Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
  • A61K31/06—Phenols the aromatic ring being substituted by nitro groups
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24011—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral endopeptidase 24.11

Definitions

• the present invention falls within the field of biotechnology applied to the medical-pharmaceutical sector for the treatment of cognitive disorders in dementias that occur with taupatias, and especially Alzheimer's.
• the present invention provides a new treatment for said diseases based on the use of 4PBA.
• AD Alzheimer's disease
• Alzheimer's disease is related to deposits of aggregate proteins: intracellular aggregates of phosphorylated tau in the neurofibrillary tangles and extracellular aggregates of ⁇ 3-amyloid (A ⁇ ) in the senile plaques. Therefore, Alzheimer's disease is a very representative example of diseases related to abnormal protein folding (Selkoe 2004).
• the endoplasmic reticulum (ER) is a site of particular importance in the process of quality control of processes related to proteins, since it regulates the synthesis, folding and trafficking of proteins in the body.
• the continuous production of proteins requires strict maintenance of quality control, which is carried out in the ER.
• ER does not always work with adequate accuracy and poorly folded proteins can accumulate inside. This situation may occur because there are proteins that are difficult to fold, such as those responsible for Alzheimer's disease, and also because the proteins are synthesized with faster than the ER can double them. This leads to a situation of "stress" of the ER.
• the sensors are activated and begin to deliver a series of signals aimed at stopping the synthesis of most of the proteins, and on the other hand to regulate the rate of doubling, so that it is possible to correctly bend all the proteins and ultimately recover the RE from your stress.
• RE stress is a key event when it comes to triggering and mediating neuronal death in Alzheimer's disease.
• Several types of cellular stress can cause the accumulation of abnormally folded proteins in the ER lumen, triggering this stress situation activating protein quality control mechanisms to prevent their toxic actions (or the response to known unfolding proteins by its acronym in English UPR "Unfolded Protein Response”).
• the activation of the UPR control mechanism produces a general reduction in protein synthesis, greater protein degradation and increasing the transcription of genes that encode molecular companions residing in the ER, such as glucose-regulated proteins (GRPs) GRP78 / Bip and GRP94, together with the protein disulphide isomerase (PDI), to facilitate protein folding (Kozutsumi et al., 1998).
• GRPs glucose-regulated proteins
• PDI protein disulphide isomerase
• the UPR is not able to resolve the stress of the ER that neuronal cells are experiencing, it is very possible that a progression of the disease will be achieved, triggering the apoptosis of neurons. Such would be the situation of the most common and devastating neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's disease, which are characterized by the accumulation and aggregation of unfolded proteins. Therefore, it is not surprising that many researchers have found evidence that under these extreme conditions the UPR is also activated. In this case this activation ends up doing more damage than benefit; The initial response is protective, but the final response is destructive.
• 4PBA is a low molecular weight fatty acid whose use has been approved in clinical practice as an ammonia sequestrant in children suffering from urea cycle disorders (Maestri et al., 1996) and in the treatment of sickle cell anemia and thalassemia due to its ability to activate transcription of fetal hemoglobulin (Dover et al., 1994; Collins et al., 1995). Its beneficial effect has also been documented in patients with Spinal Muscular Atrophy (Mercuri E, et al. 2004; Mercuri E, et al. 2007).
• 4PBA can act as a chemical companion by reversing the processes of abnormal localization and / or aggregation of proteins associated with human diseases (Rubenstein and Zeitlin, 2000; Kubota et al., 2006). Likewise, 4PBA can play a protective role in certain diseases associated with the neuroinflammatory response such as multiple sclerosis and ischemia (Dasgupta et al., 2003; Qi et al., 2004). The medical literature also cites its beneficial effects on polyglutamine toxicity (Steffan et al., 2001).
• the patent WO99 / 026657 protects the use of inducible nitric oxide synthase (iNOS) inhibitor compounds.
• iNOS inducible nitric oxide synthase
• the reference to PBA is made marginally in the context of a ratio of iNOS inhibitor compounds (lovastatin, forscolin, mevastatin, etc.).
• lovastatin, forscolin, mevastatin, etc. there is no experiment aimed at demonstrating the beneficial effect of PBA in a contrasted model of AD.
• tau phosphorylation is an integral component of neural machinery, so that a lack of regulation of this function leads to a deterioration of memory in the development of diseases such as Alzheimer's (Arendt et al., 2003; Ikeda et al., 2007).
• Histone acetylation mediates the regulation of gene transcription via chromatin modification, and therefore has recently been implicated in synaptic plasticity.
• the administration of 4PBA has been shown to increase histone acetylation in a murine model expressing amyotrophic lateral sclerosis (Petri S, et al., 2006). Consequently, it He speculated that 4PBA, by inhibiting histone deacetylase, can induce a transcriptional modification that leads to the activation of neuronal plasticity genes. Andreassi et al. (2004) directly related the administration of 4PBA with the increase in the complete expression of genes whose loss is responsible for Spinal Muscular Atrophy.
• Gene transcription requires the activation of transcription factors, but also the induction of dynamic changes in the chromatin organization that directs gene expression. Hypeacetylation of histones releases histones from their binding with chromatin, which has subsequent effects on gene transcription processes (Lea and Randolph, 1998) and protein synthesis. It has been shown that the synthesis of RNA and "de novo" proteins are necessary for the long-term memory formation process in several species of vertebrate and invertebrate animals, and that the formation of new synaptic connections will probably mediate ( Tully et al., 2003).
• the histone acetylation process controls the transcription of the genes necessary for memory consolidation and long-term synaptic potentiation (LTP) (Bailey et al., 2004, Levenson and Sweatt 2005; Fischer et al., 2007 ).
• LTP long-term synaptic potentiation
• HAT histone acetyltransferase
• HDAC histone deacetylase
• Some studies have shown that gene transcription has been deregulated in some brain regions in association with disease progression and supports the notion of a mechanism for disruption of RNA transcription in some degenerative disorders (Anderson et al., 2007).
• the GIuRl subunit of the AMPA receptor and PSD95 are postsynaptic markers of crucial importance in synapse formation and function.
• the postsynaptic deficits of PSD95 and GIuRl found in the brains of patients affected by Alzheimer's disease and in the brains of Tg2576 transgenic mice can contribute to synaptic dysfunction and cognitive function deficits (Almeida et al., 2005).
• Several studies have already outlined the spectrum of genes altered by 4PBA that prevent neurotoxicity in different experimental models (Chang and Min, 2002; Kang et al., 2002; Ryu et al., 2005).
• the pathophysiology of many degenerative disorders such as Alzheimer's disease, and also neuroprotection, probably comprise numerous mechanisms.
• these pathogenic mechanisms mainly include the aggregation and deposition of AJ3 with the development of plaques, hyperphosphorylation of tau with the formation of clews, neurovascular dysfunction, and other mechanisms such as alterations of the cell cycle , inflammatory processes, oxidative stress and mitochondrial dysfunction.
• the central hypothesis about the cause of Alzheimer's disease is the amyloid cascade hypothesis.
• Alzheimer's disease will be the great challenge of socio-health planning for the next 50 years. Since there is no effective treatment for Alzheimer's disease, this invention describes the ability of 4PBA, through its multiple mechanisms, to reverse the deterioration in the learning process and memory function characteristic of Alzheimer's disease in a model murine
• Fischer et al in 2007 is not a representative model of Alzheimer's disease, since it does not reproduce the pathogenic mechanisms related to human beta-amyloid protein or aberrant phosphorylation of tau. In particular, it only represents the deficient expression of the p25 protein, a truncated form of p35, which is a regulatory subunit of the cyclin-5 dependent kinase (CDK5).
• CDK5 cyclin-5 dependent kinase
• the inventors of the present invention using a transgenic mouse model agreed and validated for Alzheimer's disease, ie Tg2576, which expresses a variety of human beta-amyloid precursor protein (APP) related to the disease of the Alzheimer's have surprisingly found that 4-phenylbutyrate is effective in the treatment of Alzheimer's disease.
• the inventors have been able to demonstrate that clinical symptoms are improved as well as memory acquisition and retention, and most importantly, they have also shown that after administration of 4PBA, hyperphosphorylated tau levels are reduced.
• the present invention describes the potentially beneficial role of 4PBA in Alzheimer's type amnesia.
• the results obtained suggest that the effect of 4PBA is likely to be mediated, on the one hand by the protection that confers cells against the effects of endoplasmic reticulum stress by increasing levels of molecular companions and inducing tau dephosphorylation, and by another part by increasing the acetylation of histone H4 that induces the expression of synaptic markers, which in turn leads to the recovery of long-term spatial memory.
• H4 in the cerebral cortices of Tg2576 transgenic mice could be related to a very densely packed chromatin structure that would correspond to transcriptional repression.
• the present invention relates to the use of 4PBA or a pharmaceutically acceptable salt thereof for the prevention and / or treatment of Alzheimer's disease.
• the present invention relates to the use of 4PBA or a pharmaceutically acceptable salt thereof for obtaining a medicament for the prevention and / or treatment of Alzheimer's disease.
• the present invention describes a method for preventing or treating Alzheimer's disease, which comprises the administration of a pharmaceutically acceptable amount of 4PBA or a pharmaceutically acceptable salt thereof to a subject in need of such prevention or treatment.
• 4PBA used here also refers to those metabolites or compounds resulting from its metabolization in the body.
• the embodiments of the present invention related to the different therapeutic uses of this compound also comprise the use of its metabolite, that is, phenylacetate.
• the present invention relates to 4PBA or any of its pharmaceutically acceptable salts, or of a composition comprising 4PBA or any of its pharmaceutically acceptable salts, for use in the prevention or treatment of dementias that occur with taupathy, and more particularly for use in the prevention or treatment of cognitive disorders in said dementias.
• the invention also relates to the use of 4PBA or any of its pharmaceutically acceptable salts, or of a composition comprising 4PBA or any of its pharmaceutically acceptable salts, in the preparation of a medicament for prevention or treatment of dementias that occur with taupathy, and in particular of the cognitive disorders associated with these dementias.
• the invention particularly relates to the sodium salt of 4PBA for use in the prevention or treatment of dementias that occur with taupathy.
• 4PBA is 4-phenyl-butyric acid (4-phenyl-butyrate), a commercially available product, which can be obtained for example from SIGMA-ALDRICH (Product No.: P21005); It has registration number CAS 1821-12-1; Its chemical formula is C 6 H 5 (CH 2 ) 3 COOH.
• the sodium salt of 4PBA is also a commercially available product, which can be obtained for example from BIOMOL International LP (Palatine House, Matford Court, Starbucks EX2 8NL, UK; Catalog No. EI320); It has registration number CAS 1716-12-7; Its chemical formula is C 6 H 5 (CH 2 J 3 COONa; and its structural formula is:
• this compound can be obtained by known methods.
• Buphenyl ® and Ammonaps ® are trademarks of sodium phenyl butyrate compositions, authorized by the FDA and EMEA respectively, for the treatment of urea cycle disorders. It can also be found under the trademark TriButyrate ® (Triple Crown America).
• pharmaceutically acceptable means that the compound or combination of compounds must be compatible with the rest of the ingredients in a formulation, and not deleterious to the subject or patient who receives it.
• 4PBA salts are those in which the counter-ion is pharmaceutically acceptable.
• composition comprising 4PBA or a pharmaceutically acceptable salt thereof is a pharmaceutical composition, which also comprises a pharmaceutically acceptable carrier or excipient.
• the pharmaceutically acceptable salts mentioned above are considered to comprise the active non-toxic forms that 4PBA can form.
• Pharmaceutically acceptable salts can be conveniently obtained by treating the acid form of 4PBA with the appropriate cations.
• Appropriate basic salts include those formed with organic cations such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine and those formed with metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.
• salt forms can be converted into their free forms by treatment with an appropriate acid.
• salts is also intended to include the hydrates and solvates that 4PBA can form, such as, for example, alcoholates such as methanolates or ethanolates.
• solvate refers to those crystalline forms of 4PBA that comprise both stoichiometric and non-stoichiometric amounts of solvent. Since water is a solvent, solvates also include hydrates.
• pseudopolymorph is a synonym for solvate when it refers to crystalline polymorphic forms that have solvent molecules incorporated into their laminar structures. Hydrates and Alcoholates such as methanolates or ethanolates are examples of this type of solvates.
• prevention refers to the act of "preventing”, understood to prevent the occurrence, existence, or alternatively delaying, the occurrence or recurrence of a disease, disorder or condition to which said term applies, or of one or several of the symptoms associated with the disease, disorder or condition.
• treatment refers to the act of "treating”, a term that in turn is understood as reversing, alleviating or inhibiting the progression of the disease, disorder or condition to which the term applies, or of one or more of the symptoms of said disease, disorder or condition.
• “Dementias that occur with taupathy” refers to neurodegenerative diseases in which there is an alteration of the metabolism of the Tau protein and in which one of its most relevant symptoms or consequences is the appearance of cognitive disorders.
• the use of 4PBA or its salts is useful for the prevention or treatment of cognitive disorders associated with such dementias.
• the invention relates to the prevention or treatment of diseases such as Alzheimer's, frontotemporal dementia, progressive supranuclear paralysis, corticobasal degeneration, Pick's disease, Lewy body dementia, dementia with argyrophilic grains, Niemann-Pick type C disease, or pugilistic dementia.
• diseases such as Alzheimer's, frontotemporal dementia, progressive supranuclear paralysis, corticobasal degeneration, Pick's disease, Lewy body dementia, dementia with argyrophilic grains, Niemann-Pick type C disease, or pugilistic dementia.
• a more preferred embodiment of the present invention is the use of the sodium salt of 4PBA in the manufacture of a medicament for the treatment of the described diseases.
• the invention in another aspect relates to a composition comprising 4PBA or any of its pharmaceutically acceptable salts for use in the prevention or treatment of dementias that occur with taupathy, and in particular of the cognitive disorders associated with them.
• the composition comprises the sodium salt of 4PBA.
• a particular embodiment concerns a pharmaceutical composition comprising a pharmaceutically acceptable salt of 4PBA and a pharmaceutically acceptable carrier for use in the prevention and treatment of Alzheimer's disease.
• So another embodiment of the present invention to enhance its healing effect is a combination that It comprises 4PBA and / or any of its pharmaceutically acceptable salts as the first active ingredient together with a second active ingredient agent that is an inducing or facilitating agent for the clearance of cerebral ⁇ -amyloid deposits.
• a more preferred embodiment uses metal chelating agents, catabolizing enzymes of the soluble form of the ⁇ -amyloid peptide, cerebrolysin or nitrophenols as the inducing agent for said deposits.
• Another embodiment of the present invention offers an acceptable pharmaceutical combination comprising 4PBA or one of its pharmaceutical salts as the first active ingredient, a second active ingredient agent is an inducer or a facilitating agent for the clearance of ⁇ -amyloid deposits in the brain, and a pharmaceutically acceptable vehicle.
• a more preferred embodiment uses as a inducing agent for the clearance of said deposits, metal chelating agents, catabolizing enzymes of the soluble form of the ⁇ -amyloid peptide, cerebrolysin or nitrophenols.
• a more preferred embodiment is the use of clioquinol, cerebrolysin, or 2,4-dinitrophenol or 3-nitrophenol as a clearing agent.
• a more preferred embodiment is the use of clioquinol, cerebrolysin, IDE insulin degrading enzyme or neprilysin, or 2,4,4-dinitrophenol or 3-nitrophenol as a deposit clearance inducing agent.
• Another embodiment of the invention is therefore the use of a product containing 4PBA and / or any of its pharmaceutically acceptable salts as the first active ingredient, and a second active ingredient that is an inducing or facilitating agent for the clearance of ⁇ - deposits.
• cerebral amyloid as a combined preparation for simultaneous use in the prevention and / or treatment of cognitive disorders in dementias that occur with taupathy.
• this combined preparation is designed for a separate use of both active ingredients, and yet another embodiment is that it is for sequential use.
• the two active components or ingredients of the pharmaceutical combination of the invention may be part of the same unit pharmaceutical formulation; or forming part of separate pharmaceutical formulations for co-administration of the two active components or ingredients but forming part of the same therapeutic regimen.
• the two components are administered in separate formulations, they do not need to be administered at the same time, although it could be done if desired.
• the invention relates to a product or kit comprising a pharmaceutical combination comprising 4PBA and / or any of its pharmaceutically acceptable salts together with an inducing or facilitating agent for the clearance of cerebral ⁇ -amyloid deposits, in any of the specified embodiments.
• the invention relates to said pharmaceutical combination or kit comprising said pharmaceutical combination for use in the prevention or treatment of dementias that occur with taupathy, and particularly associated cognitive disorders.
• Said combination or kit is useful for the preparation of a medicament for the prevention of said dementias, in particular any of those specified above, and more particularly for Alzheimer's disease.
• the invention also relates to a method for prevention or treatment of dementias that occur with taupathy, and particularly of the cognitive disorders associated therewith, which comprises administering to a subject in need of such prevention and / or treatment a pharmaceutically effective amount of 4PBA or any of its pharmaceutically acceptable salts, or of a composition comprising 4PBA or any of its pharmaceutically acceptable salts, or one of a pharmaceutical combination comprising 4PBA and / or any of its pharmaceutically acceptable salts together with an inducing or facilitating agent for the clearance of cerebral ⁇ -amyloid deposits.
• pharmaceutically effective amount refers to an amount of compound or combination of active compounds that is effective in the treatment of the disease, which improves, attenuates or eliminates one or more of its symptoms; or prevents or delays the appearance of one or more symptoms of the disease.
• the dose of active substances of the present invention depends on each individual case and, usually, has to be adapted to the individual conditions of the case for optimal effect. . Therefore, it depends, of course, on the frequency of administration and the potency and duration of the action of the compound used in each case for therapy or prophylaxis, but also on the nature and severity of the disease and symptoms, and on sex. , age, weight, co-medication and the responsibility of the human or animal being treated and whether the therapy is acute or prophylactic.
• the therapeutically effective amount of 4PBA and / or its pharmaceutically acceptable salts should be in the following ranges, from 1 mg to 200 g per day, from 2 mg to 150 g per day, from 5 mg to 100 g per day, from 10 mg to 75 g per day, from 20 mg to 75 g per day, from 50 mg to 75 g per day, from 0.1 g to 75 g per day, from 0.2 g up to 75 g per day, from 0.5 to 75 g per day, from 1 g to 50 g per day and from 5 g to 50 g per day.
• subject means an animal, in particular a mammal such as primates, dogs, cats, bovine animals, horses, sheep and humans. It applies particularly to human mammals of both sexes.
• a preferred form of administration of the medicament preparation containing 4PBA for the uses described above is in tablets containing its sodium salt.
• the pharmaceutical preparation of these tablets may comprise excipients such as microcrystalline cellulose, magnesium stearate or colloidal silicon dioxide.
• Another preferred form of administration is a powder containing the 4PBA sodium salt and an excipient to be dissolved in water.
• the pharmaceutical preparation of this powder may comprise excipients such as calcium stearate or colloidal silicon dioxide.
• Figure IA Latency times of transgenic mice treated with 4PBA and with saline, with respect to non-transgenic mice, on different days of measurement of a Morris test in the visible platform phase.
• Figure IB Latency times of transgenic mice treated with 4PBA and saline, with respect to non-transgenic mice, on different days of measuring a Morris test in the invisible platform phase.
• Figure IC Time of permanence in the correct quadrant of the transgenic mice treated with 4PBA and with saline, with respect to non-transgenic mice, on different days of measurement of a Morris test.
• Figure 2A Levels of derivatives of peptides A ⁇ 42 and A ⁇ 40 in bark extract of transgenic mice treated with 4PBA and with saline.
• Figure 2B Plating of plaques formed by accumulation of the A ⁇ peptide in the brain of transgenic mice treated with 4PBA and with saline compared to non-transgenic mice.
• Figure 3A Relationship between phosphorylated tau levels with respect to normal protein in transgenic mice treated with 4PBA and saline, with respect to non-transgenic mice.
• Figure 3B Relationship between levels of phosphorylated GSK3 (inactive form) with respect to unphosphorylated protein (active form) in transgenic mice treated with 4PBA and saline, with respect to non-transgenic mice.
• Figure 4 GRP78 expression levels in hippocampal extracts of transgenic mice treated with 4PBA and saline, compared to non-transgenic mice.
• FIG. 5 Levels of Histones 4 (AcH4) and histone 3 (AcH3) acetylated in bark extracts of transgenic mice treated with 4PBA and saline, compared to non-transgenic mice.
• Figure 6 GIuRl, PSD95 and MAP-2 protein expression levels in membrane-enriched protein extracts obtained from the hippocampus of transgenic mice treated with 4PBA and saline, with respect to non-transgenic mice.
• Tg2576 transgenic mice with Alzheimer's disease that express the human isoform 695-aa of APP containing the Swedish double mutation APPswe [(APP695) Lys670 ⁇ Asn, Met671 ⁇ Leu] triggered by a hamster cryonic promoter.
• the content of the A ⁇ peptide in the brain accumulates exponentially between 7 and 12 months of age, and the mice so manipulated showed deterioration of the function of memory when they were subjected to the Morris water maze test when they reached ages between 12 and 15 months.
• the 16-month-old Tg2576 mouse females were treated once daily intraperitoneally with 200 mg / kg 4PBA or their vehicle for 5 weeks.
• a solution of 4PBA was prepared by titrating equimolecular amounts of 4-phenylbutyric acid (Sigma) and sodium hydroxide with a pH: 7.4.
• a control group a group of non-genetically altered mice, normal mice, of similar strains and age was used. All clinical trial procedures were performed according to European and Spanish legislation (86/609 / EEC; RD1201 / 2005).
• the labyrinth consisted of a circular pool full of water at 20 0 C. The mice received prior training in a pool with a visible platform for three consecutive days (8 tests / day) being able to swim to an elevated platform by above water level.
• the hidden platform training lasted 9 consecutive days (4 tests / day) during which the mice were allowed 60 seconds to find the submerged platform 1 cm below the water surface. The mice that found it impossible to reach the platform were guided to it. All animals were allowed a rest on the 20-second platform and then removed from it to return them to their cages. At the beginning of the 4 th, 1st and final day of the test, a pre - test during which the pool deck was removed and allowed the mice search for 60 seconds was performed. All attempts were monitored with an HVS camera and the Watermaze program to analyze the escape latencies and the percentage of occupancy time of each quadrant of the pool during the previous tests (with the Ethovision program, Wageningen, The Netherlands). Mice that were not able to learn to locate the visible platform or mice that exhibited abnormal swimming patterns or were floating persistently were excluded from the analyzes.
• mice were subjected to a previous test during which they were made to swim in the pool from which the platform had been removed for 15 seconds.
• a measure of retention memory is the percentage of 15 seconds of swimming in the quadrant in which the platform was during training sessions (target quadrant).
• the percentage of time that the vehicle-treated transgenic mice spent in the target quadrant was significantly less than that of the control group mice.
• the amount of time that the transgenic mice treated with 4PBA spent in said quadrant did not differ from that of the mice of the same age control group (Fig. IC). Although the differences were evident in the invisible platform test, the escape latencies did not differ significantly during the first training tests with visible platform (Fig. IA).
• mice were sacrificed 24 hours after they performed the last test in the Morris water maze.
• the cortex and hippocampus were dissected and used to perform biochemical analyzes.
• the levels of AJ340 and AJ342 in the cerebral cortex were then determined by a sandwich ELISA.
• extracts of frontal cerebral cortex were obtained from the mice and homogenized in a cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 mM Na 3 VO 4 , 1 mM benzamidine, 10 ⁇ g / ml leupeptin, 400 U / ml aprotinin), and se were centrifuged at 14,000 xg 4 ° C for 20 min and aliquots of the supernatant stored at -80 0 C. were obtained the total protein concentration was obtained with the Bradford BioRad Bradford (BioRad Laboratories, Hercules, California) test.
• protease inhibitors 0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 m
• Example 5 Pathological markers of Alzheimer's disease To investigate the changes that could explain the differences observed in the learning capacity of transgenic mice after treatment, phosphorylation levels of tau in the hippocampus extracted from mice were analyzed using AT8 , a phospho-specific antibody that recognizes hyperphosphorylated aberrant epitopes on Ser-202 / Thr-
• Fig. 3A The phosphorylation function of tau is regulated by several protein kinases and phosphatases. It has been shown that GSK3b participates in the pathological phosphorylation of tau. The levels of inactive GSK3b, phosphorylated in Ser 9, were measured and found to be higher in the hippocampi of transgenic mice treated with 4PBA when compared to mice treated with the vehicle, which would explain the reduction in phosphorylated form of tau observed in the treated mice ( Figure 3B).
• the next step of the investigation was to analyze the levels of the molecular companion resident in the GRP78 endoplasmic reticulum in the hippocampus of transgenic mice and bait brothers of the control group mice.
• GRP78 levels were found to be lower in the hippocampus of 16 month old transgenic mice when compared to those of mice of the same age.
• the GRP78 levels of transgenic mice treated with 4PBA did not differ from the levels of GRP78 found in non-transgenic mice, suggesting that the effect of 4PBA treatment may be due in part to an increase in the expression of the GRP78 levels (Fig. 4).
• Tg2576 16-month-old transgenic mice showed very low levels of H4 acetylation when compared with those of mice in the same age control group. Therefore, induction was found in acetylation of cortical histone 4 (H4) in transgenic mice that had been treated with 4PBA (Fig. 5).
• the GIuRl subunit of the AMPA receptor, PSD95 and MAP-2 are vitally important plasticity markers in the formation and function of the synapse.
• the microtubule association protein (MAP-2) is a protein located mainly in neuronal dendrites.
• the expression of MAP-2 coincides with the growth, branching and dendritic remodeling post-injury, suggesting that this protein plays a crucial role in plasticity.
• a quantitative analysis of MAP-2 was performed by Western Blott, and it was found that MAP-2 expression levels were significantly lower than those of the non-transgenic mouse (54.9 + 10.7) compared to non-transgenic mice (100.0 ⁇ 14.9) and partially restored after treatment with 4PBA. (77.7 ⁇ 11.6) (Fig. 6).
• Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GIuRl in synapses. Neurobiol Dis 20: 187-198.
• Kang HL Benzer S, Min KT (2002). Life extension in Drosophila by feeding a drug.
• Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response, Nat CeIl Biol 1, 479-485.
• Histone deacetylase inhibitors exhibit anti-inf lammatory and neuroprotective effects in a rat permanent ischemic model of stroke: multiple mechanisms of action. J Pharmacol Exp Ther 321: 892-901. - Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J (1998). The presence of malfolded proteins in the endoplasmic reticulum sign the induction of glucose-regulated proteins. Nature 332: 462-464.
• ASKl is essential for endoplasmic reticulum stress -induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16: 1345-55.
• Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413: 739-743.

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