• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Combined use of biotin and thiamine in the treatment of Huntington's disease. The present invention relates to the use of a combination of vitamins, more specifically it relates to the combined use of biotin and thiamine, for the treatment of Huntington's disease. More specifically, the present invention describes that treatment with a combination of biotin and thiamine is capable of improving the neurological, neuroimaging and spectroscopic symptoms associated with Huntington's disease. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2814048A1
    申请号:ES201930825
    申请日:2019-09-24
    公开日:2021-03-25
    发明作者:Lozano Jose Javier Lucas；Del Pino Sara Pico；Rodriguez Alberto Parras；Galindo Maria Santos
    申请人:Consorcio Dentro De Investig Biomedica En Red Del Area De Enfermedades Neurodegenerativas M P；Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0002] Combined use of biotin and thiamine in the treatment of Huntinqton's disease
    [0004] The present invention is encompassed in the field of medicine and pharmacy, and refers to the use of a combination of vitamins, more specifically it refers to the combined use of biotin and thiamine in the prevention and / or treatment of Huntington's disease ( HEY).
    [0006] BACKGROUND OF THE INVENTION
    [0008] Huntington's disease is an inherited neurodegenerative disorder characterized by marked striatum (St) atrophy and prominent motor symptoms caused by a repeat of CAG trinucleotides within the coding region of exon 1 of the Huntingtin gene (Htt) on chromosome 4 human, giving rise to a segment of self-aggregating polyglutamine (polyQ) in the N-terminal region of the Huntingtin protein, which leads to an accumulation of said structurally changed and modified protein and its fragments, in the brain and other organs or cells over time. In addition to the toxicity of PoliQ, there is also evidence of Htt mRNA molecule-induced toxicity with CAG expansion.
    [0010] The clinical symptoms of Huntington's disease advance in a predictable way, beginning with mood swings or cognitive problems followed by unsteady gait and motor problems, as a consequence of progressive neuronal death of different areas of the brain, which manifests itself more virulence in the medium-sized spiny neurons of the striatum and which determines the appearance of motor incoordination and the classic "chorea" type movements. Over time, physical abilities deteriorate further, with manifest movement coordination problems in combination with a further decline in mental characteristics and behavioral and psychiatric problems.
    [0012] Symptoms can vary significantly and it is well documented that age of onset is inversely correlated with the number of CAG repeats in the modified Htt gene, corroborating the causative role of modified Huntingtin.
    [0013] There is no cure or prevention for HD, nor is there a known way to stop the disease from worsening. The average life expectancy is approximately twenty years from the first clinical manifestation. The goal of treatment is to delay and reduce symptoms and to help the person with the disease to help themselves for as long as possible.
    [0015] Studies in mouse models of HD generated by genetic modification have shown that phenotypes similar to HD can be resolved if the expression of the mutant huntingtin gene is eliminated, even in advanced stages of the disease (Yamamoto et al. Cell. 2000,101: 57-66; Díaz-Hernándezetal. J. Neurosci., 2005, 25: 9773-9781). In this sense, gene therapy is an important avenue in the search for a possible treatment of the disease. Thus, the introduction of different gene constructs, at the cellular level, to inhibit the transcription or translation of the mutated protein, including replacing the defective gene with a new one, by means of gene therapy, has given good results in murine animal models of the pathology, but Unfortunately, these trials are not providing the same results in humans, since this therapy is not being effective in them, therefore this strategy is still far from being an effective strategy for its application in humans (Tabrizi SJ. et al. N Engl J Med. 2019; 380 (24): 2307-2316).
    [0017] Pharmacological therapy is the most widely used to alleviate the symptoms of Huntington's sufferers. In this sense, tetrabenazine is used for the symptomatic treatment of hyperkinetic movement disorders, such as Huntington's disease, hemiballism, senile chorea, tics, tardive dyskinesia and Tourette's syndrome. The main pharmacological effect of tetrabenazine is to reduce the supply of monoamines (eg, dopamine, serotonin, and norepinephrine) to the central nervous system by inhibiting isoform 2 of the human vesicular monoamine transporter (hVMAT2). The drug also blocks postsynaptic dopamine receptors. Tetrabenazine is an effective and safe drug for the treatment of various hyperkinetic movement disorders and, unlike typical neuroleptic agents, it has not been shown to cause tardive dyskinesia. However, tetrabenazine does exhibit a number of dose-related side effects, including depression, parkinsonism, drowsiness, nervousness or anxiety, insomnia and, in rare cases, neuroleptic malignant syndrome.
    [0018] International patent application W02006053067 describes the use of a combination of amantidine and tetrabenazine for the treatment of hyperkinetic disorders and, in particular, of Huntington's disease. There is a serious side effect of this tetrabenazine drug, which triggers or worsens depression and other psychiatric disorders.
    [0020] Despite the fact that in recent years great progress has been made in terms of understanding the etiology, genetics and pathophysiology of HD, the currently available therapies are palliative and also have multiple side effects. The absence of drugs that act to prevent or significantly delay the development of this type of disease means that new alternative therapies are required to effectively reduce the progression of the disease. Therefore, there is still a need for new therapies capable of preventing or delaying the development of HD.
    [0022] DESCRIPTION OF THE INVENTION
    [0024] The inventors have observed that the administration of a combination based on vitamins, preferably a combination of biotin and thiamine, is capable of improving motor symptoms and reducing striatal alterations, particularly reducing striatum atrophy and increasing levels of phosphocreatine, associated Huntington's disease.
    [0026] Thus, first of all, the inventors have observed that the cytoplasmic polyadenylation element binding proteins (CPEB; Cytoplasmic polyadenylation element binding proteins), more specifically the CPEB1 and CPEB4 proteins, responsible for regulating the translation of numerous mRNAs by modifying their poly (A) tail show altered expression in the striatum of subjects suffering from HD compared to healthy control subjects (Example 1). The alteration in the levels of these proteins led to the analysis of the level of transcriptome polyadenylation in striatum tissue samples from murine HD models that show similar alterations in the levels of CPEB1 and CPEB4 (Example 2). This analysis revealed that one of the genes that showed a greater shortening of the poly (A) tail is the SLC19A3 gene that codes for a thiamine transporter, specifically for the ThTr2 transporter (Example 3). A shortening of the poly (A) tail correlates with a lower level of mRNA translation and, therefore, a lower level of protein, which led to suspect a deficiency of the ThTr2 transporter (SLC19A3) in subjects with HD. In fact, this was corroborated in brain samples from striatum from subjects with HD, where a decrease in the thiamine transporter SLC19A3 was observed (Example 3).
    [0028] In the brain, the thiamine transporter SLC19A3 is located mainly in blood vessels and neurons and, as observed in Example 3, the inventors have detected a decrease in thiamine in the cerebrospinal fluid (CSF) of subjects with the EH. This decrease is mainly observed in the monophosphate form of thiamine (TMP), which is the majority in CSF. In the interior of cells and tissues, on the other hand, the majority form is thiamine pyrophosphate (TPP) which is the one that has a biological role as a cofactor of multiple metabolic enzymes and the inventors have observed a decrease in the percentage of TPP in homogenates of striatum tissue samples from HD subjects.
    [0030] It is known that subjects suffering from biotin-thiamine-sensitive basal ganglia disease (BTBGD) present mutations in the SLC19A3 gene, causing the protein encoded by said gene to not adequately fulfill its function, decreasing the absorption of thiamine in cells, with serious consequences for the health of affected individuals, such as encephalopathy (lethargy, stupor, ...), movement disorders, speech difficulties or loss of speech, swallowing difficulties, epileptic seizures, etc. These symptoms characteristically respond to treatment with high-dose biotin and thiamine, hence the name given to this form of the disease.
    [0032] As mentioned above, the inventors have discovered a decrease in SLC19A3 protein levels in the striatum of HD subjects, as well as a decrease in thiamine levels in the cerebrospinal fluid (total and TMP) and in the brain. of these subjects with HD (PPD). These results made the inventors suspect that the administration of a combination of vitamins based on thiamine and biotin would improve the symptoms of HE, since the subjects suffering from BBGD respond favorably to the administration of thiamine and biotin, since this The latter increases the expression of the transporter. Thus, as observed in Example 4, the combined administration of thiamine and biotin in murine models of HD improves the symptoms of the disease and attenuates morphological and metabolic alterations. striatum such as atrophy and increased phosphocreatine, which are also associated with the disease. Therefore, the invention relates to the use of a combination of thiamine and biotin for the treatment of HD. This discovery opens a new therapeutic window for the treatment of this pathology not previously contemplated in the state of the art.
    [0034] Compositions comprising a combination of thiamine and biotin for use in the treatment of HD are also an object of the invention. Therefore, the invention also relates to a method of treating a subject suffering from HD, comprising the step of administering a therapeutically effective amount of a combination of thiamine and biotin to said subject.
    [0036] Thus, in a first aspect the present invention relates to a composition, preferably a pharmaceutical composition comprising a therapeutically effective concentration of biotin and thiamine for use in the prevention and / or treatment of Huntington's disease. Hereinafter, this first aspect of the invention will be referred to as the composition for use of the invention.
    [0038] The term "composition", "pharmaceutical composition", or "medicine", used interchangeably throughout the document, refers to any substance or combination of substances, used for the prevention, diagnosis, relief, treatment or cure of diseases in the man or animals. In the context of the present invention it refers to a composition capable of preventing and / or treating HD. The pharmaceutical composition of the invention can be used alone or in combination with other pharmaceutical compositions.
    [0040] As used herein, "treatment", "treating" or "treating" refers to: (a) preventing the onset of the disease or condition in a subject who may be predisposed to the disease or disease but not yet diagnosed; (b) the inhibition of the disease or disease, that is, the interruption of its development; (c) the relief or improvement of the disease or disease, that is, the induction of regression of the disease or condition; or (d) curing the disease or condition, that is, the interruption of its development or progression. The population of subjects treated by the composition of the composition of the invention includes those subjects who suffer from the undesirable ailment or disease, as well as the subjects at risk of development of the disease or illness.
    [0042] The terms "disorder" and "'disease" are used interchangeably to refer to a condition in a subject or individual.
    [0044] In the present invention, the terms "subject" and "individual" are used interchangeably. As used herein, the term "subject" or "individual" refers to all animals classified as mammals and includes, but is not limited to, farm and domestic animals, primates, and humans, eg, humans, primates. non-humans, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a human, male or female, of any age or race, suffering from HD.
    [0046] Biotin (Formula I), also known as vitamin H, vitamin B7, or vitamin B8, is a water-soluble vitamin that occurs naturally in many foods, such as organ meats, eggs, and certain vegetables. In mammals, biotin acts as a cofactor for four metabolic carboxylases involved in several key stages of energy metabolism, including pyruvate carboxylase (neoglycogenesis), 3-methylcrotonyl CoA, and propionyl CoA carboxylases (catabolism of certain amino acids that supply metabolites to the Krebs cycle. intermediates), and acetyl CoA carboxylase (fatty acid synthesis). Consequently, the mechanism of action of biotin can be seen as an enhancer of energy production for the brain (ATP). There is evidence that biotin is also capable of regulating gene expression through mechanisms independent of its function as a prosthetic group of carboxylases, such as, for example, the regulation of the transcription of the SLC19A3 gene. Biotin is composed of a ureido (imidazoline) ring fused with a tetrahydrothiophene ring. A substitute valeric acid binds to one of the carbon atoms of the tetrahydrothiophene ring. There are three forms of biotin: free biotin, biocytin (e-biotin-L-Lysine) and two sulfoxides L and D of biotin. Biotin's CAS number is 58-85-5.
    [0049] Formula I: Biotin
    [0051] For the purposes of the present invention, the term "biotin", "vitamin H", "vitamin B7" or "vitamin B8", refers to the biotin compound itself, to any of its salts and / or its derivatives, which have the same biological functionality as biotin.
    [0053] Thiamine (Formula II), also known as vitamin B1 or aneurin, is a water-soluble, alcohol-insoluble vitamin that occurs naturally in many foods, such as beef, chicken, cereals, nuts, and beans. Vegetables provide free thiamine while meats provide mostly thiamine diphosphate (TDP, also called thiamine pyrophosphate or TPP). Absorption occurs in the small intestine (jejunum, ileum) being favored by the presence of vitamin C and folic acid, but inhibited by the presence of ethanol (ethyl alcohol). It is necessary in the daily diet of most vertebrates and some microorganisms. Its deficiency in the human body causes diseases such as beriberi and Korsakoff syndrome. The majority and biologically active form of thiamine is TDP, which acts as a cofactor for numerous metabolic enzymes. The CAS number for thiamine is 59-43-8.
    [0057] Formula II: Thiamine
    [0059] For the purposes of the present invention, the term "thiamine" or "vitamin B1" refers to compound of thiamine itself, to any of its salts and / or its derivatives, which have the same biological functionality as thiamine.
    [0061] The term "therapeutically effective amount" as used herein refers to the amount of a compound or compounds that, when administered, is sufficient to prevent the development of, or alleviate to some degree, one or more of the symptoms of the disease you are targeting. The particular dose of each compound administered according to this invention will of course be determined by the particular conditions surrounding the case, including the compound administered, the route of administration, the particular condition being treated, as well as considerations such as age, weight. and sex of the treated subject.
    [0063] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a human dose can be formulated to achieve a concentration that has been found to be effective in animals. Dosage amounts and ranges can be individually adjusted to provide effective administered compound levels for the particular clinical indication being treated. This will provide a therapeutic regimen commensurate with the severity of the individual's disease state. Thus, adjusting the dose to achieve maximum efficacy in humans is within the capabilities of one of skill in the art. Reagan-Shaw S.'s article " Dose translation from animal to human studies revisited." FASEB J 2007, 22: 659-661, provides the standard conversion factors used to convert mg / kg to mg / m2. The article also explains that this conversion is the basis for converting the dose in a first animal species into a second animal species (allometric dose translation). Therefore, the animal dose (DA) in mg / kg can be converted to human equivalent dose (HED) in mg / kg using the following formula:
    [0065] Animal Km
    [0066] DEH (mg / kg) = DA (mg / kg) X .................................
    [0067] Human Km
    [0069] where the Km factor for each species is shown in Table 1 (data extracted from Reagan-Shaw S. " Dose translation from animal to human studies revisited '. FASEB J 2007, 22: 659-661).
    [0070] Table 1. Km factor for the conversion of DA to DEH.
    [0075] Using the teachings provided herein, one can plan an effective therapeutic treatment regimen that does not cause substantial toxicity and yet is effective in treating the clinical symptoms demonstrated by a particular HD subject. This planning should involve the careful choice of the active compound considering factors such as the potency of the compound, the relative bioavailability, the subject's body weight, the presence and severity of adverse side effects, the preferred mode of administration, and the toxicity profile of the drug. selected agent.
    [0077] In a preferred embodiment, the concentration of biotin in the composition of the invention comprises at least 0.40 mg / kg / day. In a more preferred embodiment, the concentration of biotin in the composition of the invention is selected from the list consisting of: at least 0.40 mg / kg / day, at least 1 mg / kg / day, at least 1.5 mg / kg / day, at least 2 mg / kg / day, at least 2.5 mg / kg / day, at least 3 mg / kg / day, at least 3.5 mg / kg / day, at least 4 mg / kg / day, at least 4.5 mg / kg / day and at least 5 mg / kg / day.
    [0079] In another preferred embodiment, the thiamine concentration in the composition of the invention comprises at least 2 mg / kg / day. In a more preferred embodiment, the concentration of thiamine in the composition of the invention is selected from the list consisting of: at least 2 mg / kg / day, at least 2.5 mg / kg / day, at least 3 mg / kg / day, at least 4 mg / kg / day, at least 5 mg / kg / day, at least 10 mg / kg / day, at least 15 mg / kg / day, at least 20 mg / kg / day, at least less 25 mg / kg / day, at least 30 mg / kg / day, at least 35 mg / kg / day, at least 40 mg / kg / day, at least 45 mg / kg / day, at least 50 mg / kg / day, at least 55 mg / kg / day, at least 60 mg / kg / day, at least 65 mg / kg / day, at least 70 mg / kg / day, at least 75 mg / kg / day , at least 80 mg / kg / day, at least 85 mg / kg / day, at least 90 mg / kg / day, at least 95 mg / kg / day and at least 100 mg / kg / day.
    [0081] In another more preferred embodiment, the composition for use of the invention further comprises at least one pharmacologically acceptable excipient and / or vehicle.
    [0083] In the present invention, the term "excipient" refers to a substance that assists in the absorption of any of the components of the composition of the present invention, stabilizes said components or assists in the preparation of the composition in the sense of providing consistency. or to contribute flavors that make it more pleasant. Thus, excipients can have the function of keeping the components together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protection function against drugs such as insulation from air and / or humidity, the function of filling a tablet, capsule or other form of presentation such as, for example, dibasic calcium phosphate, a disintegration function to facilitate the dissolution of the components and their absorption in the intestine, without excluding any other type of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as the material included in the galenic forms, is added to the active ingredients or their associations to facilitate their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the pharmaceutical composition and its bioavailability. Preferred excipients for use in the present invention include sugars, starches, celluloses, gums, and proteins.
    [0085] Examples of pharmaceutically acceptable carriers are known in the state of the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, different types of wetting agents, sterile solutions, etc. Compositions comprising said carriers can be formulated by conventional procedures known in the state of the art. As used herein, the terms "pharmaceutically acceptable,""physiologicallytolerable," and grammatical variations thereof, as they relate to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the materials they are capable of being administered to a subject without producing undesirable physiological effects.
    [0086] In another more preferred embodiment, the active principles that are part of the composition for use of the invention, that is, biotin and thiamine, are adapted for their separate, simultaneous or sequential administration, or by juxtaposition, by any convenient route of administration, both in separate or combined compositions.
    [0088] By the term "active principle" is meant that substance or molecule that is biologically active. It is also known by "pharmaceutically active principle" or API ( Active Pharmaceutical Ingredient).
    [0090] By "separate administration" is understood the individual administration of each of the active principles of the composition of the invention. Said individual administration of each active principle can be simultaneous or sequential. Separate administration is understood to be "simultaneous" when the administration of the active ingredients is carried out at the same moment in time. On the contrary, it is understood that the separate administration is "sequential", when the administration of the active principles is carried out at different moments in time, that is, one active principle is administered first and then the other is administered. Thus, in a particular embodiment, the administration of biotin is prior to the administration of thiamine or vice versa.
    [0092] By "juxtaposed administration" is understood to be the joint administration of both active principles, that is, biotin and thiamine are administered together in a single composition, which is administered to the subject.
    [0094] The composition of the invention, as well as the active principles that comprise it, can be administered by any route or route of administration. Examples of routes of administration include, but are not limited to, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, and parenterally (intravenously). In a particular embodiment, the composition of the invention and / or the active principles that comprise it, are administered orally or parenterally, more preferably orally. Thus, both the composition of the invention and the active principles that comprise it will be formulated in a suitable way for the chosen route of administration, such as, for example, in a solid pharmaceutical form of administration (for example, tablets, capsules, dragees, granules. , suppositories, sterile crystalline or amorphous solids that can be reconstituted to provide liquid forms etc.) or liquid (e.g. solutions, suspensions, emulsions, elixirs, lotions, ointments etc.), preferably in unit dosage forms suitable for simple administration of precise doses.
    [0096] In another preferred embodiment, the composition for use of the invention is repeatedly administered to a subject suffering from HD.
    [0098] In another more preferred embodiment, the composition for use of the invention may additionally comprise at least one additional active ingredient to those mentioned above.
    [0100] In a more preferred embodiment, the additional active ingredient that is part of the composition for use of the invention is selected from the list consisting of: tetrabenazine, haloperidol, chlorpromazine, risperidone, quetiapine, amantadine, levetiracetam, clonazepam, citalopram, escitalopram , fluoxetine, sertraline, quetiapine, risperidone, olanzapine, valproate, carbamazepine, lamotrigine and / or any of their combinations.
    [0102] In another aspect, the present invention also provides methods for preventing and / or treating HD which comprises administering a therapeutically effective amount of the composition of the invention to a subject in need, as previously described throughout the present document.
    [0104] Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0106] BRIEF DESCRIPTION OF THE FIGURES
    [0108] FIG. 1. Analysis of the levels of the binding proteins to the cytoplasmic polyadenylation element 1, 2, 3 and 4 (CPEB1 to 4) at the protein level by means of Western blot (A, B and E) and of mRNA by qRT-PCR (C and D) in the striatum of: subjects suffering from HD (HD) and control subjects (CTRL) (A) (C), R6 / 1 mice and control mice (CTRL) ( B) (D), zQ175 mice and control mice (CTRL) (E). The bar graphs show the mean ± sem
    [0109] FIG. 2. Poly (U) chromatography. (A) Percentage of genes whose transcripts have shortened, elongated or unaltered poly (A) tails in the striatum (St) of R6 / 1 mice.
    [0110] (B) Gene ontology analysis of mRNAs with changes in their poly (A) tail in St of R6 / 1. (C) Venn diagram of altered poly (A) transcripts implicated in neurodegenerative diseases. (D) Percentage of transcripts with CPE sequences in their 3 'UTR as a function of the state of their poly (A) tail in R6 / 1 mice.
    [0111] FIG. 3. Protein and mRNA levels of proteins and genes showing poly (A) tail shortening in R6 / 1 mice. Western Blot of the AUTS2, ROCK1 and KTN1 proteins in the striatum of: (A) R6 / 1 mice and control mice (CTRL) and (B) subjects suffering from HD (HD) and control subjects (CTRL). To the right of each panel are also shown the mRNA levels obtained by RT-PCR for each of the genes that code for the aforementioned proteins.
    [0112] FIG. 4. Analysis of the gene and protein expression of SLC19A3 and thiamine in subjects with HD. (A) SLC19A3 mRNA and protein levels in striatum and cortex of HD subjects (HD) and control subjects (CTRL). (B) Immunohistochemical photographs showing SLC19A3 immunolocation in striatum (St) and cortex (Cx) sections of HD subjects (HD) and healthy control subjects (CTRL). (C) Concentration of thiamine pyrophosphate (TPP), thiamine monophosphate (TMP) and free-thiamine, as well as the sum of the three (total) in cerebrospinal fluid (CSF) and total thiamine concentration in blood of healthy control subjects (CTRL) and subjects with HD (HD). (D) Percentage of thiamine forms: thiamine pyrophosphate (TPP), thiamine monophosphate (TMP) and thiamine-free in striatum of healthy control subjects (CTRL) and HD subjects. The bar graphs show the mean ± sem
    [0113] FIG. 5. Thiamine levels in murine HD models. (A) Concentration of thiamine monophosphate (TMP) in cerebrospinal fluid (CSF) and total thiamine concentration in blood of control (CTRL) and R6 / 1 mice and control (CTRL) and zQ175 mice. (B) Percentage of thiamine forms: thiamine pyrophosphate (TPP), thiamine monophosphate (TMP) and thiamine-free in striatum of control mice (CTRL) and R6 / 1 and control mice (CTRL) and zQ175. The bar graphs show the mean ± sem
    [0114] FIG. 6. Phenotypic analysis of R6 / 1 mice treated with thiamine, biotin or both.
    [0115] Percentage of (A) distance covered in the open field test at 13 weeks and (B) time it takes to fall in the Rotarod test at 18 weeks for untreated (H 2 O) or biotin-treated ( Bio) or thiamine (Tia) relative to untreated (H20) or biotin (Bio) or thiamine (Tia) treated WT mice. (C) Percentage of time it takes untreated (H20) or biotin and thiamine (T + B) treated R6 / 1 mice to fall into the Rotarod test at 18 weeks compared to untreated WT mice (H20) or treated with biotin and thiamine (T + B).
    [0116] FIG. 7. Magnetic resonance analysis of zQ175 mice treated with thiamine and biotin. Magnetic resonance analysis of the striatum volume of zQ175 and WT mice at the ages of (A) 4 months (pretreatment) and (B) 7 months under control conditions (H20) or treated with biotin and thiamine (B + T). (C) Striatum phosphocreatine levels of untreated (H20) or biotin and thiamine (B + T) treated zQ175 mice, relative to untreated WT (H20) mice analyzed by magnetic resonance spectroscopy (MRS).
    [0118] EXAMPLES
    [0120] The invention will be illustrated below by means of tests carried out by the inventors, which show the effectiveness of the product of the invention.
    [0122] Materials and methods
    [0124] Human tissue samples
    [0126] Brain samples from subjects suffering from HD and from healthy controls were provided by the Neuropathology Institute of the Brain Bank (HUBICO-IDIBELL, Hospitalet de Llobregat, Spain), the Banco de Tejidos Fundación Cien (BT-CIEN, Madrid , Spain) and the Netherlands Brain Bank (Amsterdam, The Netherlands). Written informed consent for brain removal after death for diagnostic and research purposes was obtained from brain donors and / or close family members. Blood samples and cerebrospinal fluids were provided by the Biobank of the Ramón y Cajal University Hospital after informed consent by the subjects studied.
    [0127] Mice
    [0129] Two mouse models were used: (1) R6 / 1 mice expressing human exon-1-Htt as a transgene and having a B6CBAF1 genetic background (Mangiarini et al., Cell.
    [0130] 1996; 87 (3): 493-506) and (2) zQ175 mice with heterozygous knock-in of an expanded CAG lane in exon 1 of the Htt gene and having a C57BL / 6J genetic background (Menalled et al., PLoS One. 2012; 7 (12): e49838). Non-transgenic littermates (B6CBAF1 for R6 / 1 and C57BL / 6J for zQ175) were used as control (CTRL) or wild-type (WT) animals. Mice were housed with mixed genotypes four per cage and with food and water available ad libitum. They were kept in a controlled temperature environment in a 12/12 h light / dark cycle with light onset at 08:00. All mice were kept in the animal facility of the Centro de Biología Molecular Severo Ochoa (CBMSO). The animal housing and maintenance protocols followed the guidelines of the local authorities. The animal experiments were carried out under the protocols (P15 / P16 / P18 / P22) approved by the Institutional Committee for the Care and Use of Animals of the Severo Ochoa Molecular Biology Center (CBM Animal Experimentation Ethics Committee, CEEA-CBM) and Community of Madrid PROEX 293/15.
    [0132] The treatment based on thiamine and / or biotin was administered orally in the drinking water, with mice with normal drinking water serving as control of the treatment and non-transgenic littermates serving as control of the genotype.
    [0134] In the case of R6 / 1 mice, treatment began at 3 weeks of age, just after weaning. Biotin treatment consisted of a dose of 10 mg / kg / day, thiamine treatment began with a dose of 200 mg / kg / day that was decreased to 50 mg / kg / day after 18 weeks, and Combination treatment with thiamine and biotin consisted of a 5 mg / kg / day dose of biotin 100 mg / kg / day thiamine, which was reduced to 25 mg / kg / day at the age of 18 weeks. The different treatments were maintained until sacrifice, by cervical dislocation, at the age of 7-8 months, therefore the treatment had a total duration of approximately 32 weeks. The reason for reducing thiamine doses from week 18 is that, in a pilot group of mice in which the dose was not reduced, possible toxicity was detected in R6 / 1 evidenced by an increase in the volume of drink consumed and urination, these effects were no longer observed by reducing the thiamine concentration to a quarter of the initial concentration, after 18 weeks of treatment.
    [0136] In the case of zQ175 mice, the combined treatment with thiamine and biotin consisted of a dose of 5 mg / kg / day of biotin 100 mg / kg / day thiamine with a duration of 3 to 6 months until sacrifice, by cervical dislocation. , at the age of 7 months.
    [0138] In all cases, the number of animals included in each group is mentioned in each example.
    [0140] Analysis of chemical ontology
    [0142] Co-immunoprecipitated mRNAs of CPEB4 from RWP-1 cells were analyzed from the tables published in Ortiz-Zapater et al. (Nat Med. 2011; 18 (1): 83-90) and transcripts with changes in the length of the poly (A) tail (see section “PoliU Chromatography”) in the striatum of symptomatic R6 / 1 mice (with expressed increase in number of times the initial value, “fold change” or “fe” <-2 or> 2) with DAVID Bioinformatics Resources 6.7 ( .Laboratory of Immunopathogenesis and Bioinformatics ; LIB) using the KEGG path annotation (Huang da et al. ., NucleicAcids Res. 2009; 37 (1): 1-13).
    [0144] Sequence analysis of cytoplasmic polyadenylation elements (CPE)
    [0146] CPEs are the best characterized elements that regulate cytoplasmic polyadenylation of mRNAs. The 3'UTR (untranslated region) sequences of the gene sets whose poly (A) is shortened, elongated or unaltered in R6 / 1 mice (based on an "faith" <- 1.5 or> 1.5 ) were extracted from Ensembl (http://www.ensembl.org/) and the incidence of canonical and functional CPEs was detected using the algorithm described in Pique et al. (Cell. 2008; 132 (3): 434-48) (http://genome.crg.es/CPE/).
    [0148] Western blot
    [0150] The human brain samples were stored at -80 ° C and crushed with a mortar in a frozen environment with liquid nitrogen to avoid thawing of the samples, resulting in tissue powder. Murine brain samples they were rapidly dissected after death on a frozen plate and the different structures were stored at -80oC.
    [0152] Human and murine brain extracts were prepared by homogenizing brain samples in ice cold extraction buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 20 mM NaF, 1% Triton X-100, sodium orthovanadate 1 mM, 1 pM okadaic acid, 5 mM sodium pyrophosphate, 30 mM p-glycerophosphate, 5 mM EDTA, protease inhibitors (Complete, Roche, Cat. No. 11697498001)). The homogenates were centrifuged at 15,000 g for 15 min at 4 ° C. The resulting supernatant was collected and the protein content was determined by the Bradford assay using the Quick Start kit (Bio-Rad, 500-0203). 10 and 20pg of total protein were subjected to 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (Amersham Protran 0.45 pm, GE Healthcare Life Sciences, 10600002) and blocked in TBS-T (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20, supplemented with 5% skimmed milk powder). The membranes were incubated overnight at 4 ° C with the primary antibody in TBS-T supplemented with 5% skimmed milk powder, washed with TBS-T and then incubated with the corresponding secondary antibody, developing using the kit of ECL detection (PerkinElmer, NEL105001EA). The images were scanned with a densitometer (Bio-Rad, GS-900) and quantified with Image Lab 5.2 (Bio-Rad).
    [0154] The following primary antibodies generated in rabbit were used: CPEB1 (1: 350, Santacruz, sc-33193); CPEB2 (1: 1000, Abcam, ab51069); CPEB3 (1: 1000, Abcam, ab10883); CPEB4 (1: 1000, Abcam, ab83009); AUTS2 (1: 750, Sigma, HPA000390); KTN1 (1: 1000, Proteintech, Cat. 19841-1-AP); ROCK1 (1: 1000, Abcam, ab45171); SLC19A3 (1: 1000, Sigma, HPA038898); vinculin (1: 1000, Abcam, ab129002); as well as mouse-generated anti-p-actin (1: 25000, Sigma, A2228).
    [0156] The secondary antibodies used were: anti-rabbit or anti-mouse IgG conjugated with HRP (1: 2000, DAKO, P0447 for mouse and P0448 for rabbit).
    [0158] Poly (U) Chromatography
    [0160] After sacrifice of control WT and R6 / 1 mice (n = 4) by cervical dislocation at the age of 7-8 months, the striatum was rapidly dissected on a chilled plate. with ice and dipped into ice cold RNAIater (Sigma, R0901). The striations were then homogenized and the total RNA was purified with Ultraspec (Biotecx, BL-10050), frozen and stored at -80 ° C until use.
    [0162] The poly (A) RNA fraction was purified by poly (U) chromatography. Poly (U) -agarose (Sigma, p8563) was dissolved in 35 ml / g of expansion buffer (0.05 M Tris-HCl, pH 7.5, 11 M NaC), incubated overnight at room temperature and was loaded onto the chromatography column. An aliquot of total RNA was stored at -80 ° C ("Entry") and the remainder was incubated with sample buffer (0.01 M Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS) for 5 min at 650C and subsequently cooled on ice. Next, binding buffer (0.05 M Tris-HCl, pH 7.5, 0.7 M NaCl, 10 mM EDTA, 25% [v / v] formamide) was added and the sample was loaded onto the column. chromatography of poly (U) -agarose (Mobitec, M1002s) and incubated for 30 minutes at room temperature (250C) with shaking. Subsequently, the column was washed three times at 25 ° C and six times at 550C with washing buffer (0.05 M Tris-HCl, pH 7.5, 0.1 M NaCl, 10 mM EDTA, 25% [v / v] formamide). Washes carried out at 550 ° C were collected and stored at -80 ° C ( "WASH" or "short poly (A) tail fraction"). The remaining poly (A) RNA ( "ELUTED" or "poly (A) long tail fraction") was eluted with elution buffer (0.05 M HEPES, pH 7, 10 mM EDTA, 90% [v / v ] formamide) at 550 ° C and stored at -80 ° C.
    [0164] The RNA from the two poly (A) fractions, the short poly (A) tail fraction and the long poly (A) tail fraction, was precipitated by adding 1 volume of isopropanol, 1/10 volumes of sodium acetate 3 M pH 5.2 and 20 pg glycogen (Sigma, G1767). The samples were incubated at -200C for 20 min and centrifuged for 15 min at 14000 g at 40C. The supernatant was removed and the precipitate was washed with 750 µl of ethanol and centrifuged at 14000 g and 4 ° C for 5 min. The supernatant was removed and the precipitate was air dried for 5 min. The RNAs were resuspended in 300 µl of nuclease-free water and 300 µl of phenol acid: chloroform (5: 1) was added. The samples were vortexed and centrifuged for 10 minutes at 14000 g and 40C.
    [0166] RNA quantification was performed by Qubit fluorimeter using the Qubit RNA Hs assay kit (Thermo-Fisher Scientific, Q32852). Quality control of RNA integrity was performed with Agilent Bioanalyzer 2100, using the RNA Nano Assay (Agilent Technologies 5067-1511) and the RNA Pico Assay (Agilent Technologies 5067-1513).
    [0168] The preparation of the cDNA library and its amplification was carried out with the WTA2 kit using a template of 2 to 5 ng of total RNA and following the manufacturer's instructions (Sigma-Aldrich). The cDNA was amplified for 22 cycles and purified using the PureLink Quick PCR Purification Kit (Invitrogen, K310001). The quantification of the amplified cDNA was carried out in a Nanodrop ND-1000 (Thermo-Fisher Scientific), and 8 pg of the cDNA of each sample was fragmented and labeled with the GeneChip Mapping 250K Nsp assay kit (Affymetrix, 900753) following the instructions manufacturer.
    [0170] Hybridization was performed using the GeneAtlas Hyb, Wash and Stain kit for 3 'IVT arrays. Ready-to-hybridize samples were denatured at 960C for 10 minutes before incubation in a MG-430 PM mouse array (Affymetrix, 901570) and hybridization was performed for 16 hours at 450C in the GeneAtlas hybridization oven (Affymetrix, 00- 0331). After this time, washing and staining were carried out in the GeneAtlas Fluidics Station (Affymetrix, 00-0079), following the specific command sequence for MG-430 PM mouse arrays. Finally, the arrays were scanned with the GeneAtlas Scanner (Affymetrix) using predetermined parameters, and the generation of CEL files for their bioinformatic analysis was performed with the GeneAtlas software (Affymetrix).
    [0172] The processing of the arrays was carried out using R (R Development Core Team. R: A language and environment for statistical computing, R Foundation for Statistical Computing, 2014) and Bioconductor (Gentleman et al., Genome Biol. 2004; 5, R80) . The raw CEL files were normalized using RMA background correction and the abstract was generated as indicated in Irizarry RA. et al. (Stat Appl Genet Mol Biol. 2003; 2: Article1). Standard quality controls were performed to identify abnormal samples, as outlined in Gentleman R. et al, 2005 (Springer, New York) regarding: (a) spatial artifacts in the hybridization process (scan images and pseudo - images of probe level models); (b) intensities dependent on the differences between chips (MvA graphics); (c) RNA quality (RNA digest plot); and (d) overall intensity levels (boxplot of perfect match record intensity distributions before and after normalization and RLE plots). Probe annotation was performed using the information available on the Affymetrix website (https://www.affymetrix.com/analysis/index.affx) using version na35.
    [0174] Expression values were adjusted for technical biases as described in Eklund and Szallasi (Genome Biol. 2008; 9, R26) using a linear model and implemented with the R package "limma" (Ritchie et al., Nucleic Acids Res. 2015; 43, e47). For each biological replica, the change in log2 value was calculated between the "WASH" and " ELUTED " samples and used to find significant differences between WT and R6 / 1 control mice. Differential expression was performed using a linear model with fluidics mechanisms and amplification batches as covariates. Values of "fold change" <-1.5 in at least one probe mean that the poly (A) tail is shortened in the transcript of said gene in R6 / 1 mice,> 1.5 in at least one probe means that it is lengthened and intermediate values mean there are no changes. If the same transcript showed opposite results for different probes, it was considered unchanged.
    [0176] Quantitative real-time PCR
    [0178] Quantification was performed by real-time PCR using a CFX 384 Real Time System C1000 thermal cycler (Bio-Rad) in combination with SsoFast Eva Green (Bio-Rad, CN 172-5204) and 0.25 pM of each pair of primer oligonucleotides. The data were analyzed by GenEx 5.3.7 software (Multid analysis AB). The mRNA levels were normalized first in relation to total RNA and then in relation to the expression of the following genes: 18S ribosome subunit, p-Actin, GAPDH and p-Tubulin.
    [0180] Table 2. Human primers
    [0182]
    [0183]
    [0185] For the amplification of the GAPDH (qA-01-0101S) and p-ACTIN (qA-01-0104S) genes, commercial primers obtained from Tataa biocenter (Vestec, Czech Republic) were used.
    [0186] Table 3. Mouse primers.
    [0188]
    [0191] Immunohistochemistry
    [0193] The human brain samples (cortex and striatum) were fixed with formalin (4%, 24 h), and embedded in paraffin. Sections (5 pm thick) were mounted on superfrost-plus tissue slides and deparaffinized. Peroxidase activity was stopped with 0.3% H 2 O 2 in methanol for 30 minutes, followed by epitope unmasking with 10 mM citrate buffer pH 6.0 heated in the microwave for 15 minutes.
    [0195] The sections were immersed for 1 hour in blocking solution (PBS supplemented with 0.5% fetal calf serum, 0.3% Triton X-100 and 1% BSA) and incubated overnight at 4 ° C with rabbit anti-SLC19A3 antibody (Sigma, HPA038898) diluted 1: 1000 in blocking solution. After washing, they were incubated with biotinylated rabbit secondary antibody and then with the avidin-biotin complex using the Elite Vectastain kit (Vector Laboratories, PK-6101). Chromogen reactions were performed with diaminobenzidine (SIGMAFAST DAB, Sigma, D4293) for 10 minutes and then the sections were dehydrated and covered with DePeX (Serva). Images were captured using an Olympus BX41 microscope with an Olympus DP-70 camera (Olympus Denmark A / S).
    [0197] Thiamine detection
    [0199] The samples of cerebrospinal fluid (CSF), blood and striatal homogenates from both human and mouse were collected in a vial protected against light and stored at -80 ° C. For the striatum samples, the frozen tissue was homogenized and the protein content was determined by the Bradford test using the Quick Start kit (Bio-Rad, 500-0203).
    [0201] For human, blood and mouse striatum samples, 100 µl of extraction buffer (Chromsystems, 37003) was added to 100 µl of sample and mixed for 2 s under shaking. Next, 150 µl of precipitation reagent (Chromsystems, 37004) were added, and it was mixed again for 30 s under stirring. Later, They were centrifuged for 5 min at 10,000g and a 100 µl derivatization mixture (Chromsystems, 37005-6) was selected in a new reaction vial to which 50 µl of the sample supernatant was added for brief mixing. Then, 50 µl of neutralization buffer (Chromsystems, 37009), 50 µl of stabilization buffer (Chromsystems, 37007) were added and allowed to stand for 20 minutes.
    [0203] For mouse CSFs, a direct extraction of the cisterna magna was performed using capillaries. 2 µl of CSF were diluted in 7 µl of physiological serum. The previously described protocol scaled up was performed for a sample volume of 9 instead of 100 µl of sample.
    [0205] Then, 50 µl was injected into the HPLC system. A common chromatographic method was standardized for the analysis of thiamine derivatives (TPP, TMP and free thiamine). Finally, we calculated the percentages of each thiamine derivative from the data in nmol / g of protein for the striatum samples, while for whole blood and CSF we calculated the amount of each thiamine derivative as nmol / L.
    [0207] Open field
    [0209] Locomotive activity analysis was performed in 27.5 cm x 27.5 cm transparent Plexiglas® boxes equipped with photoelectric detectors to monitor horizontal and vertical activity. Activity levels were recorded with a MED Associates activity monitor (MED Associates, St. Albans, VT) and analyzed with MED Associates activity monitor data analysis software v.5.93.773. Control WT or R6 / 1 mice were placed in the center of the open field box and allowed to move freely. Data were recorded individually for each animal for 15 min and the distance traveled by the animal was measured.
    [0211] Rotarod
    [0213] Analysis of motor coordination was analyzed at 18 weeks in R6 / 1 mice on a rotarod acceleration apparatus (Ugo Basile). The mice were pre-trained for two days: Day 1: 4 rpm for 1 minute, four repetitions; Day 2: acceleration from 4 to 8 rpm for 1 minute followed by constant 8 rpm for 1 minute, four repetitions. On the third day, the test was performed with the rotarod adjusted to acceleration from 4 to 40 rpm for 5 min, four repetitions, and the time it takes for the mouse to fall from the bar was measured.
    [0215] T2 magnetic resonance
    [0217] Animals were placed in the center of the RF (radio frequency) volume coil and positioned on the magnet under continuous inhalation anesthesia through a nasal mask. A respiratory sensor connected to a monitoring system (SA Instruments, Stony Brook, NY) was placed under the abdomen to monitor the rate and depth of respiration. The mice were anesthetized with 2% isofluorane in 1L of oxygen in an induction chamber and the flow of anesthetic gas was constantly regulated to maintain a respiratory rate of 50 +/- 20 ppm. The temperature of the animals was maintained at approximately 37 ° C by passing hot water through a heat exchanger machine to the platform of the animals. The physiological status of the animals was monitored using an NMR compatible small animal gate system by SA Instruments (Stony Brook, NY; http://www.i4sa.com/) which monitored the respiratory rate.
    [0219] NMR experiments were performed on a Bruker Pharmascan system (Bruker Medical GmbH, Ettlingen, Germany) using a horizontal diameter 7.0-T superconducting magnet, equipped with a 23mm 1H selective cage resonator and gradient insert. 90 mm diameter bruker (maximum intensity 36 G / cm). All data were acquired using a Hewlett-Packard dashboard with Paravision 5.1 software (Bruker Medical GmbH) operating on a Linux platform.
    [0221] The spin-echo images weighted in T2 sequence (T2-W) were taken with a fast acquisition sequence with improvement in relaxation time (RARE) in axial orientations and the following parameters: TR = 3000 ms, TE = 14.7 ms , RARE factor = 8, Av = 6, FOV = 2.3cm, acquisition matrix = 256 * 256, slice thickness = 1.00mm without gap and number of slices = 16.
    [0223] Diffusion tensor (DTP
    [0225] Diffusion tensor data was obtained with a spin-echo, single shot and flat image (EPI) pulse sequence using the following parameters: TR / TE 3500 / 40ms; an average signal of 4.7 non-collinear diffusion gradient schemes with a diffusion weighting of b = 100 and 1400s / mm2, 1mm thick slices without gap, 23x23mm field of view and acquisition matrix = 128x128.
    [0227] Fractional anisotropy, mean diffusivity, tracing, eigenvalues, and eigenvector maps were calculated with a homemade software application written in Matlab (R2007a).
    [0229] Magnetic resonance spectroscopy
    [0231] The 1H nuclear magnetic resonance (NMR) spectroscopy study was performed in the striatum region of the right hemisphere. The in vivo spectroscopy protocol used Point Resolved Spatial Spectroscopy (PRESS), combined with VAPOR water suppression and the following parameters were used: TR = 3000 ms, TE = 35 ms, Av = 128, voxel volume = 3 mm3 . Before acquisition, the FASTMAP automatic shoeing procedure (Gruetter, R. Magn. Reson. Med.
    [0232] 1993; 29: 804-811) to achieve optimal magnetic field uniformity throughout the voxel volume. All 1H spectra were automatically analyzed using LC Model version 6.2 OR (Stephen Provencher, Oakville, ON; Cañada).
    [0234] Statistic analysis
    [0236] Statistical analysis was performed with SPSS 21.0 (SPSS® Statistic IBM®). Data are represented as mean ± s.e.m (standard error of the mean) with a 95% confidence interval.
    [0238] The normality of the data was analyzed using the Shapiro-Wilk test (n <50) or Kolmogorov-Smirnov (n> 50). Homogeneity of variance was analyzed using Levente's test.
    [0240] For the comparison of two independent groups of the unpaired t-Student test (data with normal distribution), Mann-Whitney-Wilcoxon or Kolmogorov-Smirnov tests (with non-normal distribution) were performed. To compare the dependent measures, we use a paired t-test (normal distribution) or Wilcoxon signed rank tests (non-normal). For multiple comparisons, data with a normal distribution were analyzed using a one-way ANOVA followed by a post hoc Tukey, Games-Howell, or Dunnet's T test. Statistical significance of nonparametric data for multiple comparisons was determined using the Kruskal-Wallis one-way ANOVA test. The enrichment tests were carried out using the one-sided Fisher's exact test. A critical value for significance of p <0.05 was used throughout the study.
    [0242] RESULTS
    [0244] Example 1. Protein levels of CPEB1 and CPEB4 are altered in Huntington's disease.
    [0246] A statistically significant increase in CPEB1 protein levels (453%, p = 0.037) and a statistically significant decrease in CPEB4 (83% decrease, p = 0.001) were found in the post-mortem striatum of subjects with HE, without changes. significant at CPEB2 or CPEB3 levels (FIG. 1A). Equivalent results were found in the striatum of the R6 / 1 transgenic mouse model (FIG. IB).
    [0248] In general, the CPEB1 / CPEB4 imbalance at the protein level did not correlate with corresponding alterations in transcript levels (FIG-1C-D), except for increased CPEB1 mRNA levels in the striatum of R6 / 1 (FIG. IC).
    [0250] In zQ175 knock-in mice, a murine model of slow disease progression, only the decrease in CPEB4 levels reached statistical significance (FIG.
    [0251] 1E), suggesting that changes in CPEB4 could precede changes in CPEB1.
    [0253] Example 2. Analysis of the polyadenylation of genes associated with Huntington's disease.
    [0255] To detect possible changes in the length of the poly (A) tail in the transcriptome of R6 / 1 mice compared to control mice, a poly (U) column chromatography was carried out followed by a gene chip analysis. .
    [0257] The results showed that in the striatum of R6 / 1 mice, the length of the poly (A) tail is increased in transcripts of 8.6% of the genome and decreased in those of 8.7% (FIG. 2A).
    [0259] Subsequently, the analysis of the gene ontology (KEGG Pathway) in the 1,467 genes with an absolute change of poly (A) (fc; fold change) above 2 (FIG. 2B) produced four terms with Benjamini <1.5e- 1; among them: HD, Alzheimer's disease (AD), Parkinson's disease (PD) and contraction of the heart muscle. Thus, these results indicate that altered polyadenylation may contribute to the pathogenesis not only in HD, but also in other neurodegenerative pathologies, such as AD and PD, since many of the genes mutated in family forms of AD / tauopathies, PD or Amyotrophic lateral sclerosis, such as the Psenl, Mapt, Snca, LrrK2, Pinkl, Dj1 and Sod1 genes, along with the Htt gene, showed altered polyadenylation (FIG. 2C).
    [0261] To corroborate that the observed changes in polyadenylation could be attributed to an altered function of the CPEBs proteins, the potential enrichment of the genes with CPE sequences in their 3'UTRs was analyzed. In fact, this was the case for transcripts with the shortened poly (A) tail (FIG. 2D). Furthermore, 93% of the genes with the most extreme shortening (f.c. <- 4.0) of their poly (A) tail harbor CPE sequences in their 3'UTRs (Table 4).
    [0263] Table 4. Genes showing greater shortening (f.c. <-4.0) of their poly (A) tail.
    [0265]
    [0266]
    [0269] The decreased polyadenylation is expected to be reflected in decreased protein levels as well. Thus, the results obtained confirm a large decrease in the levels of the Auts2 protein (the gene with the highest degree of deadenylation, fc = -5.4;
    [0270] Table 4) both in the striatal tissue of R6 / 1 mice (FIG. 3A) and in that of subjects suffering from HD (FIG. 3B), despite presenting an unaltered transcriptional level. Similar results were found for other genes with a deadenylation of fc <-4, such as the Rockl and Ktn1 genes (FIG. 3A-B and Table 4).
    [0272] Therefore, the results shown indicate the existence of an imbalance between the protein levels of CPEB1 / CPEB4 in the striatum of subjects with HD and in the murine model R6 / 1 of HD, as well as a global alteration of the length of the polytail. (A) which notably affects genes associated with neurodegeneration. Furthermore, the enrichment of the CPE sequences in the genes showing deadenylation indicates that the latter could be secondary to the alteration of the CPEB proteins. Likewise, highly deadenylated genes show a decrease in their protein levels both in the murine model R6 / 1 of HE, and in the striatum of subjects suffering from the pathology, in the absence of coincident changes in the levels of transcripts.
    [0274] Example 3. Subjects with Huntington's disease have a decreased concentration of the SLC19A3 protein in the brain along with a deficiency in thiamine levels.
    [0276] Among the genes with more prominent deadenylation shown in Table 4, the Slc19a3 gene was particularly interesting because its mutations cause a devastating basal ganglion disorder called biotin-thiamine-responsive basal ganglia disease that can be reversed with a therapy based on the administration of said vitamins. In more detail, the Slc19a3 gene encodes a thiamine transmembrane transporter (hTHTR2) and subjects with this pathology show decreased levels of thiamine in the cerebrospinal fluid, bilateral atrophy in the head of the caudate nucleus and in the putamen, as well as a variety of neurological symptoms, including lethargy, irritability, dystonia, spasticity, tremor and chorea, among others, that improve with a treatment based on thiamine and biotin. Thus, the inventors hypothesized that HD could be in part a thiamine deficiency due to decreased levels of the SLC19A3 protein.
    [0278] To corroborate his hypothesis, the protein concentration of SLC19A3 was analyzed in murine and human brain samples.
    [0280] Levels of the SLC19A3 protein were highly decreased in both the striatum and the cortex of HD subjects, despite showing a trend towards increased transcript levels (FIG. 4A). This is reflected in a strong decrease in immunohistochemical staining both in the histological samples of the striatum and cortex (FIG. 4B) which, in good agreement with the human protein atlas (www.proteinatlas.org), revealed a neuronal and endothelial localization. . Furthermore, a decrease in thiamine levels was also observed in the CSF of subjects with HD, although with unchanged blood levels (FIG. 4C). This fits with the previously observed CSF thiamine decrease in subjects with BTBGD (Ortigoza-Escobar, JD, et al. Brain. 2016; 139: 31-38).
    [0282] Inside the cell, thiamine is converted to its phosphorylated derivatives, such as thiamine monophosphate (TMP) and, more importantly, to thiamine triphosphate (TPP), which is the bioactive form that acts as a cofactor for multiple enzymes in the catabolism of sugars and amino acids. TPP represents 80-85% of the thiamine present in the brain. However, when the level of free thiamine and its phosphorylated derivatives was analyzed in the striatal tissue of subjects with HD, the results showed that TPP represents only 60% of the sum of free thiamine + TMP + TPP, with a concomitant increase in the percentage of free thiamine (FIG. 4D), which is indicative of a decrease in the intracellular availability of thiamine.
    [0284] Taken together, these results demonstrate a decreased concentration of the SLC19A3 protein together with a deficiency of thiamine levels in the brain of subjects with HD and suggest that subjects suffering from this pathology could benefit from a therapy based on thiamine and / or biotin, as do subjects with BTBGD.
    [0285] Example 4. The combined administration of thiamine and biotin in mouse models of HD improves motor symptoms and striatum atrophy.
    [0287] To test preclinically whether the administration of thiamine and biotin could be useful in the treatment of HE, it was first analyzed whether the murine models of HE tested: R6 / 1 and zQ175, also presented a thiamine deficiency, as it has been shown in subjects suffering from HD. As seen in Fig. 5A, both mouse models showed unchanged thiamine levels in blood and a trend towards lower levels of TMP in CSF. The distribution of thiamine and its phosphorylated derivatives, TPP and TMP, in the striatal tissue of these mice was also analyzed against WT control mice, showing an increase in free thiamine (FIG. 5B) in both models and a decrease in TPP in R6 / 1 mice (FIG.
    [0288] 5B) as in the samples of subjects suffering from HD (FIG. 4C and 4D, respectively).
    [0290] Since R6 / 1 mice show motor phenotypes from an early age, it was decided to administer biotin (B), thiamine (T), or a combination of both (B + T) when they finished breastfeeding (that is, , three weeks after the birth of the mice), and to analyze the effect of each treatment on hypoactivity, observed in the open field test at 13 weeks of age and on the motor coordination deficit, detected in the rotarod a test. 18 weeks of age.
    [0292] The results observed show that the treatment with biotin or thiamine alone only showed improvement trends in some of the behavioral tests (FIG. 6A-B). However, the combined biotin and thiamine treatment improved the motor coordination deficit in the rotarod test at 18 weeks (FIG. 6C).
    [0294] Compared to the R6 / 1 mouse transgene, the CAG mutation in the heterozygous zQ175 murine HD model, being in the endogenous gene, is more similar to the mutation suffered by HD subjects; despite not inducing clear motor phenotypes in the mouse lifetime. However, it is known that heterozygous zQ175 mice show striatal atrophy from an early age (Heikkinen, T., et al.
    [0295] PloS one. 2012: 7, e50717) (FIG. 7A) and in the heterozygous zQ175 murine model treated with the combined therapy of thiamine and biotin, described here, an attenuation of the atrophy suffered by the striatum after a minimum of three months after the administration of said combined treatment (FIG. 7B). Heterozygous zQ175 mice also show an increase in striatum phosphocreatine detectable by magnetic resonance spectroscopy (MRS) that is not seen in heterozygous zQ175 mice treated with the combined thiamine and biotin therapy, described herein (FIG. 7C).
    [0297] Thus, the results indicate that HD mice also show thiamine deficiency in the brain, and that HD motor symptoms, striatal atrophy and increased phosphocreatine, improve with thiamine and biotin therapy.
    [0299] In summary, the results shown in the present document demonstrate that the decrease in SLC19A3 protein levels in subjects with HD correlates with a thiamine deficiency in the brain (decrease in thiamine in the CSF and in the striatal TPP), which promotes the usefulness of treating these subjects with vitamin supplements based on a combined administration of thiamine and biotin, which is further supported by the fact that motor symptoms, striatum atrophy and increased striatal phosphocreatine in murine models suffering from HD improve with the combination of vitamin supplements based on thiamine and biotin.
    权利要求:
    Claims (9)
    [1]
    1. Composition comprising biotin and thiamine for use in the prevention and / or treatment of Huntington's disease.
    [2]
    Composition for use according to claim 1, wherein the biotin concentration is at least 0.40 mg / kg / day, and the thiamine concentration is at least 2 mg / kg / day.
    [3]
    Composition for use according to any of claims 1 to 2, further comprising at least one pharmacologically acceptable excipient and / or vehicle.
    [4]
    Composition for use according to any one of claims 1 to 3, further comprising at least one active principle.
    [5]
    5. Composition for use according to claim 4 where the active principle is selected from the list consisting of: tetrabenazine, haloperidol, chlorpromazine, risperidone, quetiapine, amantadine, levetiracetam, clonazepam, citalopram, escitalopram, fluoxetine, sertraline, quetiapine, risperidone , olanzapine, valproate, carbamazepine, lamotrigine and / or any of their combinations.
    [6]
    6. Composition for use according to any of claims 1 to 5, characterized in that it is administered by any of the following routes: oral, sublingual, parenteral, intravenous, intraperitoneal and / or intramuscular.
    [7]
    7. Composition for use according to claim 6, wherein the route of administration is the oral route.
    [8]
    Composition for use according to any one of claims 1 to 7 wherein the compounds of said compositions are formulated or administered together, separately or sequentially.
    [9]
    Composition for use according to any one of claims 1 to 8 wherein said composition is repeatedly administered to a subject.
    类似技术:
    公开号 | 公开日 | 专利标题
    Guo et al.2017|Protocatechualdehyde protects against cerebral ischemia-reperfusion-induced oxidative injury via protein kinase Cε/Nrf2/HO-1 pathway
    Cordero et al.2013|Is inflammation a mitochondrial dysfunction-dependent event in fibromyalgia?
    Lu et al.2012|Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice
    ES2640777T3|2017-11-06|Anaplerotic therapy for Alzheimer&#39;s disease
    Li et al.2008|Isoflurane preconditioning increases B-cell lymphoma-2 expression and reduces cytochrome c release from the mitochondria in the ischemic penumbra of rat brain
    Zhang et al.2014|Endoplasmic reticulum stress is involved in restraint stress-induced hippocampal apoptosis and cognitive impairments in rats
    Stojković et al.2012|Risperidone reverses phencyclidine induced decrease in glutathione levels and alterations of antioxidant defense in rat brain
    Freire et al.2016|Wheat gluten intake increases weight gain and adiposity associated with reduced thermogenesis and energy expenditure in an animal model of obesity
    Jeong et al.2016|Effect of alpha-lipoic acid on radiation-induced small intestine injury in mice
    Hwang et al.2017|Parkin deficiency exacerbate ethanol-induced dopaminergic neurodegeneration by P38 pathway dependent inhibition of autophagy and mitochondrial function
    US11160776B2|2021-11-02|Compositions and methods for neuroprotection and treatment of neurodegeneration
    Yang et al.2017|Pioglitazone ameliorates Aβ42 deposition in rats with diet-induced insulin resistance associated with AKT/GSK3β activation
    ES2659763T3|2018-03-19|Compositions and procedures for the treatment of obesity and related disorders
    ES2882954T3|2021-12-03|Medical uses comprising methods for the measurement of inhibition of c-Jun N-terminal kinase in the skin
    Sofic et al.2015|Brain catalase in the streptozotocin-rat model of sporadic Alzheimer’s disease treated with the iron chelator–monoamine oxidase inhibitor, M30
    Xu et al.2019|Curcumin prevents brain damage and cognitive dysfunction during ischemic-reperfusion through the regulation of miR-7-5p
    Tang et al.2017|Potassium oxonate induces acute hyperuricemia in the tree shrew |
    Tran et al.2018|PKCδ knockout mice are protected from dextromethorphan-induced serotonergic behaviors in mice: involvements of downregulation of 5-HT1A receptor and upregulation of Nrf2-dependent GSH synthesis
    Li et al.2018|Deletion of Mst1 attenuates neuronal loss and improves neurological impairment in a rat model of traumatic brain injury
    Wan et al.2020|Erythromycin has therapeutic efficacy on muscle fatigue acting specifically on orosomucoid to increase muscle bioenergetics and physiological parameters of endurance
    ES2814048B2|2021-08-12|COMBINED USE OF BIOTIN AND THYAMINE IN THE TREATMENT OF HUNTINGTON DISEASE
    Huang et al.2021|Everolimus inhibits PI3K/Akt/mTOR and NF-kB/IL-6 signaling and protects seizure-induced brain injury in rats
    Li et al.2020|A Glimmer of Hope: Maintain Mitochondrial Homeostasis to Mitigate Alzheimer’s Disease
    Sari et al.2012|Activity-dependent neurotrophic factor-derived peptide prevents alcohol-induced apoptosis, in part, through Bcl2 and c-Jun N-terminal kinase signaling pathways in fetal brain of C57BL/6 mouse
    Okusha et al.2017|Effects of intraperitoneally administered L-histidine on food intake, taste, and visceral sensation in rats
    同族专利:
    公开号 | 公开日
    ES2814048B2|2021-08-12|
    WO2021058847A1|2021-04-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US6746678B1|1991-02-22|2004-06-08|Howard K. Shapiro|Method of treating neurological diseases and etiologically related symptomology using carbonyl trapping agents in combination with medicaments|
    法律状态:
    2021-03-25| BA2A| Patent application published|Ref document number: 2814048 Country of ref document: ES Kind code of ref document: A1 Effective date: 20210325 |
    2021-08-12| FG2A| Definitive protection|Ref document number: 2814048 Country of ref document: ES Kind code of ref document: B2 Effective date: 20210812 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201930825A|ES2814048B2|2019-09-24|2019-09-24|COMBINED USE OF BIOTIN AND THYAMINE IN THE TREATMENT OF HUNTINGTON DISEASE|ES201930825A| ES2814048B2|2019-09-24|2019-09-24|COMBINED USE OF BIOTIN AND THYAMINE IN THE TREATMENT OF HUNTINGTON DISEASE|
    PCT/ES2020/070570| WO2021058847A1|2019-09-24|2020-09-23|Combined use of biotin and thiamine in the treatment of huntington's disease|
    [返回顶部]