Composition for preventing and treating tauopathies

Abstract

The present invention relates to the delivery of a composition, preferably a pharmaceutical composition, comprising D-pinitol, D-chiro-inositol or myo-inositol, or any pharmaceutically acceptable salt thereof, for the treatment or prevention of a condition, disease or condition in a subject in need thereof in response to stimulation of a starvation receptor. In particular, for the treatment and / or prevention of conditions responsive to positive modulation (stimulation) of the starvation receptor, such as diabetes, obesity-related conditions, and most preferably for the treatment or prevention of age-related conditions or diseases, such as obesity-related conditions. Enhancing muscle viability or fragility, and treating or preventing sarcopenia, improving cognition (and / or treating or preventing diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease and huntington's disease), for example by promoting appetite, inhibiting insulin secretion and reducing insulin resistance, increasing growth hormone release, by increasing net muscle mass and treating or preventing age-related hypertension.

Description

Technical Field

[0001] This invention relates to the delivery of compositions comprising D-pineol, D-chiro inositol or myo-inositol or any pharmaceutically acceptable salt thereof, preferably pharmaceutical compositions, for use in treating or preventing symptoms, diseases or conditions in response to stimulation of ghrelin receptors in subjects of need. Background Technology

[0002] Human aging is associated with physical decline and impaired metabolic homeostasis (1). While physical decline is associated with increased fragility and the development of sarcopenia (a general loss of muscle mass and muscle cell performance), dysregulation of the metabolic network leads to an increase in age-related obesity, insulin resistance, diabetes, and hypertension (2). Aging impairs the activity of key metabolic signaling pathways, and the resulting metabolic dysregulation accelerates aging. Therefore, the association of frailty, impaired metabolic homeostasis, and hypertension increases vulnerability and limits the quality of life and even lifespan of older adults.

[0003] The key signaling system impaired during aging is insulin signaling. Impaired activity of the age-related insulin signaling pathway leads to insulin resistance (1). Due to dysregulation of glucose clearance, the resulting hyperglycemia promotes the formation of advanced glycation end products (AGEs), which in turn leads to tissue damage, further exacerbating metabolic disorders and accelerating the aging process. The critical hepatic muscle circulation is greatly affected by the development of obesity and insulin resistance, in which the binding between hepatic glucose production and muscle glucose consumption is dysregulated as a result of impaired insulin signaling, which leads to excessive hepatic glucose production and inhibits muscle glucose utilization, being affected by age-related weakness (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, is a potential target for age-related metabolic disorders and frailty (3-5). Ghrelin can promote appetite, inhibit insulin secretion, increase growth hormone release, enhance muscle vitality by increasing net muscle mass, and improve cognition (6-9). If we consider aging to cause loss of appetite, excessive insulin secretion, insulin resistance, muscle weakness, and cognitive impairment, then the physiological effects of ghrelin could counteract all these age-related conditions. Therefore, methods to enhance ghrelin release or administer ghrelin receptor agonists have been proposed as a means to combat the aforementioned age-related conditions (5) (including hypertension), since decreased ghrelin levels are positively correlated with age-related hypertension (10). Summary of the Invention

[0005] This invention relates to a composition, preferably a pharmaceutical composition, nutritional or food composition, comprising D-pinel, D-chiral inositol and / or myo-inositol or any pharmaceutically acceptable salt, ester, tautomer, solvate and hydrate thereof, for use in treating or preventing in a subject of need an impaired ghrelin secretion condition characterized by a bloodstream (preferably plasma) level of active ghrelin below a normal pre-meal bloodstream (preferably plasma) reference level. The condition, disease, or condition characterized by impaired ghrelin secretion resulting in a bloodstream level of active ghrelin below a normal pre-meal bloodstream reference level can be determined by establishing whether such condition, disease, or condition is characterized by an altered concentration of active ghrelin circulating levels, as measured by standard and conventional methods (such as ELISA or radioimmunoassay).

[0006] In the context of this invention, "normal pre-meal reference level" is understood to be the pre-meal reference level for healthy subjects. Plasma levels of ghrelin are discussed in *European Endocrinology*, 2015; 11(2):90-5 DOI:10.17925 / EE.2015.11.02.90.

[0007] In the context of this invention, "active ghrelin" is understood to be the acylated (typically n-octanoyl) form of ghrelin. It is produced by the post-translational esterification of a fatty acid (octanoic acid or, to a lesser extent, decanoic acid) at the serine residue at position 3 in secreted ghrelin. This acylation is essential for the activity of ghrelin.

[0008] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion, resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin, is an age-related condition.

[0009] In the context of this invention, "age-related condition" is understood to mean any physiological changes or pathological conditions most commonly associated with aging. Normal physiological changes associated with aging include muscle loss and increased muscle weakness or mild cognitive impairment. Pathological conditions 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 condition, disease, or symptom that responds to regulation of the ghrelin receptor is an age-related condition, and the composition is preferably used to enhance muscle vitality and reduce fragility by increasing net muscle mass.

[0011] Preferably, the condition, disease, or symptom that responds to regulation 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 this invention, "sarcopenia" is defined as the loss of skeletal muscle mass and strength due to aging.

[0013] Preferably, the symptom, disease, or condition responding to regulation of the ghrelin receptor is hypertension, wherein the composition is used to treat or prevent hypertension, preferably for the treatment or prevention of age-related hypertension.

[0014] In the context of this invention, "age-related hypertension" is understood as an age-related increase in systolic blood pressure, which results from an age-related increase in total (and renal) vascular resistance, as a result of the gradual loss of viscoelastic properties of ductal vessels, an increase in atherosclerotic arterial disease, and hypertrophy and hardening of muscular arteries and arterioles.

[0015] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion, resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin, is an age-related condition, wherein the composition is preferably used to inhibit insulin secretion and reduce insulin resistance to prevent the progression of insulin resistance syndrome (a condition in which cells gradually lose their response to insulin, leading to progressively elevated insulin levels and subsequent endocrine pancreatic failure). Diseases that may benefit from reducing excess insulin include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, non-alcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease.

[0016] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin blood circulation is an age-related condition, wherein the composition is used to increase circulating growth hormone release to compensate for physiological decline in age-related growth hormone release, and for physiological decline in conditions in which we need to increase exercise capacity, muscle mass, and bone density due to low growth hormone secretion such as infection with human immunodeficiency virus (HIV).

[0017] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin is an age-related condition, wherein the composition is preferably used to treat or prevent cognitive impairment or to improve cognition.

[0018] In the context of this invention, "cognitive impairment" is defined as a deficiency in acquired cognitive abilities (including learning, memory, perception, and problem-solving) (as opposed to "cognitive decline" associated with normal development and aging) and may involve underlying brain lesions; "improved cognition" is understood as amplifying or expanding the core mental capabilities through improvements or information processing systems. Cognitive improvement can be achieved through interventions including the application of compounds included in this patent.

[0019] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin is selected from the list of the following: Alzheimer's disease, vascular dementia, Parkinson's disease, and Huntington's disease.

[0020] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal blood circulation reference level is selected from the list of: diabetes, preferably type II diabetes.

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

[0022] Preferably, the composition is administered orally or via the stomach to the recipient.

[0023] Furthermore, the present invention also relates to:

[0024] - Compositions containing D-pineol, D-chiral inositol, or myo-inositol or any acceptable salt thereof are used for non-therapeutic purposes to promote appetite.

[0025] - Compositions containing D-pineol, D-chiral inositol, or myo-inositol or any acceptable salt thereof are intended for non-therapeutic purposes to increase net muscle mass.

[0026] - Compositions containing D-pineol, D-chiral inositol or myo-inositol or any acceptable salt thereof are intended for non-therapeutic purposes to improve cognition. Attached Figure Description

[0027] Figure 1The structures and relationships of D-pinel ((1S,2S,4S,5R)-6-methoxycyclohexane-1,2,3,4,5-pentaol), D-chiral inositol ((1R,2R,3S,4S,5S,6S)-cyclohexane-1,2,3,4,5,6-hexane), and D-myo inositol ((1R,2S,3r,4R,5S,6s)-cyclohexane-1,2,3,4,5,6-hexane) were investigated. Inositol is a polyol with insulin-like properties. D-pinel from a natural source (carob fruit) can be demethylated in the acidic environment of the stomach, thus converting to D-chiral inositol. Additionally, another dietary inositol is an isomer of D-chiral inositol, which can be converted to D-chiral inositol via epimerase.

[0028] Figure 2 :like Figure 2 As shown, oral administration of pinoxazone (100 mg / kg) dissolved in sterile water to adult male Wistar rats resulted in: a) an increase in circulating plasma ghrelin concentration; which was associated with B) a decrease in insulin release into the bloodstream; C) a decrease in insulin resistance as measured by the HOMA index; and D) inhibition of pyruvate kinase expression, a key enzyme that converts phosphoenolpyruvate to glucose for production.

[0029] Figure 3 :like Figure 3 As shown, oral administration of pinoxazone (500 mg / kg) to adult male Wistar rats resulted in: A) an increase in circulating plasma ghrelin concentration; which was associated with B) a decrease in insulin release into the bloodstream; 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 its phosphorylation.

[0030] Figure 4 :like Figure 4 As shown, oral administration of D-chiroinositol (500 mg / kg) enhances ghrelin secretion, as measured by monitoring circulating plasma ghrelin concentration.

[0031] Figure 5A proposed model of the role of inositol in metabolic aging is presented. D-pineol or D-chiral inositol enhances ghrelin secretion and promotes metabolic conditions characterized by reduced insulin demand from the endocrine pancreas, new glucose production in the liver, increased glucose utilization by muscles associated 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 of the pancreas from age-related insulin resistance and obesity-induced deterioration, enhanced muscle vitality (preventing age-specific sarcopenia and weakness), and better guidance of the body in processing glucose.

[0032] Figure 6 (A) In this invention, kinases and phosphatases are analyzed by Western blotting to assess their activity and / or expression. (C) Schematic diagram of tau phosphorylation due to dysfunction of the Akt-GSK3 pathway or activation of other kinases such as protein kinase A (PKA) or cyclin-dependent kinase (CDK5). Under physiological conditions, Akt is phosphorylated and thus activated. Under pathological conditions, such as brain insulin resistance, Akt activates kinase GSK-3β, leading to tau phosphorylation. This can occur when other kinases (PKA, CDK5) are overactivated. When tau protein is overphosphorylated, it dissociates from microtubules and forms insoluble aggregates called neurofibrillary tangles (NFTs) in neurons and glial cells. Microtubule instability and NFT aggregation both lead to apoptosis. (B) Chemical structure of D-pineol (DPIN). DPIN is the 3-O-methyl form of D-chiral inositol (DCI) and has been found to be a cyclic polyol. It is a known antidiabetic agent, isolated from the pulp of carob fruit (Ceratonia siliqua). The schematic timeline of the experimental design involved long-term oral administration of DPIN and DCI to 20 male Wistar rats for 10 consecutive days. Plasma and brain samples were collected from both experimental groups. Abbreviations: DPIN: D-pinenolide; DCI: D-chiroinositol.

[0033] Figure 7Effects of oral administration of DPIN or DCI for 10 days on tau dephosphorylation in the hippocampus of Wistar rats. A) Bar graphs show the ratio between tau phosphorylation (AT8:Ser202,Thr205) and total tau in Wistar rats compared to α-adaptor proteins, and the amount of total tau. Histograms show mean ± SEM (n=8 for Wistar rats). B) Protein blot membranes after each antibody incubation (target protein to the right of the recognition band). Molecular weight (MW) is expressed in kilodaltons (kD). Blots show results for four of eight independent samples from the Wistar rat group. Corresponding expression of α-adaptor proteins is shown as the loading control for each lane. One-way ANOVA and Tukey tests were performed: (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 compared to the solvent group.

[0034] Figure 8 Effects of oral administration of DPIN or DCI for 10 days on tau kinase cyclin-dependent kinase 5 (CDK5) activity in the hippocampus of Wistar rats. A) Bar graphs show the ratios of p25, p35, and total CDK5 tau kinase levels relative to α-adaptor proteins in Wistar rats. Histograms represent mean ± SEM (n=8 for Wistar rats). B) Protein blot membranes after each antibody incubation (target protein to the right of the recognition band). Molecular weight (MW) is expressed in kilodaltons (kD). Blots show results from three of eight independent samples from the Wistar rat group. Corresponding expression of α-adaptor proteins is shown as loading controls for each lane. Unpaired t-tests were performed on the Wistar rat analyses, with (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 compared to the solvent group.

[0035] Figure 9 Effects of oral administration of DPIN or DCI for 10 days on glycogen synthase kinase-3β (GSK-3β) phosphorylation in the hippocampus of Wistar rats. A) Bar graphs show the ratio of GSK-3β phosphorylation (Ser9 / Tyr216) to total GSK3β in Wistar rats, relative to α-adaptor proteins, and the amount of total GSK3β. B) Protein blot membranes after each antibody incubation (target protein to the right of the recognition band). Molecular weight (MW) is expressed in kilodaltons (kD). Blots show results from three of eight independent samples from the Wistar rat group. Corresponding expression of α-adaptor proteins is shown as loading controls for each lane. Unpaired t-tests were performed on the analysis of Wistar rats, with (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 compared to the solvent group.

[0036] Figure 10 Effects of oral administration of DPIN or DCI for 10 days on other hippocampal tau kinases in Wistar rats: mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), and protein kinase A (PKA). A) Bar graphs show the ratios of total tau kinase levels to α-adaptor proteins in Wistar rats. Histograms represent mean ± SEM (n=8 for Wistar rats). B) Protein blot membranes after each antibody incubation (target protein to the right of the recognition band). Molecular weight (MW) is expressed in kilodaltons (kD). Blots show results from three of eight independent samples from the Wistar rat group. Corresponding expression of α-adaptor proteins is shown as loading controls for each lane. Unpaired t-tests were performed on the Wistar rat analysis, with (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 compared to the solvent group. Detailed Implementation

[0037] This invention relates to a method for increasing ghrelin levels by oral administration of D-pinel (a natural cyclic polyol derived from plants including the carob tree pods) or related inositols (such as D-chiral inositol or its epimer myo-inositol). Figure 1 Other stereoisomers of inositol may be used, such as cis-inositol, epi-inositol, iso-inositol, muco-inositol, neo-inositol, L-chiral inositol, acyllo-inositol, or any pharmaceutically acceptable salt, ester, tautomer, solvate, and hydrate thereof, or any combination thereof. As illustrated in the examples included throughout this specification, oral administration of D-pineol (100 mg / kg) Figure 2 Oral administration of 500 mg / kg of D-chiral inositol ( Figure 3 It enhances ghrelin secretion and reduces circulating insulin, and further activates phosphorylation of mTOR in the hypothalamus, a metabolic sensor required to increase appetite. Figure 3 The net effect is a reduction in insulin demand, as reflected by a decrease in the insulin resistance index HOMA (Homo simulacrum). Figure 2 and Figure 3 However, it does not cause hypoglycemia or hyperglycemia. This is likely due to the direct effect of D-pineol and D-chiral inositol on glucose uptake by muscle cells, combined with the reduction in net hepatic glucose production (NGH) resulting from the inhibition of pyruvate kinase, which redirects glycolysis back to glucose production in the liver (neo-glucose production). Figure 2 The administration of D-chiroinositol also enhanced the secretion of ghrelin (glucagon). Figure 4 ).

[0038] In addition, such as Figure 5 The proposed use of D-pinel and D-chiral inositol to promote ghrelin secretion coordinates insulin and glucagon secretion, reduces insulin requirements, and redirects glucose production from the liver to its utilization by muscles. Furthermore, given the known effects of ghrelin, D-pinel or related inositols such as D-chiral inositol or its epiomer myo-inositol are expected to increase muscle mass and vitality and reduce muscle weakness. Since ghrelin concentrations decline with age (16), these compounds may contribute to promoting healthy metabolic aging.

[0039] On the other hand, the authors of this invention have surprisingly discovered a role for D-pinesol in preventing cognitive impairment in aging and tau pathologies. In this sense, the insulin-sparing properties of D-pinesol and its ability to enhance ghrelin secretion have a pro-cognitive effect and could be used as a preventative strategy for cognitive impairment associated with neurodegenerative diseases. Insulin resistance in the brain is associated with cognitive impairment, particularly through alterations in hippocampal function. Ghrelin, on the other hand, is a unique hormone that enhances cognition by crossing the blood-brain barrier and interacting with growth hormone secretagogue receptors. Therefore, the novel effects described in this invention may explain the use of D-pinesol in aging or neurodegenerative diseases, particularly Alzheimer's disease, to reduce cognitive impairment in the early stages of the disease (when symptoms allow for diagnosis).

[0040] However, recent research highlights the need to focus on a molecular target that is the best-known correlator for the onset of mild cognitive impairment and the development of major dementia: tau protein. Aberrant phosphorylation or acetylation of tau produces tau protein deposition, leading to neurofibrillary tangles (NFTs), a histopathological biomarker for a group of diseases collectively known as tau-pathopathies. The formation of NFTs is more strongly associated with cognitive decline than the distribution of senile plaques, which are formed from polymorphic β-amyloid (Ax) protein deposits, a pathological marker of Alzheimer's disease, one of the major dementias. Therefore, tau deposition is a key factor in the cognitive impairment observed in Alzheimer's disease (where β-amyloid deposition is insufficient to produce dementia and the progression to dementia observed in mild cognitive impairment and chronic traumatic encephalopathy).

[0041] Therefore, tau proteinopathy has been recognized as an important marker of neurodegenerative diseases and normal brain aging, and its prevention before the onset of clinical symptoms represents an unmet medical need. In light of this, we examined the effect of D-pinesol administration on tau phosphorylation status, a complex process regulated by multiple proteins. Figure 6 And surprisingly, it was found that oral administration of D-pineol works by reducing cyclin-dependent kinase 5 (CLP). Figure 8One of its main phosphorylases, it significantly reduces the phosphorylation of Tau through the mechanism of its activity. Figure 7 The effects of D-pineols are specific because they do not affect other tau regulatory proteins. Figure 9 This gives this natural inositol a unique pharmacological character.

[0042] Based on the above findings, in this invention, we further propose that the application of D-pineol will:

[0043] a) If a condition is promoting tau hyperphosphorylation and there is an intention to prevent or delay its clinical symptoms, then prevention or delay of the onset of mild cognitive impairment and its further development into dementia is desired. These conditions include Pick's disease, progressive supranuclear palsy, corticobasal degeneration, auricula-satellite disease, glioblastoma, primary age-related tau lesions including neurofibrillary tangles dementia, chronic traumatic encephalopathy (CTE), and age-related tau astrogliopathy.

[0044] b) Treatment of diagnosed tau diseases, including Pick's disease, progressive supranuclear palsy, corticobasal degeneration, auricula-satellite disease, glioblastoma, primary age-related tau diseases including neurofibrillary tangles dementia, chronic traumatic encephalopathy (CTE), and age-related tau astropathy.

[0045] Therefore, this invention relates to inositols for increasing ghrelin levels, such as D-pineol, D-chiral inositol, and their epimer myo-inositol (from the compounds of this invention described below). The compounds of this invention are used... Figure 1 The invention relates to the structural formula defined herein or any pharmaceutically acceptable salt derived therefrom. Furthermore, the invention further relates to compounds of the invention for reducing Tau phosphorylation and thus preventing or slowing the progression of clinical manifestations of tau proteinopathy or mild cognitive impairment in subjects, preferably in subjects prior to the appearance of such clinical manifestations, more preferably in human subjects.

[0046] In this invention, the term "asymptomatic subject" is understood to mean a subject that does not exhibit clinical manifestations of disease, particularly tau proteinosis.

[0047] In this invention, the term "onset of clinical manifestations of tau proteinopathy" is understood to mean 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 indicate the diagnosis of tau proteinopathy.

[0048] In this invention, the term "onset of clinical manifestations of mild cognitive impairment (MCI)" is understood as a stage between the expected cognitive decline of normal aging and more severe dementia. It is characterized by problems with memory, language, thinking, or judgment.

[0049] In this invention, the term "prevention" refers to avoiding the occurrence of a disease or pathological condition in an individual, particularly when an individual has a predisposition to such a pathological condition but has not yet been diagnosed. In this invention, the disease or pathological condition is preferably "tau proteinosis".

[0050] In this invention, the term "tau proteinopathy" refers to a class of neurodegenerative diseases involving the aggregation of tau protein in the human brain into neurofibrils or glial fibrillary tangles (NFTs). These tangles are formed by the hyperphosphorylation of a microtubule protein called tau, causing the protein to dissociate from the microtubules and form insoluble aggregates (also known as paired helical filaments). Examples of tau proteinopathy are selected from the following list: Pick's disease, progressive supranuclear palsy, corticobasal degeneration, auricula-sagittosis, glioblastoma, primary age-related tau proteinopathy including neurofibrillary tangles dementia, chronic traumatic encephalopathy (CTE), and age-related tau astropathy.

[0051] As already noted, the compounds of the present invention are effective in increasing ghrelin levels. Therefore, they can be used to treat and / or prevent conditions characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin below the normal pre-meal reference level, such as age-related conditions where normal physiological changes associated with aging include muscle loss and increased muscle weakness or mild cognitive impairment. Pathological conditions whose prevalence increases exponentially with age further include: atherosclerosis, cardiovascular disease, type 2 diabetes, osteoporosis, hypertension, Alzheimer's disease, arthritis, cataracts, and cancer. Preferably, the condition, disease, or condition characterized by impaired ghrelin secretion resulting in circulating levels of active ghrelin below the normal pre-meal reference level is hypertension, preferably age-related hypertension.

[0052] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion, resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin, is an age-related condition, wherein the composition of the present invention is preferably used to inhibit insulin secretion and reduce insulin resistance to avoid the progression of insulin resistance syndrome (a condition in which cells gradually lose their responsiveness to insulin, leading to progressively elevated insulin levels and subsequent endocrine pancreatic failure). Diseases that may benefit from reducing excess insulin include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, non-alcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease.

[0053] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin blood circulation is an age-related condition, wherein the composition of the present invention is used to increase circulating growth hormone release to compensate for physiological decline in age-related growth hormone release, and physiological decline in conditions in which we need to increase exercise capacity, muscle mass, and bone density due to low growth hormone secretion such as infection with human immunodeficiency virus (HIV).

[0054] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal reference level of active ghrelin is an age-related condition, wherein the composition of the present invention is preferably used for the treatment or prevention of cognitive impairment or for the improvement of cognition.

[0055] Preferably, the condition, disease, or symptom characterized by impaired ghrelin secretion resulting in a blood circulation level of active ghrelin below the normal pre-meal blood circulation reference level of active ghrelin is selected from the list of the following: Alzheimer's disease, vascular dementia, Parkinson's disease, and Huntington's disease.

[0056] Therefore, one aspect of the present invention provides a method for treating or preventing any of the aforementioned diseases, conditions, or states in a subject in need, the method comprising administering to the subject a therapeutically or preventively effective amount of D-pineol, D-chiral inositol, and / or myo-inositol, or any pharmaceutically acceptable salt thereof. The present invention also relates to a method for treating or preventing any of the aforementioned diseases, conditions, or states in a subject in need by combining D-pineol, D-chiral inositol, and / or myo-inositol, or any pharmaceutically acceptable salt thereof with a therapeutically or preventively effective amount of another known agent known for treating or preventing any of the aforementioned conditions or diseases. Another aspect of the present invention provides a pharmaceutical composition comprising a compound having the structural formula D-pineol, D-chiral inositol, and / or myo-inositol, and a pharmaceutically acceptable carrier.

[0057] Another aspect of the invention relates to the use of D-pinel, D-chiral inositol, and / or myo-inositol, or any pharmaceutically acceptable salt thereof, in the preparation of medicaments for treating, preventing, or inhibiting diseases characterized by impaired ghrelin secretion resulting in a lower than normal pre-meal reference level of active ghrelin circulating in the subject of need. Specifically, it is intended for treating, preventing, or inhibiting any of the aforementioned diseases, conditions, or circumstances.

[0058] Another aspect of the invention relates to the non-therapeutic use of a certain amount of D-pineol, D-chiroinositol and / or myo-inositol or any salt thereof for the purpose of promoting appetite, inhibiting insulin secretion and reducing insulin resistance, increasing growth hormone release, enhancing muscle vitality or fragility by increasing net muscle mass, improving cognition, and preventing age-related hypertension in subjects in need.

[0059] Furthermore, as already noted, the compounds of the present invention can be used to significantly reduce Tau phosphorylation, and thus prevent or slow the onset or progression of clinical manifestations of tau proteinopathy in subjects, preferably in subjects before or after the onset of such clinical manifestations. Therefore, another aspect of the present invention provides a method for preventing or slowing the onset or progression of clinical manifestations of tau proteinopathy in subjects. More preferably, the present invention provides a method for preventing or slowing the onset of clinical manifestations of tau proteinopathy in subjects, i.e., a method for preventing or slowing the occurrence of such clinical manifestations in said subjects, preferably in healthy subjects. Furthermore, another aspect of the present invention provides a method for preventing or slowing the onset or progression of clinical manifestations of mild cognitive impairment in subjects. More preferably, the present invention provides a method for preventing or slowing the onset of clinical manifestations of mild cognitive impairment in subjects, i.e., a method for preventing or slowing the occurrence of such clinical manifestations in said subjects, preferably in healthy subjects.

[0060] Another aspect of the invention relates to the use of D-pinel, D-chiral inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, in the preparation of a medicament for preventing or slowing the progression of clinical manifestations of tau proteinosis in subjects, i.e., for preventing or slowing the occurrence of such clinical manifestations in said subjects, preferably in healthy subjects. Furthermore, another aspect of the invention relates to the use of D-pinel, D-chiral inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, in the preparation of a medicament for preventing or slowing the progression of clinical manifestations of mild cognitive impairment in subjects. More preferably, the invention relates to the use of D-pinel, D-chiral inositol and / or myo-inositol, or any pharmaceutically acceptable salt thereof, in the preparation of a medicament for preventing or slowing the onset of clinical manifestations of mild cognitive impairment in subjects, i.e., for preventing or slowing the occurrence of such clinical manifestations in said subjects, preferably in healthy subjects.

[0061] In addition to the foregoing, this invention also encompasses the possibility that the compositions of this invention are in the form of dietary supplements or nutritional compositions comprising D-pinel, D-chiral inositol, and / or myo-inositol or any salt thereof. In this sense, when the compositions of this invention are formulated into nutritional compositions, the nutritional compositions may be food or incorporated into food or food products intended for human or animal consumption. Therefore, in a particular embodiment, the nutritional composition is selected from foods (which may be foods or medicinal foods for specific nutritional purposes) and nutritional supplements.

[0062] In this invention, the term "nutritional composition" refers to a food that, regardless of whether it provides nutrition to the person consuming it, beneficially affects one or more bodily functions, thereby providing better health and wellness.

[0063] The term "supplement," synonymous with any of the terms "dietary supplement," "nutritional supplement," "food supplement," or "food and digestive supplement" or "food and digestive complement," refers to a product or preparation intended to supplement a normal diet consisting of concentrated nutrients or other sources of substances with nutritional or physiological effects. In this invention, the "substance" that has a nutritional or physiological effect on an individual when ingested as food and digestive complement is D-pinel, D-chiral inositol, or myo-inositol, or any salt thereof, which are part of any composition of this invention. Food supplements may be in single or combined forms and are sold in dosage forms, i.e., capsules, pills, tablets and other similar forms, powder sachets, liquid ampoules and drop dispensing bottles, and other similar forms of liquids and powders designed for single-dose administration. Food supplements may be any plant-derived supplement containing D-pinel, including carob syrup.

[0064] Preferably, the nutritional composition or dietary supplement is intended or used to prevent or slow the progression of clinical manifestations of tau proteinosis in subjects, preferably in subjects before or prior to the onset of such clinical manifestations, and more preferably in healthy subjects. Furthermore, the nutritional composition or dietary supplement may be further intended or used to prevent or slow the progression of clinical manifestations of mild cognitive impairment in subjects. More preferably, it is used to prevent or slow the onset of clinical manifestations of mild cognitive impairment in subjects, i.e., in subjects before or prior to the onset of such clinical manifestations, and more preferably in healthy subjects.

[0065] A wide range of nutrients and other elements may be found in food and digestive complement, including vitamins, minerals, amino acids, essential fatty acids, fiber, enzymes, plants, and plant extracts. Because their role is to supplement the nutritional supply in the diet, they should not be used as a substitute for a balanced diet, and intake should not exceed the daily dose explicitly recommended by a doctor or nutritionist.

[0066] Examples of food products that may contain the compositions of the present invention include, but are not limited to, animal feed, dairy products, vegetable products, meat products, snacks, chocolate, beverages, baby food, cereals, fried foods, industrially baked products, and biscuits. Examples of dairy products include, but are not limited to, products derived from fermented milk (e.g., but not limited to yogurt or cheese) or non-fermented milk (e.g., but not limited to ice cream, butter, margarine, or whey). Vegetable products are, for example, but not limited to, snacks and any form of fermented (e.g., soy yogurt, oat yogurt, etc.) or unfermented cereals. Beverages may be, but are not limited to, non-fermented milk. In one particular embodiment, the food product or food is selected from the group consisting of: fruit or vegetable juice, ice cream, baby formula, milk, yogurt, cheese, fermented milk, milk powder, freeze-dried or air-dried products (suitable for reconstitution with liquid solvents), cereals, baked foods, dairy products, meat products, and beverages.

[0067] D-Pineol, D-Chiral Inositol, and / or myo-inositol, or any pharmaceutically acceptable salt or ester of these compounds, may be provided in a kit. Such kits typically contain the active compound in a dosage form for administration. The dosage form contains sufficient amounts of the active compound to achieve a beneficial effect when administered to the subject at regular intervals, such as 1, 2, 3, 4, 5, or 6 times daily over a period of 1 day or more. Preferably, the kit includes instructions for use of the dosage form and the amount of dosage form to be taken over a specific time period.

[0068] The term "object" refers to a mammal. One embodiment of the term "mammal" is "human," meaning male or female. The compounds of this invention are also used in cats and dogs to treat or prevent age-related conditions or diseases, such as promoting appetite, inhibiting insulin secretion and reducing insulin resistance, increasing growth hormone release, enhancing muscle vitality or fragility by increasing net muscle mass, and treating or preventing sarcopenia, improving cognition, and treating or preventing age-related hypertension. Therefore, the term "mammal" includes companion animals such as cats and dogs. The term "mammal in need" refers to a mammal that researchers, veterinarians, physicians, or other clinicians have determined to require treatment or prevention.

[0069] As in pharmaceutical compositions, the term "composition" is intended to cover products comprising an active ingredient and an inert component constituting a carrier, as well as any product directly or indirectly resulting from any combination, complexation or aggregation of any two or more components, or dissociation of one or more components, or other types of reactions or interactions of one or more components. Therefore, the pharmaceutical compositions of the present invention cover any composition prepared by mixing the compounds of the present invention with a pharmaceutically acceptable carrier. The term "composition" is also intended to cover nutritional or food compositions. A nutritional product is a food or food product that provides health and medical benefits, including the prevention and treatment of minor human ailments.

[0070] It should be understood that the compounds of the present invention include hydrates, solvates, polymorphs, crystals, hydrated crystals, and amorphous forms of the compounds of the present invention, as well as pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali or acid, including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, trivalent manganese salts, divalent manganese salts, potassium salts, sodium salts, zinc salts, etc. Particularly preferred are ammonium salts, calcium salts, lithium salts, magnesium salts, potassium salts, and sodium salts. Salts derived from pharmaceutically acceptable, non-toxic organic bases include 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-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compounds of this invention are basic, the salts can be prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids. These 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, hydroxyethylsulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, malonic acid, mucoic acid, nitric acid, pyric 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.

[0071] Preferably, and as indicated throughout this specification, it should be understood that, as used herein, references to the compounds D-pinel, D-chiral inositol, and myo-inositol mean that they also include pharmaceutically acceptable salts, such as hydrochloride. The compounds of the present invention can be used to treat, control, or prevent diseases, conditions, or states characterized by impaired ghrelin secretion resulting in blood circulation levels of active ghrelin below normal pre-meal reference levels of active ghrelin. In particular, the compounds of the present invention can be used to promote healthy metabolic aging. This promotion is achieved by treating or preventing diseases, conditions, or states including, but not limited to, pathological conditions whose prevalence increases exponentially with age, such as: atherosclerosis, cardiovascular disease, type 2 diabetes, osteoporosis, hypertension, Alzheimer's disease, arthritis, cataracts, and cancer. Preferably, the condition, disease, or state responding to regulation of ghrelin receptors is hypertension, preferably age-related hypertension. Preferably, the condition, disease, or symptom responsive to regulation of the ghrelin receptor is an age-related condition, wherein the compositions of the present invention are preferably used to inhibit insulin secretion and reduce insulin resistance to prevent the progression of insulin resistance syndrome (a condition in which cells gradually lose their responsiveness to insulin, leading to progressively elevated insulin levels and subsequent endocrine pancreatic failure). Diseases that will benefit from reducing excess insulin may include type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, non-alcoholic steatohepatitis, and brain insulin resistance associated with Alzheimer's disease. Preferably, the condition, disease, or symptom responsive to regulation of the ghrelin receptor is an age-related condition, wherein the compositions of the present invention are used to increase circulating growth hormone release to compensate for physiological decline in age-related growth hormone release, and for physiological decline in conditions in which we need to increase exercise capacity, muscle mass, and bone density due to low growth hormone secretion such as infection with human immunodeficiency virus (HIV). Preferably, the condition, disease, or symptom responsive to regulation of the ghrelin receptor is an age-related condition, wherein the compounds of the present invention are preferably used to treat or prevent cognitive impairment or to improve cognition. Preferably, the condition, disease, or symptom that responds to the regulation of ghrelin is selected from the list of the following: Alzheimer's disease, vascular dementia, Parkinson's disease, and Huntington's disease.

[0072] 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 used to treat and / or prevent gestational diabetes. Treatment of diabetes refers to the administration of the compounds or combinations of the present invention to treat diabetes. One result of treatment may be increased insulin levels and increased insulin sensitivity. Another result of treatment may be reduced insulin resistance in subjects with increased 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 subjects at risk of developing diabetes.

[0073] The terms "application of the compound" and / or "application of the compound" should be understood as referring to providing a subject requiring treatment with the compound of the present invention or a prodrug of the compound of the present invention. The application of the compound of the present invention for carrying out this treatment method is performed by administering a therapeutically effective amount of the compound to a subject requiring such treatment or prevention. The need for prophylactic application according to the method of the present invention is determined by using well-known risk factors.

[0074] As used herein, the term "therapeutic effective amount" refers to the amount of an active compound that will elicit a biological or medical response (including relief of symptoms of the condition being treated) in a tissue, system, object, mammal, or human being sought by a researcher, veterinarian, physician, or clinician. The novel treatment methods of this invention are for diseases known to those skilled in the art. As used herein, the term "preventive effective amount" refers to the amount of an active compound that will elicit a biological or medical response in a tissue, system, object, mammal, or human being sought by a researcher, veterinarian, physician, or clinician to prevent the onset of a condition in the object as a risk factor for obesity or other diseases. Ultimately, the therapeutic or preventive effective amount or dosage of a single compound is determined by the physician in charge of the case, but depends on factors such as the exact disease to be treated, the severity of the disease and other diseases or conditions the patient has, the chosen route of administration, other drugs and treatments the patient may require concurrently, and other factors in the physician's judgment.

[0075] Application and Dosage Range. The compounds of the present invention can be administered to a subject or mammal, particularly a human, via any suitable route of administration. For example, they can be administered orally, rectally, topically, parenterally, ocularly, pulmonaryly, or nasally. Dosage forms include tablets, lozenges, dispersants, suspensions, solutions, capsules, creams, ointments, aerosols, etc. Preferably, the compounds of the present invention are administered orally. The effective dose of the active ingredient used can vary depending on the specific compound used, the method of administration, the condition being treated, and the severity of the condition. Such a dose can be readily determined by those skilled in the art.

[0076] Of course, the preventive or therapeutic dosage of the compounds of this invention will vary depending on the specific compound used, the method of administration, the condition being treated, and the severity of the condition. The dosage will also vary depending on the individual patient's age, weight, and response. Those skilled in the art can readily determine such a dosage.

[0077] As already described, the compounds of the present invention can be used in combination with other medicaments for treating / preventing / inhibiting or improving diseases or conditions for which the compounds of the present invention are applicable. Such other medicaments may be administered simultaneously or sequentially with the compounds of the present invention via the same route and amount normally used for that medicament. When the compounds of the present invention are used simultaneously with one or more other medicaments, a pharmaceutical composition comprising, in addition to the compounds of the present invention, such other medicaments is preferred.

[0078] While the most suitable route of administration in any given case will depend on the nature and severity of the condition being treated and the nature of the active ingredient, the compositions of the present invention include those suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic artery), pulmonary (nasal or oral inhalation), or nasal administration. They can be conveniently present in unit dosage forms and prepared by any method well known in the pharmaceutical field. In practical applications, the compounds of the present invention can be combined as active ingredients with drug carriers into close mixtures using conventional pharmaceutical compounding techniques. The carriers can take various forms depending on the desired formulation for administration, e.g., oral or parenteral (including intravenous) administration. When preparing compositions for oral dosage forms, any commonly used pharmaceutical medium can be used. For example, in the case of oral liquid dosage forms such as suspensions, elixirs, and solutions, water, ethylene glycol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc., can be used; or in the case of oral solid dosage forms such as powders, hard capsules, soft capsules, and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used, with solid oral dosage forms being preferred over liquid dosage forms.

[0079] Tablets and capsules represent typical oral unit dosage forms due to their ease of administration, in which case a solid drug carrier is typically used. Tablets can be coated using standard aqueous or non-aqueous techniques if desired. Such compositions and formulations should contain at least 0.1% of the active compound. Of course, the percentage of the active compound in these compositions can vary and can conveniently be from about 2% to about 60% by weight. The amount of active compound in such therapeutically useful compositions ensures an effective dose. The active compound can also be administered intranasally, for example, as drops or sprays. Tablets, pills, capsules, etc., may also contain: binders such as gum arabic, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginate; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin. When the unit dosage form is a capsule, it may also contain a liquid carrier, such as fatty oil, in addition to the materials of the types described above. Various other materials may be present as coating agents or to alter the physical form of the dosage unit. For example, tablets can be coated with shellac, sugar, or both. In addition to the active ingredient, syrups or elixirs may also contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings, such as cherry or orange flavorings.

[0080] The compounds of this invention can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water with a suitable mixture of surfactants such as hydroxypropyl cellulose. Dispersants can also be prepared in oils with glycerol, liquid polyethylene glycol, or mixtures thereof. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0081] Finally, the compounds of the present invention can also be used as nutritional supplements or food compositions such as beverages.

[0082] Suitable injectable drug forms include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be a fluid to achieve injectability. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0083] The following examples are used to illustrate the present invention but do not limit the invention.

[0084] Example

[0085] Example 1.

[0086] Method of Example 1

[0087] Animal and ethical statements.

[0088] The experimental procedures involving animals were conducted in strict accordance with the recommendations for the care and use of laboratory animals in European Community Directive 2010 / 63 / EU and Spanish regulations (RealDecreto 53 / 2013, BOE 34 / 11370–11421, 2013). This protocol was approved by the Ethics Committee for Animal Experiments of the University of Malaga. All studies involving animals were reported in accordance with the ARRIVE Guidelines for Reporting Animal Research (Kilkenny, C., Browne, WJ, 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)). Every effort was made to minimize animal suffering and reduce the number of animals used. The experiment was conducted on 4- to 5-week-old male Wistar rats (Crl:WI; Charles River Laboratories, Barcelona, ​​Spain). Animals were kept in a temperature- and humidity-controlled room under a standard 12-hour light-dark cycle. All rats were provided with water and commercially available standard rat pellets (STD) (3.02 kcal / g, containing 30 kcal% protein, 55 kcal% carbohydrates, and 15 kcal% fat) on an ad-hoc basis.

[0089] Drug preparation and dosage.

[0090] D-Pineol (3-O-methyl-D-chiral-inositol) is generously supplied by EURONUTRA SL (https: / / www.euronutra.com / , Málaga, Spain) in the form of fine crystalline powder (batch number: PPN-M0201). For acute treatment, D-Pineol is dissolved in water and administered via tube feeding (orally) at different concentrations (100 mg / kg and 500 mg / kg), with the drug administered at a volume of 1 ml / kg.

[0091] Treatment guidelines.

[0092] After an 18-hour fast, D-pinesol dissolved in water was acutely administered via tube feeding (100 mg / kg in the first study and 500 mg / kg in subsequent studies). Animals were sacrificed at different time points following administration (D-pinesol administration time was 0 minutes). For the first study (100 mg / kg), 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 only water (via tube feeding).

[0093] Sample collection.

[0094] Animals were anesthetized with sodium pentobarbital (50 mg / kg, ip), and blood, brain, and liver samples were collected. Blood samples were centrifuged (2100 g for 8 minutes, 4°C), and plasma was retained for further analysis. Liver and brain samples were rapidly frozen in liquid nitrogen and then stored at -80°C until further analysis.

[0095] Measurement of metabolites in plasma.

[0096] Plasma insulin and ghrelin levels were measured using enzyme-linked immunosorbent assay (ELISA) with commercial kits from EMD Millipore Corporation (Billerica, MA, USA), Cat.#EZRMI-13K and Cat.#EZRGRT-91K, respectively. Plasma glucagon levels were measured using an EIA kit: Cat.#RAB0202Sigma-Aldrich (Saint Louis, MO, USA). All serum samples were tested in duplicate in a single assay, and results are expressed as specific standard hormones.

[0097] RNA isolation and cDNA synthesis.

[0098] According to the manufacturer's instructions (GIBCO BRL Life Technologies), use Total RNA was extracted from liver tissue fractions (100 mg to 300 mg). To ensure mRNA sequence purity, RNA samples were isolated using the RNeasy Minelute Cleanup Kit (Qiagen) according to the manufacturer's instructions, including digestion with a DNase I column (RNase-free DNase setting, Qiagen). Total RNA was quantified using a spectrophotometer (Nanodrop 1000 spectrophotometer, ThermoScientific) to ensure an A260 / 280 ratio of 1.8 to 2.0. Reverse transcription was performed from 1 μg of RNA using a Transcriptor Reverse Transcriptase kit and random hexamer primers (Transcriptor RT, Roche Diagnostic GmbH). A negative control included a reverse transcription reaction without reverse transcriptase.

[0099] Real-time quantitative polymerase chain reaction (qPCR) and gene expression analysis.

[0100] Real-time qPCR was performed according to the MIQE guidelines (Bustin, SA et al. The MIQE guidelines: minimization for publication of quantitative real-time PCR experiments. Clin Chem. 55, 611–622 (2009)). On a CFX96Touch... TM Polymerase chain reaction (PCR) was performed on each cDNA template using a real-time PCR detection system (Bio-Rad, Hercules, CA), and amplification was performed in a 20 μl reaction volume containing 9 μl of cDNA (1 / 100 dilution) and 11 μl of a premix containing primers (TaqMan, Life Technologies). The target rat gene was pyruvate kinase liver / RBC (Pklr) (Supplementary Table). Primers were based on... Gene expression assay ( Gene Expression Assays and FAM TMDye-labeled data were obtained from Life Technologies. Each reaction was performed in duplicate. Cycling parameters were as follows: 50°C for 2 min to inactivate single-stranded and double-stranded DNA containing dUTP, 95°C for 10 min to activate Taq DNA polymerase, followed by 40 cycles at 95°C for 15 sec for cDNA melting, and 60°C for 1 min to allow primer annealing and extension, during which fluorescence was obtained. Raw fluorescence data were submitted to the online Miner tool (http: / / www.miner.ewindup.info / ) to calculate Cq and efficiency values ​​for each experimental set (Zhao, S. & Fernald, RD Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction. J Comput Biol. 12, 1047–1064 (2005)). Using at least two reference rat genes: β-actin (Actb) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Cq values ​​were converted to relative expression values ​​that took into account amplification efficiency, inter-run variability, and normalization factors using Biogazelle's qbasePLUS software (Biogazelle, Zwijnaarde, Belgium) (Supplementary Table). For all reference and target gene studies, two independent biosamples for each experimental condition were evaluated as technical replicas. Reproducibility between replicates was accepted when the ΔCq value was ≤0.7. Finally, calibrated normalized relative quantity (CNRQ) values ​​were derived from qbasePLUS software and statistically analyzed.

[0101] Table 1. Primer reference for gene expression assays (Applied Biosystems).

[0102]

[0103] Protein extraction and Western blot analysis.

[0104] Brain extract. Frozen brain samples (17 mg each) 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 mixture (Roche). The suspension was incubated at 4 °C for 2 h, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to new clean centrifuge tubes, and the total protein concentration was determined using the Bradford colorimetric method. The protein extract was diluted 1:1 in loading buffer (DTT 2X) and heated at 99 °C for 5 min, followed by electrophoresis.

[0105] Western blot analysis. Expression of proteins including mTOR (289 kDa) and adaptor proteins (100 kDa) was analyzed by Western blot. Tissue proteins (10 μg to 15 μg) were electrophoresed on 4%–12% Criterion XT Precast Bis-Tris gels (Bio-Rad, Hercules, CA, EE.UU.) at 80 V for 30 min and then at 150 V for 2 h. Proteins were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules, CA, EE.UU.) at 80 V for 1 h using a wet transfer apparatus. The membranes were washed twice with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 5 min each time and blocked on a shaker platform at room temperature with 5% BSA-TBST for 1 h. Subsequently, the membranes were incubated overnight at 4°C with their 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); the α-adaptor protein was from Abcam (Cambridge, United Kingdom). The following day, the membranes were washed three times with TBST for 5 minutes each time. Appropriate HRP-binding rabbit / mouse secondary antibodies (Promega, Madison, WI, EE.UU.) were diluted 1:10000 in 2% BSA-TST and incubated with the membranes at room temperature with shaking for 1 hour. Finally, the membranes were washed as above and exposed to a chemiluminescent reagent (Santa Cruz, Biotechnology Inc., CA, EE.UU.) for 5 minutes. The individual membrane-binding proteins were then observed using chemiluminescence (ChemiDoc Imaging System, Bio-Rad). The bands were quantified using optical density analysis using ImageJ software (Rasband, WS, ImageJ, National Institutes of Health, Bethesda, MD, USA).

[0106] All data are presented as mean ± SEM. Statistical analyses were performed in GraphPad Prism version 8 (GraphPad Software, Inc., LaJolla, CA). One-way ANOVA was evaluated, followed by Turkey's Multiple Comparisons Test where appropriate. A p-value less than 0.05 was considered significant.

[0107] Results of Example 1

[0108] 1.1. Results of oral administration of pine alcohol (100 mg / kg) dissolved in sterile water to adult male Wistar rats

[0109] like Figure 2 As shown, oral administration of pinoxazone (100 mg / kg) dissolved in sterile water to adult male Wistar rats resulted in: a) an increase in circulating plasma ghrelin concentration; which was associated with B) a decrease in insulin release into the bloodstream; C) a decrease in insulin resistance as measured by the HOMA index; and D) inhibition of pyruvate kinase expression, a key enzyme that converts phosphoenolpyruvate to glucose for production.

[0110] In addition, such as Figure 3 As shown, oral administration of pinoxazone (500 mg / kg) to adult male Wistar rats resulted in: A) an increase in circulating plasma ghrelin concentration; which was associated with B) a decrease in insulin release into the bloodstream; 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 its phosphorylation.

[0111] 1.2. Results of oral administration of D-chiral inositol (500 mg / kg) dissolved in sterile water to adult male Wistar rats

[0112] like Figure 4 As shown, oral administration of D-chiroinositol (500 mg / kg) enhances ghrelin secretion, as measured by monitoring circulating plasma ghrelin concentration.

[0113] 1.3. Proposed Model of the Role of Inositol in Metabolic Aging

[0114] D-Pinol or D-Chiroinositol enhances ghrelin secretion and promotes metabolic processes, characterized by reduced insulin demand from the endocrine pancreas, increased new glucose production in the liver, increased glucose utilization by muscles associated 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 of the pancreas from age-related insulin resistance and obesity-related deterioration, enhanced muscle vitality (preventing age-specific sarcopenia and weakness), and better guidance of the body in processing glucose.

[0115] Example 2

[0116] method

[0117] Animal and ethical statements.

[0118] Animal experimental procedures were conducted in accordance with European Community Directive 2010 / 63 / EU and Spanish Regulation (Real Decreto 53 / 2013). The protocol was approved by the research department and the Animal Ethics Committee of 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. A long-term drinking experiment was conducted on 20 male Wistar rats. All adult rats, aged 2 months (approximately 300g body weight), were provided by Charles River Laboratories (Barcelona, ​​Spain). Animals were housed individually in temperature- and humidity-controlled rooms under a standard 12-hour light-dark cycle. Water and rat feed pellets were provided freely throughout the study.

[0119] Preparation and application of inositol

[0120] -D-Pinol (3-O-methyl-d-chiral-inositol, DPIN, 98% purity) and -D-chiral inositol (cis-1,2,3-trans-3,5,6-cyclohexanehexol, DCI) was provided by Euronutra SL (Málaga, Spain). They were dissolved in water and administered orally to Zucker and Wistar rats daily via drinking and / or tube feeding at a dose of 100 mg / kg BW in a volume of 1 ml / kg body weight (BW) for 4 weeks (28 days) and 10 days (3 days) respectively. Figure 1 During the drinking water treatment, the concentration of inositol in the water was updated to account for the daily increase in rat body weight (BW) and possible water loss due to evaporation.

[0121] Protein extraction and Western blot analysis.

[0122] - Brain extract. Frozen brain samples (17 mg each) 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 mixture (Hoffmann-Roche). The suspension was incubated at 4 °C for 2 h, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to new clean centrifuge tubes, and the total protein concentration was determined using the Bradford colorimetric method. The protein extract was diluted 1:1 in loading buffer (DTT 2X) and heated at 99 °C for 5 min, followed by electrophoresis.

[0123] - Western blot analysis. Tissue proteins (10 μg to 15 μg) were electrophoresed on 4%–12% Criterion XTPrecast Bis-Tris gels (Bio-Rad, USA) at 80 V for 30 min and then at 150 V for 2 h. Proteins were then transferred to 0.2 μm nitrocellulose membranes (Bio-Rad, USA) at 80 V for 1 h using a wet transfer apparatus. The membranes were washed twice with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 5 min each time, and blocked on a shaker platform at room temperature with 2% bovine serum albumin-Tris buffered saline Tween 0 (BSA-TBST) for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with their respective primary antibodies diluted in 2% BSA-TBST (see Table 2 for other information). The following day, the membranes were washed three times with TBST for 5 min each time. A suitable rabbit / mouse secondary antibody (Promega) binding to HRP was diluted 1:10000 in 2% BSA-TST and incubated with the membrane at room temperature with shaking for 1 hour. Finally, the membrane was washed as above and exposed to a chemiluminescent reagent (SantaCruz, Biotechnology Inc.) for 5 minutes. A reproving step was performed if necessary. Individual membrane-bound proteins were then observed using chemiluminescence (ChemiDoc Imaging System, Bio-Rad). Bands were quantified by densitometry using ImageJ software (Rasband, WS, ImageJ, National Institutes of Health, Bethesda, MD, USA). Normalization was performed using a reference protein, α-adaptor protein, from the same membrane. Results are expressed as protein / α-adaptor protein ratios or phosphorylated protein / total protein ratios and normalized relative to the control group (Y-axis represents the fold-mean of the control value).

[0124] Data analysis and statistics

[0125] All data are presented as mean ± SEM. Statistical analysis was performed for studies with a group size of at least n = 5. Statistical analysis was performed in GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA, USA). One-way and two-way ANOVAs were evaluated, followed by Turkey's post-hoc multiple comparisons test. Post-hoc tests were performed only if the F-value in the ANOVA was less than 0.05 and there was no statistically significant heterogeneity of variance. Student's unpaired t-test was used to analyze two separate groups. Results were considered statistically significant at P < 0.05.

[0126] Table 2. Primary antibodies used for protein expression via Western blotting.

[0127]

[0128]

[0129] Abbreviations: GSK-3β: Glycogen synthase kinase 3β; AMPK-α: Adenosine monophosphate activated protein kinase α; PTEN: Phosphatase and tensin homolog; PP2A C unit: Protein phosphatase 2A catalytic unit; PKAαcat: cAMP-dependent protein kinase α catalytic subunit; PP2C: Protein phosphatase 2C; MAPK: Mitogen-activated protein kinase; GFAP: Glial fibrous acidic protein; COX-2: Cyclooxygenase 2; Iba1: Ion calcium-binding linker 1.

[0130] result

[0131] 2.1. Effects of long-term administration of D-pineol or D-chiral inositol for 10 days on the phosphorylation status of tau protein in the hippocampus of Wistar rats.

[0132] Wistar rats were orally treated with DPIN and DCI for 10 days. Figure 6 Here, we analyzed the expression and phosphorylation of tau protein, a marker of neurodegenerative diseases such as Alzheimer's. For this purpose, we used two specific antibodies: a phosphorylated tau (AT8) antibody, which recognizes phosphorylated tau protein at serine 202 and threonine 205; and a total tau (Tau46) antibody, which recognizes both phosphorylated and non-phosphorylated isoforms. Long-term administration of DPIN and DCI had a statistically significant effect on tau protein dephosphorylation. Compared to the solvent group, the amount of phosphorylated tau decreased by more than half after administration of both compounds (one-way ANOVA: F). (2,23)=49.97, P value < 0.0001; Tukey test: P value < 0.0001, Figure 7 A.1).

[0133] Next, we quantified the total tau protein level in the same samples to assess whether the decrease in phosphorylated tau was due to a lower total tau level. A statistically significant difference was found in the relative protein levels of total tau after DPIN and DCI treatment (one-way ANOVA: F0). (2,23) =11.72, P value = 0.0003; Tukey test: P value < 0.01, Figure 7 A.2), which showed an increase compared to the solvent group. Therefore, long-term administration of DPIN and DCI for 10 days in Wistar rats not only significantly reduced phosphorylated tau but also increased total protein content.

[0134] 2.2. DPIN administration-induced inhibition of cyclin-dependent kinase 5 (CDK5) is a putative explanation for hippocampal tau dephosphorylation.

[0135] Cyclin-dependent kinase 5 (CDK5) is thought to be involved in the phosphorylation of tau protein. We investigated the protein expression levels of CDK5, its activating subunit p35, and truncated forms of p35 and p25. In Wistar rats, oral administration of DPIN via drinking significantly reduced p25 levels (unpaired t-test: t = 4.869, df = 14; p = 0.0002). Figure 8 A) and p35 subunit (unpaired t-test: t = 2.245, df = 15; p = 0.0402; Figure 8 The amount of A) was unchanged. The total amount of CDK5 remained unchanged (unpaired t-test: t = 1.623, df = 14; p = 0.1268; Figure 8 A). These results indicate that CDK5 kinase was significantly inactivated in Wistar rats after administration of DPIN.

[0136] 2.3. DPIN treatment did not affect the activity of glycogen synthase kinase 3β (GSK-3β) in the hippocampus of Wistar.

[0137] Due to the interaction between the kinase activity of GSK-3β and tau protein phosphorylation, we analyzed the expression and phosphorylation of this kinase protein. It is well known that serine 9 (S9) phosphorylation in GSK-3β results in the N-terminal tail acting as a pseudo-substrate, which hinders the binding of the actual substrate, thereby inhibiting GSK-3. This inhibition of activity translates into a reduction in tau phosphorylation, as GSK-3β is one of the major tau kinases. In this embodiment, our aim was to investigate the role of this tau kinase (GSK-3β) in Wistar rats.

[0138] Oral administration of DPIN or DCI to phosphorylate serine 9 (one-way ANOVA: F) (2,22) =1.809, P-value =0.1874; Figure 9 A.1) or tyrosine 216 (one-way ANOVA: F (2,22) =1.088, P-value =0.3543; Figure 9 A.2) had no effect. This means that there was neither enhancement nor inhibition of kinase activity (one-way ANOVA: F). (2,22) =2.473, P-value =0.1074; Figure 9 A.3). No statistically significant differences were found in the relative protein levels of total GSK-3β after administration of the three compounds (one-way ANOVA: F). (2,22) =0.5308, P-value =0.5955; Figure 9 A.4). Therefore, neither drinking DPIN or DCI for 10 days had any effect on the effect of GSK-3β in the hippocampus of Wistar rats.

[0139] 2.4. AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK / ERK1 / 2) were not associated with hippocampal tau dephosphorylation.

[0140] The kinase activation and total expression of mitogen-activated protein kinases (MAPK / ERK1 / 2), AMP-activated protein kinases (AMPK), and protein kinase A (PKA) were assessed to analyze whether these kinases were involved in the aforementioned tau results. Figure 10 As shown, the total PKA level in Wistar rats was significantly increased after administration of DPIN (unpaired t-test: t = 3.795, df = 14; P = 0.0020). Figure 10 A.2). No other changes were observed in other kinases.

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Claims

1. Use of D-pineol, D-chiral inositol and / or myo-inositol or any salt thereof in the manufacture of a composition, pharmaceutical composition or nutritional or food composition or dietary supplement for the prevention or mitigation of the onset of clinical manifestations of mild cognitive impairment or the prevention or mitigation of the onset of clinical manifestations of tau proteinosis in a subject.

2. The use according to claim 1, wherein, The composition is used to prevent or slow the onset of clinical manifestations of mild cognitive impairment in subjects in need.

3. The use according to claim 1, wherein, The composition is used to prevent or slow the onset of clinical manifestations of tau proteinosis in subjects.

4. The use according to claim 3, wherein, The tau diseases are selected from the following groups: Pick's disease, progressive supranuclear palsy, corticobasal degeneration, auricula-sagittosis, glioblastoma, primary age-related tau diseases including neurofibrillary tangles dementia, chronic traumatic encephalopathy (CTE), and age-related tau astropathy.

5. The composition for use according to any one of claims 1 to 4, wherein, The composition contains D-pineol.

6. The composition for use according to any one of claims 1 to 4, wherein, The composition is a pharmaceutical composition that also contains a pharmaceutically acceptable carrier.

7. The compound for use according to any one of claims 1 to 4, wherein, The composition is a dietary supplement.

8. The compound for use according to any one of claims 1 to 4, wherein, The composition is a nutritional composition.

9. The compound for use according to any one of claims 1 to 4, wherein, The composition is administered orally or via the stomach.

10. The composition for use according to any one of claims 1 to 4, wherein, The subjects were healthy individuals who did not exhibit any clinical signs of tau proteinosis or mild cognitive impairment.

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