to melt the cDNA, and 60°C for 1 minute to allow primer annealing and extension, during which fluorescence was acquired. Raw fluorescence data were submitted to the online Miner tool (http: / / www.miner.ewindup.info / ), and Cq and efficiency values ​​were calculated for each experimental set (Zhao, S. & Fernald, RD Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction. J Comput Biol. 12, 1047-1064 (2005)). At least two reference rat genes were included: β-actin (Actb) and glycine. Ceraldehyde-3-phosphate dehydrogenase (Gapdh) (Supplementary Table) was used and analyzed by Biogazelle's qbasePLUS software (Biogazelle, Zwijnaarde, Belgium). Cq values ​​were converted to relative expression values, taking into account amplification efficiency, inter-run variability, and normalization factors. For all reference and target gene studies, two independent biological samples of each experimental condition were evaluated in technical duplicates. Reproducibility between replicates was recognized when the ΔCq value was 0.7 or less. Finally, calibrated normalized relative quantity (CNRQ) values ​​were exported from qbasePLUS software and statistically examined.

[0077] [Table 1]

[0078] Protein extraction and Western blot analysis Brain extract. Frozen brain samples (17 mg per sample) were lysed in 1 mL of cold RIPA lysis buffer (50 mM Tris-HCl ( The cells were homogenized in a buffer solution (pH 7.4), 150 mM NaCl, 0.5% NaDOC, 1 mM EDTA, 1% Triton, 0.1% SDS, 1 mM Na3VO4, and 1 mM NaF). The suspension was incubated at 4°C for 2 hours and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new clean centrifuge tube, and the total protein concentration was determined using the Bradford colorimetric method. The protein extract was diluted 1:1 with loading buffer (DTT 2X) and heated at 99°C for 5 minutes before electrophoresis.

[0079] Western blot analysis. Expression of proteins including mTOR (289 kDa) and adaptin (100 kDa) was analyzed by Western blot. Tissue proteins (10 μg–15 μg) were run on a 4–12% Criterion XT Precast Bis-Tris gel (Bio-Rad, Hercules, CA, EE1UU) at 80 V for 30 min, 150 V for 30 min, and 100 V for 30 min. The electrophoresis was carried out for 2 hours. The proteins were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, EE.UU.) using a wet transfer device at 80 V for 1 hour. The membrane was then washed with TBST ( The membranes were washed twice for 5 min each with 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20 (pH 7.6) and blocked with 5% BSA-TBST for 1 h at room temperature on a shaker platform. Subsequently, the membranes were incubated overnight at 4°C with the respective primary antibodies diluted in 2% BSA-TBST. Antibodies against p-mTOR (Ser2448) and m-TOR were purchased from Cell Signaling Technology (Danvers, Massachusetts, United States), and antibodies against α-adaptin were purchased from Abcam (Cambridge, United Kingdom). The following day, the membranes were washed three times for 5 min each with TBST. The appropriate HRP-conjugated rabbit / mouse secondary antibodies (Promega, Madison, WI, EE.UU.) were diluted 1:10,000 in 2% BSA-TST and incubated with the membranes for 1 h at room temperature with shaking. Finally, the membranes were washed as described above and exposed to chemiluminescence reagent (Santa Cruz, Biotechnology Inc., CA, EE.UU.) for 5 minutes. Each membrane-associated protein was then visualized by chemiluminescence (ChemiDoc Imaging System, Bio-Rad). Bands were quantified by densitometric analysis using geJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA).

[0080] All data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, Inc., La Jolla, CA). One-way variance analysis was performed. Analysis was performed using ANOVA followed by Tukey's multiple comparison test where appropriate. A P value of less than 0.05 was considered significant.

[0081] Results of Example 1 1.1. Oral administration of pinitol (100 mg / kg) dissolved in sterile water to adult male Wistar rats As shown in Figure 2, oral administration of pinitol (100 mg / kg) dissolved in sterile water to adult male Wistar rats resulted in a) an increase in circulating plasma ghrelin concentrations, which was associated with B) a decrease in insulin release into the circulation, C) a decrease in insulin resistance as measured by the HOMA index, and D) an inhibition of the expression of pyruvate kinase, a key enzyme in the conversion of phosphoenolpyruvate to glucose production.

[0082] Furthermore, as shown in Figure 3, oral administration of pinitol (500 mg / kg) to adult male Wistar rats resulted in A) an increase in circulating plasma ghrelin concentrations, which was associated with B) a decrease in insulin release into the circulation, C) maintenance of plasma glucose levels, D) a decrease in insulin resistance as measured by the HOMA index, E) an increase in glucagon secretion, and F) activation of hypothalamic mTOR signaling in the hypothalamus via phosphorylation.

[0083] 1.2. Oral administration of D-chiro-inositol (500 mg / kg) dissolved in sterile water to adult male Wistar rats As shown in Figure 4, oral administration of D-chiro-inositol (500 mg / kg) enhances ghrelin secretion as measured by monitoring circulating plasma ghrelin concentrations.

[0084] 1.3. Proposal of a model for the role of inositol in metabolic aging Both D-pinitol and D-chiro-inositol enhance ghrelin secretion, promoting a metabolic profile characterized by reduced insulin demand from the endocrine pancreas, hepatic gluconeogenesis, increased muscle glucose utilization with muscle growth, and enhanced mTOR signaling in the hypothalamus, leading to increased appetite. The overall results of this unique pharmacological profile may include protection from pancreatic fatigue due to age-related insulin resistance and obesity, increased muscle vitality (preventing sarcopenia and frailty characteristic of the elderly), and better directing glucose disposal by the body.

[0085] Example 2 methodology Animals and ethical guidelines Animal testing procedures were carried out in accordance with the European Commission Directive 2010 / 63 / EU and Spanish law (Real The study was conducted in accordance with Decreto 53 / 2013. The protocol was approved by the Ethics Committee for Animal Experiments and Research at the University of Malaga, Spain. In accordance with the ARRIVE guidelines, every effort was made to minimize animal suffering and reduce the number of animals used per experimental group. Chronic drinking experiments in Wistar rats were conducted on 20 male rats. All 2-month-old adult rats (weighing approximately 300 g) were provided by Charles River Laboratories (Barcelona, ​​Spain). Animals were housed individually in a temperature- and humidity-controlled room under a standard 12-hour light-dark cycle. Water and rat chow were available ad libitum throughout the study.

[0086] Inositol Preparation and Administration Caromax™-D-pinitol (3-O-methyl-d-chiroinositol, DPIN, 98% purity) and Caromax™-D-chiroinositol (cis-1,2,3-trans-3,5,6-cyclohexanehexaol, DCI) were provided by Euronutra SL (Malaga, Spain). Inositol was administered orally by drinking and / or gavage at a dose of 100 mg / kg body weight (BW) in a volume of 1 ml / kg BW daily to rats and Wistar rats for 4 weeks (28 days) and 10 days, respectively (Fig. 1). In the drinking treatment, the concentration of inositol in the water was updated to account for the daily increase in the rats' BW and the potential loss of water due to evaporation.

[0087] Protein extraction and Western blot analysis Brain extract. Frozen brain samples (17 mg per sample) were homogenized in 1 mL of cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NaDOC, 1 mM EDTA, 1% Triton, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF) supplemented with a protease cocktail (Hoffmann-Roche). The suspension was incubated at 4 °C for 2 h and then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to a new clean centrifuge tube, and the total protein concentration was determined using the Bradford colorimetric method. The protein extract was diluted 1:1 with loading buffer (DTT 2X) and heated at 99 °C for 5 min before electrophoresis.

[0088] Western blot analysis. Tissue proteins (10–15 μg) were electrophoresed on a 4–12% Criterion XT Precast Bis-Tris gel (Bio-Rad, USA) at 80 V for 30 min and 150 V for 2 h. Proteins were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad, USA) using a wet transfer apparatus at 80 V for 1 h. The membrane was washed twice with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 5 min each and blocked with 2% bovine serum albumin-Tris-buffered saline Tween 20 (BSA-TBST) for 1 h at room temperature on a shaker platform. The membrane was then incubated overnight at 4°C with the respective primary antibodies diluted in 2% BSA-TBST (see Table 2 for further information). The next day, the membrane was washed three times for 5 minutes with TBST. The appropriate HRP-conjugated rabbit / mouse secondary antibody (Promega) was diluted 1:10,000 in 2% BSA-TST and incubated with the membrane for 1 hour at room temperature. Finally, the membranes were washed as above and exposed to chemiluminescence reagent (Santa Cruz, Biotechnology Inc.) for 5 min. Stripping / reprobing steps were used as necessary. Each membrane-bound protein was then visualized by chemiluminescence (ChemiDoc Imaging System, Bio-Rad). ImageJ software was used. Bands were quantified by densitometric analysis using software (Rasband, WS, ImageJ, (US National Institutes of Health, Bethesda, MD, USA). Normalization was performed using the reference protein α-adaptin from the same membrane. Results were expressed as protein / α-adaptin or phosphorylated / total protein ratios and normalized to the control group (y-axis represents "fold mean of control value").

[0089] Data Analysis and Statistics All data are expressed as mean ± SEM. Statistical analysis was performed on studies with a group size of at least n = 5. Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA, USA). One-way and two-way analysis of variance were performed. Analysis (ANOVA) was performed followed by Tukey's post hoc multiple comparison test. Post hoc tests were performed only if the F in the ANOVA achieved a P value of less than 0.05 and no statistically significant heterogeneity of variance was observed. Analysis of two single groups was performed using Student's unpaired t-test. Results were considered statistically significant at P < 0.05.

[0090] [Table 2]

[0091] result 2.1. Effect of chronic administration of D-pinitol or D-chiro-inositol for 10 days on the phosphorylation status of tau protein in the hippocampus of Wistar rats Wistar rats were orally treated with DPIN and DCI for 10 days (Figure 6). Here, we analyzed the expression and phosphorylation of tau protein, which is a hallmark of neurodegenerative disorders such as Alzheimer's disease. Two specific antibodies were used for this purpose: a phospho-tau (AT8) antibody that recognizes phosphorylated tau protein at serine 202 and threonine 205; and a total tau (Tau46) antibody that recognizes both phosphorylated and non-phosphorylated isoforms. Chronic administration of DPIN and DCI had a statistically significant effect on tau protein dephosphorylation. The amount of phospho-tau after both compounds was reduced by more than half compared to the vehicle group (one-way ANOVA: F (2,23) = 49.97, P value < 0.0001; Tukey's test: P value < 0.0001, Figure 7A.1).

[0092] Next, to assess whether the decrease in phospho-tau was due to a decrease in total tau, the amount of total tau protein was quantified in the same samples. There was a statistically significant difference in the relative protein levels of total tau after DPIN and DCI treatment, reflecting an increase compared to the vehicle group (one-way ANOVA: F (2,23) = 11.72, P value = 0.0003; Tukey's test: P value < 0.01, Figure 7A.2). Thus, chronic administration of DPIN and DCI in Wistar rats over 10 days resulted in a significant decrease in phospho-tau as well as an increase in total protein.

[0093] 2.2. Cyclin-dependent kinase 5 (CDK5) inhibition after DPIN administration as a possible explanation for hippocampal tau dephosphorylation Cyclin-dependent kinase 5 (cdk5) is thought to be involved in the phosphorylation of tau protein. We investigated the protein expression of CDK5, its activator subunit p35, and the truncated form of p35, p25. In Wistar rats, oral administration of DPIN significantly reduced the levels of p25 (unpaired t-test: t = 4.869, df = 14; P = 0.0002; Figure 8A) as well as the p35 subunit (unpaired t-test: t = 2.245, df = 15; P = 0.0402; Figure 8A). The total amount of CDK5 remained unchanged (unpaired t-test: t = 1.623, df = 14; P = 0.1268; Figure 8A). These results indicate a clear inactivation of CDK5 kinase in Wistar rats after DPIN administration.

[0094] 2.3. Glycogen synthase kinase-3β (GSK-3β) activity in the Wistar hippocampus after DPIN treatment is unaffected To investigate the interaction between the kinase activity of GSK-3β and the phosphorylation of tau protein, we analyzed the expression and phosphorylation of this kinase protein. It is well recognized that serine-9 (S9) phosphorylation of GSK-3β inhibits GSK-3 by causing the N-terminal tail to act as a pseudosubstrate, preventing the binding of the actual substrate. Because GSK-3β is one of the major tau kinases, inhibition of its activity leads to decreased tau phosphorylation. In this example, we aimed to investigate the role of this tau kinase (GSK-3β) in Wistar rats.

[0095] Oral administration of either DPIN or DCI significantly reduced serine-9 phosphorylation (one-way ANOVA: F (2,22) = 1.809, P value = 0.1874; Figure 9A.1) or tyrosine-216 (One-way ANOVA: F (2,22) = 1.088, P value = 0.3543; Figure 9A.2). That is, there was no enhancement or inhibition of kinase activity (One-way ANOVA: F (2,22)= 2.473, P value = 0.1074; Figure 9A.3). Regarding the relative protein levels of total GSK-3β, no statistically significant differences were observed after administration of the three compounds (One-way ANOVA: F (2,22) = 0.5308, P value = 0.5955; Figure 9A.4). Thus, the administration of either DPIN or DCI for 10 days had no effect on GSK-3β in the hippocampus of Wistar rats.

[0096] 2.4. AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK / ERK1 / 2) are not involved in hippocampal tau dephosphorylation We assessed the kinase activation and total expression of mitogen-activated protein kinases (MAPK / ERK1 / 2), AMP-activated protein kinase (AMPK), and protein kinase A (PKA) to analyze whether these kinases are involved in the above tau results. As shown in Figure 10, Wistar rats showed a significant increase in total PKA levels following oral DPIN administration (unpaired t-test: t = 3.795, df = 14; P = 0.0020; Figure 10A.2). No other changes were observed in other kinases.

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Claims

1. A composition, pharmaceutical composition, nutritional supplement composition or food composition, or dietary supplement for the prevention or delay of the onset of clinical symptoms selected from the group consisting of Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic granule dementia, spheroidal glial tauopathy, primary age-related tauopathy including neurofibrillary tangle dementia, chronic traumatic encephalopathy (CTE), and age-related tauastrogliopathies, comprising D-pinitol, D-chiro-inositol and / or myo-inositol, or any salt thereof.

2. The composition according to claim 1, comprising D-pinitol.

3. The composition according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier.

4. The composition according to claim 1 or 2, which is a dietary supplement.

5. The composition according to claim 1 or 2, which is a nutritional supplement food composition.

6. A composition according to any one of claims 1 to 5, which is administered orally or intragastricly.

7. The composition according to any one of claims 1 to 6, to be used to administer to a healthy subject who shows no clinical symptoms, selected from the group consisting of Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic granule dementia, spheroidal glial tauopathy, neurofibrillary tangle dementia, and other primary age-related tauopathies, chronic traumatic encephalopathy (CTE), and age-related tauastrogliopathies.

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