JP2016530291A - Method for treating fragile X syndrome and related disorders - Google Patents

Abstract

The present invention provides a method for alleviating the signs or symptoms of fragile X syndrome and related disorders such as autism spectrum disorders.

Description

Related Applications This application includes provisional application USSN 61 / 875,384 filed on September 9, 2013, USSN 14/038258 filed on September 26, 2013, and provisional application filed on May 9, 2014. The priority and benefit of USSN 61 / 991,351 are claimed, each of which is hereby incorporated by reference in its entirety.

The present invention relates generally to methods for treating or alleviating symptoms of fragile X syndrome and related disorders.

  Fragile X Syndrome (FXS), as its name implies, is associated with a fragile site represented as a metaphase chromosome iso-arm chromatid gap at map position Xq 27.3. Fragile X syndrome is a genetic disorder caused by mutations in the 5 'untranslated region of the fragile X mental retardation 1 (FMR1) gene located on the X chromosome. The mutation that causes FXS is associated with a CGG repeat in the fragile X mental retardation gene FMR1. For most healthy individuals, the total number of CGG repeats ranges from less than 10 to 40, with an average of about 29. In fragile X syndrome, this CGG sequence is repeated more than 200 to 1,000 times. If a subject has more than about 200 CGG repeats, the fragile X gene becomes over-methylated and the gene is silenced. As a result, fragile X mental retardation protein (FMRP) is not produced or produced only at low levels, and the subject shows signs of FXS.

  Pre-mutation expansion of the FMR1 gene (55-200 CGG repeat) is frequently seen in the general population, with an estimated prevalence of 1 in 259 women and 1 in 812 men. Premutation holders share a common emotional problem such as anxiety, but generally have a normal IQ. Elderly male premutation carriers (over 50 years old) develop progressive intention tremor and ataxia. These movement disorders are often accompanied by progressive cognitive / behavioral problems including memory loss, anxiety, and executive function deficits, withdrawal or hypersensitivity behavior, and dementia. This disorder is called fragile X-related tremor / ataxia syndrome (FXTAS). From the magnetic resonance imaging of subjects with FXTAS, an increase in T2-weighted signal intensity in the middle cerebellar limb and adjacent cerebellar white matter is evident.

  FXS is isolated as an X-linked dominant disorder with low penetrance. Any gender can show intellectual disability if it has a fragile X mutation, but the severity varies. Children and adults with FXS have varying degrees of intellectual or learning disabilities and behavioral and emotional problems, including autism-like features and trends. Infants with FXS often have delays in developmental milestones such as learning how to sit, walk and speak. Affected children may have frequent tantrums, attention deficits, frequent seizures (eg, temporal lobe seizures), often show high anxiety, are easily beaten, and exhibit hyperesthesia and gastrointestinal disorders Occasionally, it may exhibit abnormal behavior such as speech disability and fluttering, biting hands.

  FXS can be diagnosed by established genetic tests performed on samples from subjects (eg, blood samples, oral samples). This test determines whether there is a mutation or premutation in the subject's FMR1 gene based on the number of CGG repeats.

  Subjects with FXS may also have autism. About 5% of all children diagnosed with autism have a mutation in the FMR1 gene and also have fragile X syndrome (FXS). Autism spectrum disorder (ASD) is found in approximately 30% of men with FXS and 20% of women, with an additional 30% of people with FXS showing autistic symptoms without ASD diagnosis. Although intellectual disability is a prominent feature of FXS, people with FXS range from shyness, disagreement, and social anxiety in mild cases to flirting, chewing hands, and sustained conversation in severe illness Often exhibits autistic features. FXS individuals also exhibit other symptoms associated with autism such as attention deficit and hyperactivity, seizures, hypersensitivity to sensory stimuli, obsessive-compulsive behavior and changes in gastrointestinal function. FMR1 mutations suppress or greatly reduce the expression of a single protein (FMRP). Brain development in the absence of FMRP appears to give rise to the main symptoms of FXS.

  In addition to core symptoms, children with FXS often have severe behavioral disorders such as irritability, aggression and self-harm. A recent study of men with FXS (8-24 years) reported 79% self-injurious behavior and 75% aggressive behavior in a 2-month observation period.

  Currently available treatment regimens for humans with FXS include, for example, treatment with behavior modification and a range of medications (not approved by the FDA for the treatment of FXS) including antidepressants and antipsychotics. Cognitive behavioral therapy has been used to improve language and socialization in FXS and autistic individuals. In recent years, pharmacological treatment with the atypical antipsychotic drug risperidone has been commonly used to enhance non-pharmacological approaches in the treatment of autistic individuals. A randomized placebo-controlled trial of risperidone in children with autism showed significant improvements in the abnormal behavior checklist and the irritability subscale of improving clinical general impressions (McCracken, JT, et al., N. Engl. J. Med. 347: 314-321 (2002)). However, adverse events included weight gain, increased appetite, fatigue, somnolence, dizziness, and fluency. Social isolation and communication were not improved by the administration of risperidone, and adverse side effects such as extrapyramidal symptoms and dyskinesia were associated with risperidone in autistic children. Current treatment regimens are often ineffective or, especially in the case of antipsychotics, there is a need for new treatments because long-term use can cause undesirable side effects.

  In various aspects, the present invention provides a method of treating or alleviating symptoms of fragile X syndrome or related disorders by administering a composition comprising metadoxine to a subject in need thereof. Symptoms are, for example, learning disabilities or social behavior disorders. The subject has fragile X syndrome or autism spectrum disorder. A related disorder is autism spectrum disorder.

  In some aspects, a total daily dose of metadoxine of between 100 and 3000 mg is administered, with metadoxine being administered daily, every other day, or weekly.

  Optionally, metadoxine is administered in one, two, or three dosage forms per day. In some embodiments, metadoxine is administered in a sustained release oral dosage form, wherein metadoxine is a combination of slow release and immediate release form. forms).

  For example, the sustained release form provides a sustained release of metadoxine for at least 8 hours. The relative proportion of the slow release metadoxine to the immediate release metadoxine is between about 60:40 and 80:20. Preferably, the relative ratio of sustained release metadoxine and immediate release metadoxine is about 65:35.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

  Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

FIG. 1 shows vehicle (V) or metadoxine (M) (100, 150, or 200 mg / kg) for contextual fear conditioning in 2-month-old Fmrl knock-out (KO) or wild-type (WT) mice. The effect of intraperitoneal (ip) administration once a day for 7 days is shown. Specifically, Panel A shows the effect of vehicle or 150 mg / kg metadoxine. Panel B shows the effect of vehicle or 100 mg / kg metadoxine. Panel C shows the effect of vehicle or 200 mg / kg metadoxine. Data shown are mean ± standard error of the mean (sem), N = 10 mice per group. ^* P <0.05, ^****** p <0.0001, and NS = not significant. FIG. 2 shows intraperitoneal administration of vehicle (V) or 150 mg / kg metadoxin (M) once daily for 7 days for social access behavior in 2-month-old Fmrl knockout (KO) or wild type (WT) mice. The effect of Data shown are mean ± sem, mouse N = 10 per group. ^* P <0.05 and ^****** p <0.0001. FIG. 3 shows Y maze spontaneous alternation behavior (Panel A), Y maze reward alternation behavior (Panel B) or Y maze water maze spatial discrimination (Panel) in 2-month-old Fmrl knockout (KO) or wild type (WT) mice. The effect of intraperitoneal administration of vehicle (V) or 150 mg / kg metadoxine (M) once daily for 7 days to C) is shown. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.001, ^****** p <0.0001, and NS = not significant. FIG. 4 shows vehicle (V) or 150 mg / kg metadoxin (M) once daily for 7 days intraperitoneally for T maze reward alternation behavior in 2 month old Fmrl knockout (KO) or wild type (WT) mice. The effect of administration is shown. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.0001. Once daily with vehicle (V) or 150 mg / kg metadoxine (M) for behavior in a continuous alleys task in a group of N = 10 wild type (WT) or Fmrl knockout (KO) 2 month old mice, The effect of 7 day treatment is shown. The continuous path of this device provided an environment that gradually increased anxiety for the exploring mouse. Therefore, it is evaluated as anxiety when getting off the alley. In addition, the overall activity level could be quantified with this device. FIG. 6 shows the total brain phosphorylation levels of ERK (an indicator of ERK activity) (panel A) and Akt (an indicator of Akt activity) (panel B) in 2 month old Fmrl knock-out (KO) or wild type (WT) mice. The effect of intraperitoneal administration of vehicle (V) or 150 mg / kg metadoxine (M) once a day for 7 days is shown. Data shown are mean ± sem, mouse N = 10 per group. ^{** p <0.01, *** p <} 0.001, **** p <0.0001, and NS = not significant. FIG. 7 shows vehicle (V) or 150 mg / kg metadoxin (M) once daily for 7 days ip administration for contextual fear conditioning in 6 month old Fmrl knockout (KO) or wild type (WT) mice. Show the effect. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.0001 and ns = not significant. FIG. 8 shows social access behavior in 6 month old Fmrl knockout (KO) or wild type (WT) mice as measured by the number of sniffing bouts or duration of sniffing (Panel A and C) and the effect of ip administration of vehicle (V) or 150 mg / kg metadoxine (M) once daily for 7 days on social memory behavior (panels B and D). Data shown are mean ± sem, mouse N = 10 per group. ^* P <0.05, ^****** p <0.0001, and ns = not significant. FIG. 9 shows vehicle (V) or 150 mg / kg metadoxine (M) versus total brain phosphorylation levels of ERK (panel A) and Akt (panel B) in 6 month old Fmrl knockout (KO) or wild type (WT) mice. ) Shows the effect of ip administration once a day for 7 days. Data shown are mean ± sem, mouse N = 10 per group. ^* P <0.05, ^** p <0.01, ^****** p <0.0001, and ns = not significant. FIG. 10 shows once daily ip administration or vehicle (V) of metadoxine (M) at 150 mg / kg for 7 days for contextual fear conditioning in 2 month old Fmrl knockout (KO) or wild type (WT) mice. Alternatively, the effect of oral administration (po) of 150 and 300 mg / kg metadoxine is shown. Data shown are mean ± sem, mouse N = 10 per group. Specifically, Panel A shows ip and oral treatment with vehicle in Fmrl knockout and wild type mice. Panel B shows ip and oral treatment with metadoxine in wild type mice. Panel C shows ip and oral treatment with metadoxine in Fmrl knockout mice. ^** p <0.01, ^****** p <0.0001, and ns = not significant. FIG. 11 shows 7 days of vehicle (V) 150 or 300 mg / kg for social access (panel A) and social memory (panel B) in 2 month old Fmrl knock-out (KO) or wild type (WT) mice. The effects of ip administration or oral administration (po) of metadoxine (M) once daily are shown. Data shown are mean ± sem, mouse N = 10 per group. ^** p <0.01, ^****** p <0.0001, and ns = not significant. FIG. 12 shows a 7-day vehicle (V) or 150 or 300 mg / kg for lymphocyte biomarkers assessed using flow cytometry in 2 month old Fmrl knock-out (KO) and wild type (WT) mice. The effects of ip administration or oral administration (po) of metadoxine (M) once daily are shown. The biomarkers shown are pAkt (panel A) and pERK (panel B) in Fmrl knockout or wild type mice. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.0001 and ns = not significant. FIG. 13 shows 7-day vehicle (V) or 150 mg / kg metadoxine (M) once daily ip administration to pERK levels in brain regions of 2 month old wild type (WT) and Fmrl knockout (KO) mice. Show the effect. The analysis areas were the hippocampus (panel A), prefrontal cortex (panel B), and striatum (panel C) in Fmrl knockout or wild type mice. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.0001 and ns = not significant. FIG. 14 shows 7-day vehicle (V) or 150 mg / kg metadoxine (M) once daily ip administration to pAkt levels in brain regions of 2 month old wild type (WT) and Fmrl knockout (KO) mice. Show the effect. The analysis areas were the hippocampus (panel A), prefrontal cortex (panel B), and striatum (panel C) in Fmrl knockout or wild type mice. Data shown are mean ± sem, mouse N = 10 per group. ^*** p <0.0001 and ns = not significant. FIG. 15 shows vehicle (V) versus filopodia density (panel A), length (panel B), and width (panel C) in neuronal hippocampal cultures from Fmrl knockout (KO) or wild type (WT) mice. Alternatively, the effect of in vitro treatment with 300 μM metadoxine (M) for 5 hours is shown. Data shown are mean ± sem (wild type, N = 20 neurons and Fmrl knockout mice, N = 20 neurons). ^** p <0.01, ^*** p <0.001, and ns = not significant. FIG. 16 shows the effect of in vitro treatment with vehicle (V) or 300 μM metadoxin (M) on basal de novo protein synthesis in 400 μM hippocampal slices of Fmrl knockout (KO) or wild type (WT) mice. Data shown are mean ± sem, intercept N = 6 per group. ^* P <0.001 and ^****** p <0.0001.

Detailed description of the invention

  The present invention relates to the discovery that metadoxine significantly improves cognitive and social function in validated animal models of fragile X syndrome.

  Specifically, metadoxine significantly improves memory and learning during the contextual fear paradigm in a dose-dependent manner, with the two highest dose levels (150 and 200 mg / kg) being Fmrl KO. Mouse learning and memory deficits were fully rescued to the same extent as WT mouse levels. Furthermore, a significant improvement in memory was observed in behavioral tests such as the T-maze in Fmrl KO mice treated with 150 mg / kg metadoxine, indicating a significant improvement in cognitive results. These findings were complemented by improved social interaction in KO mice treated with 150 mg / kg metadoxine. Importantly, improvements in cognitive executive function, working memory and social interaction after metadoxine treatment in a validated fragile X mouse model correlate with normalization of biochemical markers reflecting neuronal signaling pathways and oxidative stress doing.

  Fragile X syndrome is the most prevalent monogenic cause of autism and an inherited cause of mental retardation among boys. A person with an FMR1 gene mutation may be handed over to a child. According to the Centers for Disease Control and Prevention (CDC), approximately 1 in 4,000 men and approximately 1 in 8,000 women have fragile X syndrome. Not all people with this mutation show signs or symptoms of fragile X, and the disorder ranges from mild to severe, with physical features such as long faces, large or overhanging ears, and large testes (macrochiosis) With behavioral features such as stereotypic movements (eg, fluttering hands) and social anxiety. Fragile X is caused by a change or mutation in the Fragile X Mental Retardation 1 (FMR1) gene found on the X chromosome. This gene usually produces a protein called fragile X mental retardation protein or FMRP. This protein is important for creating and maintaining connections between cells of the brain and nervous system. This mutation causes the body to make only a small piece of this protein or no protein at all, often resulting in symptoms of fragile X.

  Fragile X syndrome (FXS) often coexists with other conditions such as autism spectrum disorder. Autism spectrum disorder (ASD) is a group of developmental disorders that can cause serious social, communication and behavioral problems. People with ASD have different information handling in the brain than others.

  ASD is a “spectrum failure”. This means that ASD has different effects from person to person and can range from very mild to severe. People with ASD have several similar symptoms in common, such as problems with social interaction. However, there are differences in when symptoms begin, how severe they are, and the exact nature of the symptoms. ASD includes autistic disorders (also called “classical” autism), Asperger's syndrome and pervasive developmental disorders.

  Currently, the Food and Drug Administration (FDA) does not specifically approve drugs for the treatment of fragile X or its symptoms. There are drugs that are used off-label to treat certain symptoms of fragile X syndrome, but the results vary widely from patient to patient, some of these drugs have significant risks and initially worsen the symptoms It may take a few weeks to be effective. The present invention presents an unmet need for drugs for treating fragile X or its symptoms.

  Thus, the present invention provides a method for treating, preventing or alleviating signs or symptoms of fragile X syndrome and / or autism spectrum disorder by administering to a subject a composition comprising metadoxine.

  In general, signs and symptoms of fragile X fall into five categories, for example, intelligence and learning; physical disabilities, social and emotional disorders, conversational and language disorders, and sensory disorders that generally accompany or share characteristics with fragile X including. Individuals with fragile X have a sensitivity to intellectual dysfunction, social anxiety, language impairment and specific sensations. Treatment with metadoxine improves learning and enhances sociality in subjects with fragile X syndrome.

  Autism spectrum disorders are commonly associated with individuals with fragile X syndrome. Signs and symptoms of autism include significant language lag, social and communication problems, and abnormal behavior and interest. Many people with autistic disorders also have intellectual disabilities. Individuals with Asperger's syndrome usually have some mild symptoms of autistic disorder. For example, these people may have social problems and abnormal behavior and interest. Individuals with pervasive developmental disorder (PDD-NOS) may meet some, but not all, criteria for autistic disorder or Asperger syndrome and be diagnosed with PDD-NOS. People with PDD-NOS are usually less symptomatic and milder than those with autistic disorders. These symptoms may only cause social and communication problems. Treatment with metadoxine improves the symptoms of autism.

  Metadoxine is an ion pair between pyrrolidone carboxylate (PCA) and pyridoxine (vitamin B6), and these two compounds are linked as a single product by salification. Pairing with PCA synergistically enhances the pharmacological activity of pyridoxine (see, eg, US Pat. No. 4,313,952). Metadoxine is soluble in water and gastric juice without limitation. The oral absorption of this drug is fast and has high bioavailability (60-80%). The half-life of metadoxine in human serum is short (40-60 minutes) and there is no visible difference between oral and intravenous administration (Addolorato et al., Supra; Lu Yuan et al., Chin. Med). 1 2007 120 (2) 160-168).

  Metadoxine is marketed in several countries as a prescription drug in the form of 500 mg tablets and 300 mg injections. The tablets contain 500 mg metadoxine, microcrystalline cellulose and magnesium stearate. Ampoule contains 300 mg of metadoxine, sodium metabisulfite, sodium EDTA, methyl-p-hydroxybenzoate and water.

  In certain embodiments, a metadoxine composition of the invention formulated, for example, fully or partially for sustained or controlled release, is an indication of its associated pathology / disorder, such as fragile X syndrome and autism spectrum disorder Or allows more efficient use of metadoxine in the treatment, prevention and / or alleviation of symptoms.

  In certain of the above methods of the invention, metadoxine or an acceptable derivative thereof may be formulated to be released immediately upon administration to a subject. In some of the above methods of the invention, metadoxine or an acceptable derivative thereof may be formulated for sustained release and / or controlled control, and in some cases immediate release characteristics and sustained release upon administration to a subject. And / or may be formulated to have both controlled release characteristics. In certain embodiments, metadoxine or a physiologically acceptable derivative thereof is formulated for non-chronic administration. Metadoxine formulations useful in the methods of the present invention are described in further detail below.

  In certain embodiments, the present invention provides a subject for improvement, treatment, prevention and / or alleviation of signs or symptoms of its associated pathology / disorder, such as fragile X syndrome and / or autism spectrum disorder. Provided is a composition comprising metadoxine or a derivative thereof formulated to be sustained release and / or controlled release upon administration.

  In certain embodiments, the present invention comprises metadoxine or a derivative thereof, wherein the metadoxine or a portion of the derivative is formulated for sustained and / or controlled release when administered to a subject, Or improvement, treatment, prevention and / or alleviation of signs or symptoms of its associated pathology / disorder, such as fragile X syndrome and / or autism spectrum disorder, where a portion of the derivative is formulated for immediate release A composition is provided.

  In certain embodiments, the effective serum level of the active ingredient is about 10 minutes to about 20 minutes or 30 minutes or 40 minutes or 50 minutes or 60 minutes, 90 minutes, 2 hours, 3 hours after administration of metadoxine or a metadoxine derivative. It is achieved within 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours and 10 hours. In certain embodiments, the effective serum level of the active ingredient in the subject is about 5 minutes to about 20 minutes or 30 minutes or 40 minutes or 50 minutes or 60 minutes, 90 minutes, 2 minutes after administration of metadoxine or a metadoxine derivative. Within 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours and 10 hours. In certain embodiments, the effective serum level of the active ingredient is about 20 minutes to about 20 minutes or 30 minutes or 40 minutes or 50 minutes or 60 minutes, 90 minutes, 2 hours, 3 hours after administration of metadoxine or a metadoxine derivative. It is achieved within 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours and 10 hours. In certain embodiments, the effective serum level of the active ingredient is about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours. It is achieved within 5 hours, 6 hours, 7 hours, 8 hours, 9 hours and 10 hours.

  The inventors have developed an innovative approach for the administration of metadoxine or metadoxine derivatives based on the intestinal route (via the gastrointestinal tract) and / or the parenteral route (the route other than the gastrointestinal tract) (W02009 / 004629, The contents of which are incorporated herein by reference in their entirety). These approaches include carriers for the intended delivery system, such as micro / nanoparticles (eg, liposomes or nanoliposomes) encapsulating the appropriate surfactant / co-surfactant composition or active ingredient, or others It provides a rational design of a delivery system with the desired properties based on careful selection of additives or excipients. Intestinal delivery systems can be administered orally (tablets, sachets, troches, capsules, gelcaps, drops, or other palatable forms) or rectal (suppositories or (mini) enema forms). May be designed for In addition, the targeted delivery system may be in liquid form, for example, a drop solution, syrup. Further, the targeted delivery system may be in the form of a beverage or food. Thus, the active ingredients used according to the present invention are contained in beverages, in particular soft drinks such as juices, nectars, water, carbonated water and other carbonated beverages, shakes, milk shakes and other milk-based beverages etc. It's okay. The liquid formulation may also be in the form of a concentrated syrup for dilution with water or carbonated water. Alternatively, the active ingredient may be contained in foods such as snack bars, health bars, biscuits, cookies, sweets, confectionery products, ice cream, ice candy.

  Furthermore, the delivery system may be a food or beverage product comprising a physiologically active pyridoxine derivative, in particular pyridoxol L, 2-pyrrolidone-5 carboxylate (metadoxine). In certain embodiments, ingestion of a food or beverage product of the present invention can result in achieving serum levels of the active ingredient within about 10 minutes to about 40-60 minutes after ingestion. Examples include sweets, chocolate, candy and candy bars, energy bars, ice cream, pastry products and the like.

  The parenteral route of administration is subcutaneous, transferal (diffusion through intact skin), transmucosal (diffusion through mucosa), sublingual, buccal (absorption through the cheek near the gingival line), or inhalation Administration is included. In certain embodiments, the compositions used in accordance with the present invention are not administered in an invasive mode of treatment (ie, are non-invasive). In certain embodiments, the metadoxine or metadoxine derivative composition is not administered by intravenous injection.

  In a particular embodiment, the composition used according to the invention is delivered as a microcrystalline powder or solution suitable for spraying; for vaginal or rectal administration, delivered as a vaginal suppository, suppository, cream or foam Is done. A preferred formulation is a formulation for oral administration. Another preferred formulation is for topical administration. Another preferred formulation is for transmucosal administration, sublingual, buccal (absorption via the cheek near the gingival line), inhalation or intraocular administration, eg for eye drops.

  Administration of metadoxine or metadoxine derivatives for medical use requires a safe and efficient delivery system. The present invention provides a delivery system for the safe delivery of various substances with special physicochemical characteristics, in particular direct absorption by non-invasive means, and avoidance of the resulting side effects. The delivery system is based on its unique physicochemical characteristics that allow for a reduction in the concentration or amount of a substance delivered in a biologically active form to a subject, and the efficiency and quality of absorption of metadoxine or metadoxine derivatives. Is significantly increased. The delivery system of the present invention provides direct access of the active substance to the tissue and thus provides an immediate or near immediate effect of metadoxine or a metadoxine derivative on the treated subject. Thus, in certain embodiments, the present invention provides improved administration of a physiologically active pyridoxine, particularly pyridoxol L, 2-pyrrolidone-5 carboxylate (metadoxine), or a physiologically acceptable derivative thereof. Therefore, a non-invasive pharmaceutical delivery system comprising the physiologically active pyridoxine as an active ingredient in a suitable carrier is used. In certain embodiments, the serum level of the active ingredient is achieved within about 10 minutes to about 40 minutes to 60 minutes after administration. In another embodiment, the present invention provides a physiologically active pyridoxine derivative, in particular pyridoxol L, 2-pyrrolidone-5 carboxylate (intended for use in improving cognitive behavior in a subject in need thereof. For improved administration of (metadoxine), a non-invasive pharmaceutical delivery system comprising said pyridoxine derivative as active ingredient in a suitable carrier is used. In certain embodiments, the serum level of the active ingredient is achieved within about 10 minutes to about 40 minutes to 60 minutes after administration.

  In certain embodiments, the drug delivery system used in accordance with the present invention may be designed for oral, nasal, intraocular, rectal, subcutaneous, transferal, transmucosal, sublingual, buccal or inhalation administration. it can. The drug delivery system may provide the active substance in a controlled release manner. In certain embodiments, the drug delivery system of the present invention may further comprise at least one additional pharmaceutically active agent. The delivery system used in accordance with the present invention generally comprises a buffer, an agent that modulates its osmotic pressure, and optionally one or more pharmaceutically acceptable carriers, excipients and / or known in the art. An additive may be included. Supplementary pharmaceutically acceptable active ingredients can also be incorporated into the compositions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. As used herein, “pharmaceutically acceptable carrier” includes any and all of solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutically effective substances is well known in the art. Except where a conventional vehicle or drug is incompatible with the active ingredient, its use in a therapeutic composition is contemplated. It is contemplated that the active agent can be delivered by any pharmaceutically acceptable route and in any pharmaceutically acceptable dosage form. Oral forms include, but are not limited to, tablets, capsules, pills, sachets, troches, drops, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions It is. Also included are oral rapid release, controlled release, and delayed release pharmaceutical dosage forms. The active drug ingredients can be administered in a single dosage form or can be administered in separate dosage forms that are administered together or independently. The active drug ingredient can be administered as a mixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carriers”), with the materials selected as appropriate for the intended dosage form. Is done. When the delivery system is for oral administration and is in a form such as a tablet or capsule, the active drug ingredient is lactose, starch, sucrose, glucose, modified sugar, modified starch, methylcellulose and its derivatives, dicalcium phosphate, Combine with non-toxic pharmaceutically acceptable inert carriers such as calcium sulfate, mannitol, sorbitol, and other reducing and non-reducing sugars, magnesium stearate, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc. Can do. For oral administration in liquid form, the active drug ingredient can be combined with non-toxic pharmaceutically acceptable inert carriers such as ethanol, glycerol, water and the like. If desired or necessary, suitable binders, lubricants, disintegrating agents, coloring agents, and flavoring agents can be incorporated into the mixture. Antioxidants, propyl gallate, sodium ascorbate, citric acid, calcium metabisulfite, hydroquinone, and anti-degradation agents such as 7-hydroxycoumarin can also be added to stabilize the dosage form. Other suitable compounds may include natural and synthetic gums such as gelatin, sweeteners, gum arabic, tragacanth, or alginate, carboxymethylcellulose, polyethylene, glycols, waxes, and the like.

  Further suitable pharmaceutically acceptable carriers that can be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, human serum albumin and the like. Serum proteins, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated plant fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, hydrogen phosphate Potassium, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer Mer, polyethylene glycol and wool fat. In some embodiments, the pharmaceutically acceptable carrier is magnesium stearate. Additional pharmaceutical excipients commonly recognized and used can be found, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed., Mack Pub., 1990).

  For parenteral administration, solutions in suitable oils such as sesame oil or peanut oil or in aqueous propylene glycol, as well as sterile aqueous solutions of the corresponding water-soluble salts can be used. Such an aqueous solution may be appropriately buffered as necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, all sterile aqueous media used can be readily obtained by standard techniques well known to those skilled in the art. Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art in view of the present disclosure. The half-life of metadoxine in human serum is very short. Lu Yuan et al. (Chin. Med. J 2007 120 (2) 160-168) shows an average half-life of about 0.8 hours. One way to prolong the serum level of the active moiety is by administering the material as a sustained release formulation. Since metadoxine is soluble in water and various body fluids without limitation, it is difficult to maintain its release and extend its absorption time. Therefore, it was not expected that sustained release could be achieved. Controlled release dosage forms of metadoxine or metadoxine derivatives may be based on a predetermined sustained release of the active ingredient into bodily fluids that provides a sustained action with small fluctuations in plasma levels over a long period of time.

  In certain embodiments, the delivery system used in accordance with the present invention can be administered in a controlled release formulation. In certain embodiments, the method of administration is determined by the attending physician or other person skilled in the art after assessment of the subject's condition and needs. An embodiment of the method of the invention is to administer the therapeutic compound described herein in sustained release form. Any controlled or sustained release method known to those skilled in the art can be used with the compositions and methods of the present invention, such as those described in Langer, Science 249 (4976): 1527-33 (1990). Such methods include sustained release compositions, suppositories, or coated implantable medical devices in which a therapeutically effective dose of the composition of the invention is continuously delivered to the subject of such methods. Administering. Sustained release may also be achieved using patches designed and formulated for this purpose. The compositions of the invention may be delivered by capsules that allow sustained release of the drug over a period of time. Controlled or sustained release compositions include formulations in lipophilic depots (eg, fatty acids, waxes, oils). Also encompassed by the present invention are particulate compositions coated with a polymer (eg, poloxamer or poloxamine). The sustained release formulation or device, or any topical formulation, may further comprise a composition to stabilize the composition or to penetrate a physiological barrier such as the skin or mucosa. Examples of additional ingredients may include any physiologically acceptable detergent or solvent such as dimethyl sulfoxide (DMSO).

  In all embodiments of the present invention, the methods and uses of the present invention may employ compositions comprising a salt adduct as defined by the present invention formulated as a single dose. The single formulation may be an immediate release formulation, burst formulation, extended release formulation, sustained release formulation or other controlled release formulation known to those skilled in the art.

  In other embodiments of the methods and uses of the invention, a composition comprising a salt adduct as defined by the invention is associated with different types of formulations, i.e., in a single dose or individually ( concomitantly) or any combination of immediate release formulation, burst formulation, extended release formulation, sustained release formulation or any other controlled release formulation known to those skilled in the art given in individual doses given sequentially or administered to the subject Wherein the time interval between administration of the individual doses is defined based on the condition and severity of the subject's disease or disorder or the physical condition of the subject.

  In some embodiments, the composition used by the method of the present invention is formulated as a combination dosage form, wherein at least one dosage form of the salt adduct as defined by the present invention is immediate release. In form, at least one dosage form of the salt adduct defined by the present invention (same or different from the salt adduct formulated in the immediate release formulation) is formulated as a controlled (slow and / or sustained) release formulation. The In another embodiment, the weight ratio of the salt adduct defined by the present invention contained in said at least one immediate release formulation and at least one controlled release formulation is 1: 1, 1: 2, 2: 1, 3: 2, 2: 3, 1: 3, 3: 1, 4: 1, 1: 4, 5: 2, 2: 5, 1: 5, 5: 1. When using such a combination dosage form in the method or use of the present invention, said at least one immediate release form and at least one controlled release form of the salt adduct as defined above are individually associated with and sequentially Alternatively, it may be administered to the subject concurrently, sequentially, and the like. In some embodiments, the at least one immediate release form is administered first. In another embodiment, the at least one controlled release formulation is administered first.

  In certain embodiments, the metadoxine or metadoxine derivative in the composition of the invention has a duration of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. Can be formulated for sustained or controlled release over a wide range. In a particular embodiment, the metadoxine or metadoxine derivative in the composition used according to the invention is about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 It can be formulated for sustained or controlled release over a period of time. In a particular embodiment, the metadoxine or metadoxine derivative in the composition used according to the invention is from about 0.5 or 1 or 2 or 3 or 4 hours to about 5, 6, 7, 8, 9, 10, It can be formulated for sustained or controlled release over a period of between 11 or 12 hours. In certain embodiments, the metadoxine or metadoxine derivative in the composition used according to the invention is sustained release or controlled over a period of between about 5 or 6 or 7 or 8 hours to about 9, 10, 11 or 12 hours. Can be formulated for release.

  In certain embodiments, the metadoxine or metadoxine derivative in the composition used according to the present invention may be in an immediate release form, a rapid release form or an immediate, fast of burst release form.

  In certain embodiments, the metadoxine or metadoxine derivative in the composition used according to the present invention is about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours of total metadoxine or metadoxine derivative. Release up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% Can be prescribed as follows. In certain embodiments, the metadoxine or metadoxine derivative in the composition used according to the present invention is about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours of total metadoxine or metadoxine derivative. Release 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% or more Can be prescribed as follows.

  In certain embodiments, the metadoxine or metadoxine derivative in the composition used according to the present invention may be a combination of sustained release form or sustained release form and immediate release form or rapid release form. In certain embodiments, the relative ratio of sustained or sustained release metadoxine or metadoxine derivative to immediate or rapid release metadoxine or metadoxine derivative is, for example, 1 to 99, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to 65, 40 to 60, 45 to 55, 50 to 50, 55 to 45, 60 to 40, 65 to 35, 70 to 30, 75 to 25, 80 to 20, 85 to 15, 90 to 10, 95 to 5, or 99 to 1.

  In certain embodiments, polymeric materials are used to sustain or control the release of metadoxine or metadoxine derivatives. In certain embodiments, the type of polymeric material and the amount used thereof has a strong impact on the release rate of metadoxine or metadoxine derivatives from the products of the present invention. Examples of polymers include both hydrophobic and hydrophilic polymers. Examples of hydrophobic polymers include, but are not limited to, ethyl cellulose and other cellulose derivatives, glycerol palmitosterate, beeswax, glycowax, castor wax, carnauba wax, glycerol monosterate or stearyl alcohol, etc. Fatty, hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acid derivatives, and mixtures of these polymers are included. Hydrophilic polymers include, but are not limited to, hydrophilic cellulose derivatives such as methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium and hydroxyethyl methyl cellulose polyvinyl alcohol, polyethylene, polypropylene , Polystyrene, polyacrylamide, ethylene vinyl acetate copolymer, polyacrylate, poly-urethane, polyvinyl pyrrolidone, polymethyl methacrylate, polyvinyl acetate, polyhydroxyethyl methacrylate, and mixtures of these polymers. In addition, any mixture of one or more hydrophobic polymers and one or more hydrophilic polymers can optionally be used.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is microcrystalline cellulose such as “Avicel PH 101” manufactured by FMC BioPolymer's.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is a hydroxypropyl methylcellulose such as “Metrose” produced by Shin-Etsu Chemical Co.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is ethylcellulose such as “Ethocel ™” manufactured by The Dow Chemical Company.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is an acrylic polymer such as “Eudragit RS ™” produced by Rohm GmbH.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is colloidal silicon dioxide such as “Aerosil ™” manufactured by Degussa.

  In a particular embodiment, the polymeric material used in the composition of the invention or used according to the invention is a poly (vinyl acetate) such as “Kollicoat SR” manufactured by BASF.

  In a particular embodiment, the polymeric material used in the compositions of the invention or used according to the invention can be obtained from Delasco Dermatologic Lab & Supply, Inc. Ethyl acetate and vinyl acetate solutions such as “Duro-Tak” manufactured by

  In certain embodiments, the compositions of the invention or used in accordance with the invention have about 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg to about 1000, Comprise or consist essentially of 1500, 2000, 2500 or 3000 mg of metadoxine or a metadoxine derivative. In certain embodiments, compositions of the invention or used in accordance with the invention have from about 5, 100, 500, or 1000 mg to about 2000, 4000, 10,000, 15,000, or 20,000 mg of Avicel. PH 101 (TM) comprises or consists essentially of. In certain embodiments, the compositions of the invention or used in accordance with the invention have about 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 mg to about 650. 700, 750, 800, 850, 900, 950, 1000, 5000, 10,000, 15,000 or 20,000 mg of polymeric material or consist essentially of. In certain embodiments, the polymeric material is Methorose, Ethocel E10 ™ or Eudragit RS ™. In certain embodiments, the Methorose comprises or consists essentially of between 1-90%, preferably between 5-70% of the formulation. In certain embodiments, Ethocel ™ comprises or consists essentially of between 1-30%, preferably between 2-20% of the formulation. In certain embodiments, Eudragit ™ comprises or consists essentially of between 1-90% of the formulation, preferably between 5-70%.

  In certain embodiments, the delivery system of or used in accordance with the present invention comprises a delivery device. In certain embodiments, the compositions of the invention or used in accordance with the invention are delivered by an osmotic process at a controlled rate, such as by an osmotic pump. This system can be constructed by coating an osmotically active agent with a rate-controlling semipermeable membrane. The membrane may include a critical size orifice through which the drug is delivered. After the dosage form comes into contact with an aqueous fluid, it absorbs water at a rate determined by the fluid permeability of the membrane and the osmotic pressure of the core formulation. This osmotic absorption of water results in the formation of a saturated solution of active material within the core, which is dispensed at a controlled rate from the delivery orifice in the membrane.

  In certain embodiments, the compositions of or used in accordance with the present invention are delivered using biodegradable microparticles. In a particular embodiment, the system for preparing the microparticles consists of a material emulsified and encapsulated in an organic and aqueous phase composed of a volatile solvent and a dissolved polymer. In certain embodiments, the biodegradable polymer that can be used in the particulate matrix comprises polylactic acid (PLA) or a copolymer of lactic acid and glycolic acid (PLAGA). PLAGA polymers degrade over time into their monomer components by hydrolysis, which are easily removed from the body by natural metabolism.

  The formulations of or used in accordance with the present invention may also contain absorption enhancers and other optional ingredients. Examples of absorption enhancers include, but are not limited to, cyclodextrin, phospholipid, chitosan, DMSO, Tween, Brij, glycocholate, saponin, fusidate and energy-based absorption enhancers. .

  Optional ingredients present in the dosage form include, but are not limited to, diluents, binders, lubricants, surfactants, colorants, flavoring agents, buffering agents, preservatives, anti-degradation agents. Etc. are included.

  Diluents, also called “bulking agents” include, for example, dicalcium phosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, hydrolyzed starch, silicon dioxide, colloidal silica, oxidation Titanium, alumina, talc, microcrystalline cellulose, and powdered sugar are included. For administration in liquid form, diluents include, for example, ethanol, sorbitol, glycerol, water and the like.

  Binders are used to impart cohesive quality to the formulation. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugar (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, wax, natural And synthetic gums such as gum arabic, gum tragacanth, sodium alginate, cellulose, and bee gum, and synthetic polymers such as polymethacrylate and polyvinylpyrrolidone.

  Lubricants are used to facilitate manufacture; examples of suitable lubricants include, for example, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, and polyethylene glycol.

  The surfactant can be an anionic, cationic, amphoteric or nonionic surfactant, with an anionic surfactant being preferred. Suitable anionic surfactants include, but are not limited to, those containing carboxylate ions, sulfonate ions, and sulfate ions that are associated with cations such as sodium ions, potassium ions, and ammonium ions. Is included. Particularly preferred surfactants include, but are not limited to, long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinate such as sodium bis- (2-ethylhexyl)- Sulfosuccinates; and alkyl sulfates such as sodium lauryl sulfate.

  Degradation inhibitors such as antioxidants include, but are not limited to, propyl gallate, sodium ascorbate, citric acid, calcium metabisulfite, hydroquinone, and 7-hydroxycoumarin.

  If desired, the compositions of the invention or used according to the invention may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and the like.

  Any of the compositions of or used in accordance with the present invention can be used alone or in combination with one or more additional therapeutic agents for improving cognitive behavior. Examples of additional therapeutic agents are amphetamine, methylphenidate HCl, dexmethylphenidate hydrochloride, atomoxetine, reboxetine, fluoxetine, sertraline, paroxetine, fluoroxamine, citalopram, venlafaxine, bupropion, nefazodone and mirtazapine is there.

  The amount of both compound and additional therapeutic agent that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated so that doses between 0.1-1 g / kg body weight / day, preferably 0.1-300 mg / kg body weight can be administered. The dose of the compound depends on the patient's condition and disease and the desired daily dose. For human therapy, the oral daily dose is 10 to 3000 mg, or preferably 100 to 3000 mg. For example, the daily dose is 10, 25, 50 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 mg. These doses are administered in unit dosage form and may be administered in a single daily dose or, in certain cases, divided into two or three smaller doses each day.

  In certain embodiments, the compositions of the present invention may be synergistic in combination with each other and may be further synergistic in the presence of additional therapeutic agents. Accordingly, the amount of one or more compounds and one or more additional therapeutic agents in such compositions is less than that required for monotherapy using only that therapeutic agent. In such compositions, doses of additional therapeutic agent between 0.1-1 g / kg body weight / day can be administered.

Definitions For convenience, certain terms used in the specification, examples, and appended embodiments are collected here. Unless defined otherwise, all technical and scientific terms used herein are generally understood by those skilled in the art to which this invention belongs. Have the same meaning.

  The articles “a” and “an” are used herein to refer to one or more (ie, at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

  The term “including” is used herein to mean and is used interchangeably with the phrase “including but not limited to”.

  The term “or” is used herein to mean and interchangeably with the term “and / or” unless the context clearly indicates otherwise.

  The term “such as” is used herein to mean and is used interchangeably with the phrase “such as, but not limited to”.

  The term “prophylactic” or “therapeutic” treatment means administering to a subject one or more compositions of the invention. If administered prior to clinical manifestation of an undesirable condition (e.g., a host animal clinical condition or other undesirable condition), the treatment is prophylactic, i.e., it prevents prevention of the development of an undesirable condition, That is, it contributes to the protection of the subject, whereas if administered after the development of an undesirable condition, the treatment is therapeutic (i.e., resolves, ameliorates or avoids the progression of the undesirable condition or its side effects) Intended to).

  The term “therapeutic effect” means a local or systemic effect in an animal, particularly a mammal, more particularly a human, caused by one or more pharmacologically effective substances. Thus, this term refers to any substance intended for use in diagnosing, curing, alleviating, treating or preventing a disease, or promoting desirable physical or mental development and conditions in an animal or human. The term “therapeutically effective amount” means the amount of such a substance that produces any desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, the therapeutically effective amount of a compound or composition depends on its therapeutic index, solubility, and the like. For example, a particular metadoxine or metadoxine derivative formulation of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to the chosen treatment, as can be determined by one skilled in the art.

  The term “effective amount” means an amount of a therapeutic reagent that produces at least one desired result when administered to a subject in a suitable dose and dosing regimen.

  A “subject” or “patient” to be treated by the methods of the invention can refer to either a human or non-human animal, preferably a mammal. The term “subject” as used herein may refer to a healthy individual or a subject suffering from fragile X syndrome or an autism spectrum disorder. In another embodiment, the terms “subject” and “healthy subject” and “subject in need” and “patient in need” as used herein are in any form after alcohol consumption. Excludes subjects affected by alcohol, alcohol addicts (alcoholics), and alcohol addicts who are not drinking alcohol.

  As used herein, the term “salt adduct” is intended to encompass the salt product of the direct addition of two or more different ions, where the overall charge of the salt adduct is zero. It is. In certain embodiments, the salt adduct comprises a single positively charged moiety having a single positively charged functional group (ie, the positively charged moiety has a +1 net charge) and a single negatively charged functional group. Having one negative charge portion (ie, this negative charge portion has a net charge of −1). In certain embodiments, the salt adduct is one positively charged moiety having two positively charged functional groups that may be the same or different (ie, the positively charged moiety has a +2 net charge); It may be the same or different and comprises two negatively charged moieties each having a single negatively charged functional group (ie, each negatively charged moiety has a net charge of −1). In certain embodiments, the salt adducts can be the same or different and each has two positively charged moieties (ie, each positively charged moiety has a +1 net charge) with one positively charged functional group. ) And one negatively charged moiety having two negatively charged functional groups that are the same or different (ie, the negatively charged moiety has a net charge of -2). In certain embodiments, the salt adduct has a positive charge moiety having a net charge of + n (derived from one or more positively charged functional groups, which may be the same or different) and a net charge of -n (same A negatively charged moiety having one or more negatively charged functional groups, which may be different or different, wherein n may correspond to 1, 2, 3, 4, 5 or 6 It is an integer.

As used herein, a “positively charged moiety of a salt adduct” of the present invention is the corresponding acid of pyridoxine, or any derivative thereof. In certain embodiments, the positive charge of the positively charged moiety is derived from the protonated basic nitrogen atom of pyridoxine (eg, in the case of compound (2)) or from any derivative thereof (eg, the formula ( Compounds of I)). In certain embodiments, the positively charged pyridoxine derivative is, for example, —NH ₃ ⁺ , —CH ₂ NH ₃ ⁺ , NH ₂ R ⁺ , —NHR ₂ ⁺ (wherein each R is independently C ₁ -C ₆ alkyl In some embodiments, in addition to the positively charged protonated basic aromatic nitrogen atom in the pyridine ring.

  Each of the salt adduct moieties of the present invention may contain at least one chiral center and thus any of its enantiomers, diastereomers, or any mixture thereof including but not limited to racemic mixtures. It should be understood that they exist as stereoisomers and may be isolated. The invention includes any possible stereoisomers (eg enantiomers, diastereomers) of any of the moieties of the salt adducts of the invention, any mixture thereof including but not limited to racemic mixtures including. If the process described herein for the preparation of each of the salt adduct portions of the present invention produces a mixture of stereoisomers, these isomers are separated by conventional techniques such as preparative chromatography. be able to. The portions of the salt adducts of the present invention can each be prepared in any mixture of its stereoisomers, including but not limited to its racemic mixtures, or individual stereoisomers ( For example, enantiomers, diastereomers) can be prepared by enantiospecific synthesis or by chiral chromatographic separation of racemates. Whenever amino acids are referred to, the present invention should be understood to encompass natural and unnatural amino acids or any derivatives thereof.

  Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” are intended to encompass the stated integer or group of integers. Is understood not to mean the exclusion of any other integer or group of integers.

  The term “bioavailable” means that at least some amount of a particular compound is present in the systemic circulation. Formal calculations of oral bioavailability have been described in terms of F values ("Fundamentals of Clinical Pharmacokinetics," John G. Wegner, Drug Intelligence Publications; Hamilton, Ill. 1975). The F value is derived from the ratio of the parent drug concentration in the systemic circulation (eg, plasma) after intravenous administration to the parent drug concentration in the systemic circulation after administration by a non-venous route (eg, oral). Accordingly, oral bioavailability within the scope of the present invention contemplates the ratio or F value of the amount of parent drug detectable in plasma after oral administration compared to intravenous administration.

  The term “treating” or “treatment” relates to at least sedation, amelioration, reduction or alleviation of symptoms, symptoms or disorders, or conditions, diseases or disorders in mammals such as humans. Improve the measured value that can be confirmed. Treatment as used herein also includes in healthy individuals.

  The term “acceptable derivative” with respect to metadoxine or a metadoxine derivative may provide metadoxine or a metabolite or functional residue thereof or measurable metadoxine activity (directly or indirectly) when administered to a subject. It means any salt, conjugate, ester, complex or other chemical derivative of metadoxine, or a moiety comprising said. The term “physiologically compatible metadoxine derivative” is used herein interchangeably with the term “acceptable derivative” and refers to a functional and active pharmaceutically acceptable derivative of metadoxine.

  The term “excipient” means an inert substance used as a carrier for the active ingredient in a formulation.

  The term “controlled release” means any formulation designed to deliver a drug at a controlled rate for an extended period of time to achieve a desired drug level profile.

  The term “sustained release”, in its conventional sense, provides a gradual release of active material over time, in a specific embodiment, a blood level that is substantially constant over time, i.e. controlled release. Is also used to mean a formulation that can further result.

  The term “immediate release” is used in its conventional sense to mean a formulation that provides non-delayed or controlled release of the active material upon administration.

  The term “half-life” of a substance is the time it takes for a substance to lose half of its pharmacological, physiological, or other activity. Biological half-life is an important pharmacokinetic parameter and is usually represented by the abbreviation tin.

  The term “non-invasive” means a mode of treatment that does not puncture the skin.

  The term “non-chronic administration” can be used interchangeably herein with the term “acute administration” and means that a subject is given a measured or non-measured amount or portion of an agent in an irregular manner. Non-chronic administration may be a single dose treatment or a multiple dose treatment, optionally given over time. In general, but not always, non-chronic administration is given to treat or prevent non-chronic conditions. Certain chronic conditions may also benefit from non-chronic administration of the metadoxine or metadoxine derivative compositions described herein.

  The term “chronic administration” means giving a subject a measured amount of drug in a regular manner. In some embodiments, chronic administration is intended for the treatment or prevention of one or more chronic conditions, disorders or diseases. Chronic disease has one or more of the following characteristics: chronic disease is permanent, leaves behind disability, results from irreversible pathological changes, requires special patient training in rehabilitation, or long-term guidance , Need to be observed, or care.

  The term “single dose treatment” means giving a measured amount of drug taken at one time. This is given to treat non-chronic conditions in a regular way, depending on the needs of the individual.

The term “t _max ” means the time for the concentration to peak. Calculation of the time at which the maximum concentration occurs after a single dose administration is performed according to

Where λα and λz are the apparent absorption and discharge rate constants, respectively.

Example 1 General Methods The examples described herein were generally performed using the following reagents and methods.

Test animals First, knockout mice (KO2) mice (The Dutch-Belgium Fraggil X Consortium, 1994) were obtained from Jackson Laboratory and wild-type (WT) littermates were created in the C57BL / 6J background, over 8 generations. Repeated backcrossing against C57BL / 6J background. Fmrl knockout mice were housed in groups of the same genotype in a temperature and humidity control room with a 12-hour light-dark cycle (lights on from 7 am to 7 pm; the test was conducted during the light period). Room temperature and humidity were continuously recorded in the breeding room with free access to food and water. The study was performed in healthy Fmrl knockout mice and their wild type littermates (mouse N = 10 per treatment group) 2 or 6 months of age during behavioral testing. Mice were housed in commercially available plastic cages and testing was performed according to the requirements of UK Minals (Scientific Procedures) Act, 1986. All studies were performed blind to genotype and drug treatment. Animals were given a minimum environmental acclimation period of 1 week prior to experimentation. No prophylactic or therapeutic treatment was administered during the environmental acclimatization period.

In Drug Test 1 (Example 2), metadoxine was dissolved in physiological saline and administered intraperitoneally at a dose of 100, 150, or 200 mg / kg / day for 7 days. In the in vivo test of Test 2 (Example 3), metadoxine was dissolved in physiological saline, and once a day for 7 days, an intraperitoneal dose of 150 mg / kg / day or 150 or 300 mg / kg / day (volume 0. 1 ml) oral dose. In study 2 in vitro, metadoxine was administered at a concentration of 300 μM for 5 hours. In all cases, saline was used as the vehicle (control).

Behavioral testing Social interaction and social cognitive memory: mice score easily, such as approaching, tracking, sniffing, grooming, aggressive encounters, mating, nurturing behavior, nesting, and snuggling together It is a social species that conducts social behavior. Social access in mice was assessed by the time of sniffing for first-time mice.

  Mice in test arena / cage (40 x 23 x 12 cm cage, with a perspex lid to help the mouse observe) of the same size as an adult home cage, with fresh wood chips on the floor placed. Prior to the test, a few mice not used in the test were placed on the device to smell the background mice. Mice were transferred to this laboratory 10-15 minutes prior to testing. Test subjects and young individuals were placed in the test cage at the same time. The total time and number of face-to-face social surveys defined as sniffing and proximity tracking (<2 cm from the tail) of young stimulants with test mice were evaluated for 3 minutes. After 30 minutes, the test was repeated using the same stimulated young individuals. The data parameters collected were the total time and total number of face-to-face sniffing for acquisition and recognition. A social memory ratio defined as trial 2 / trial 1 + 2 was derived. Thus, no memory (eg, 20 / (20 + 20) = 0.5 and memory (eg, 10 / (20 + 10) = <0.5).

Y maze replacement behavior : Two tasks were implemented. The first was an unlearning assessment of voluntary alternation between arm selections. The second was a spatial reference memory task, where the animal had to learn which of the two arms had a food reward. The day before the start of the training, the mice were allowed to explore this maze freely for 5 minutes. These mice then underwent two trials, one with food on the left arm and one with food on the right arm. This procedure prevented the biasing of one of these arms.

Y water maze : A transparent perspex Y maze was charged with 20 ° C. water up to 2 cm. This led the mouse to leave the maze after paddling to the exit tube at the distal end of one arm. This maze was placed in the middle of a room surrounded by prominent visual stimuli.

Rewarded T-maze replacement behavior : A T-shaped device with an elevated or wall (placed horizontally) was used. The mouse is placed at the base of T and one of the goal arms adjacent to the other end of the stem is selected. Two trials were performed in quick succession, and the second trial required the mouse to select an arm that had never been before, reflecting the memory of the first choice (spontaneous alternation behavior). This trend was enhanced by making the mice hungry and rewarding the food of their choice if the mice took turns. Specifically, after a 4-day acclimation period to this T maze, mice were trained to change their arm selection and receive sweet condensed milk as a reward.

Consecutive alleys : This device is arranged on four consecutive straight lines, which increase the anxiety made of painted wood (each successive alley is lightly colored, has a low wall and / or is less than the one in front. It was narrow). Each section or alley was 25 cm long. The alley 1 was 25 cm high and 8.5 cm wide and painted black. Down 0.5 cm, there was a path 2 which was also 8.5 cm wide but with a 1.3 cm high wall and was gray. Down 1.0 cm, it became a path 3, which was 3.5 cm wide, with 0.8 cm high walls and was white. It fell 0.4 cm to a path 4, which was also white, but was 1.2 cm wide and 0.2 cm high. This device was elevated by fixing the back of the alley 1 to a stand at a height of 50 cm. Padding was provided under arms 3 and 4 when the mouse fell. Each mouse was placed at the closed end of Path 1 facing the wall. The timer was started and 1) the total length of the test (5 minutes) + the latency to enter each arm, and 2) the time spent on alley 1 was measured. A mouse was considered to have entered the alley if all four legs were placed on the next alley. The total time spent on each alley (all four legs) was recorded.

Contextual fear conditioning : In the contextual fear conditioning test, mice are placed in a new environment (dark chamber) and cue and electric foot shock (0.2 mA 1 second (Test 1) or 0.7 mA 0.5 seconds (Test 2)) pair was given. Next, when tested as an initial training, mice exhibited a natural protective response called freezing (Blanchard, 1969) or contextual fear conditioning. Immobility reaction time was defined as the time the mouse spent in immobile behavior without breathing. This data was expressed as a percentage of the test period. 24 hours after the training session, the mice were tested for 5 minutes in a non-shocked training chamber and observed for immobile reaction behavior.

Statistics : Multivariate analysis of variance was used to assess differences in groups of data. Repeated measures ANOVA were performed on behavioral data. Statistically significant effects in each ANOVA were followed by post hoc comparison using the Newman-Coils test (Test 1) or Tukey test (Test 2). A p value of less than 0.05 was considered significant.

Biochemical studies Phosphorylated ERK and Akt: The Ras-Mek-ERK and PI3K-Akt-mToR signaling pathways are involved in mediating activity dependent on gene transcription underlying the changes in synaptic plasticity ( Klann and Dever, 2004). The expression of phosphorylated ERK and Akt proteins was measured by Western blot analysis as previously described by Lopez Verrilli (Lopez Verrilli et al., 2009). The antibodies used were anti-phosphate specific antibodies against Akt (1/1000) and kinase (ERK) 1/2 (1/2000) (Cell Signaling Technology, Danvers, MA, USA). The antibody against phospho-ERK detects phosphorylation at phospho-ERK1 / 2 (Thr202 / Tyr204), and the antibody against phospho-Akt detects phosphorylation at phospho-Akt (Thr308). Total Akt and ERK1 / 2 protein content and phosphorylated ERK and Akt were membraned with anti-phospho-Akt antibody (1/1000) and anti-phospho-ERK antibody (1/2000) (Cell Signaling Technology, Danvers, MA, USA). Were evaluated by blotting. Akt or ERK phosphorylation was normalized to protein content in the same sample and expressed as% change relative to ground state, with basal levels as 100%. Protein loading was assessed by exfoliating the membrane and replotting with β-actin antibody (1/1000) (Sigma-Aldrich, St. Louis, MO, USA). Expression of phosphorylated ERK and Akt protein in blood lymphocytes was measured by flow cytometry. For the determination of lymphocyte biomarkers, FACStar plus (Becton Dickinson) was used with an excitation laser tuned to 488 nm, and green fluorescence (GST) from FITC was collected through a 515-545 nm bandpass filter. Mean FITC fluorescence intensity was calculated relative to the fluorescence of the reference cells. Mean cell fluorescence intensity (MFI) is directly proportional to the average number of Ab molecules bound per cell.

Neuronal morphology : Hippocampal cell cultures were prepared from wild-type and Fmrl KO fetal mice on day 17.5 of gestation (E17.5). Mice were killed by cervical dislocation and excised hippocampal cells were seeded into 15 mm multiwell vials (Falcon Primaria). After 5 days in vitro, green fluorescent protein (GFP) was transfected to help monitor dendritic spine morphogenesis after drug treatment (Ethell and Yamaguchi, 1999; Ethell et al., 2001, Henkemeyer et al. , 2003). Dendritic spines were seen around day 16 (DIV) in vitro. On day 17 in vitro, the cultures were treated with metadoxine at a concentration of 300 μM for 5 hours.

  The filopodia density of GFP-transfected neurons was quantified by performing a Sholl analysis of images (40 × objective, 20 × 0.2 μm stack) generated with stacked Zeiss confocals. Using Metamorph software, concentric equally spaced circles (total 20 μm) were drawn before and after the cell body of each neuron, and then the amount of filopodia was counted for each circle. The mean counts were compared using an unpaired two-tailed Student's T test.

  The maturity of process spines of GFP-transfected neurons was analyzed using Metamorph software (Molecular Devices, Sunnyvale, CA). Two distal dendritic segments of 70-100 μm were selected for each neuron for process of spinous morphometry analysis. The length and width were measured for each process spine. The length was defined as the distance from the base of the process to the tip; the width was defined as the maximum distance perpendicular to the long axis of the process spine. Measurements were compared using unpaired two-sided Student's T test and ANOVA corrections for multiple comparisons were made.

de novo hippocampal protein synthesis : Hippocampal cross-sections (400 μm) were obtained from 6-week-old Fmrl knockout and WT mice. The protein synthesis assay is a surface sensing of translation (SUNSET) method, an assay based on non-radioactive, fluorescence activated cell sorting that allows global protein synthesis monitoring and quantification in individual mammalian cells and in heterogeneous cell populations. (Hoeffer, 2011) and performed as previously described. The concentration of metadoxine used in this test was 300 μM.

Example 2: Effect of metadoxine (100-200 mg / kg) treatment on learning and memory deficits and biochemical abnormalities in Fmrl knockout mouse model of fragile X syndrome (Study 1)
Behavior analysis
Contextual fear conditioning : In the first study, the effect of intraperitoneal administration of vehicle or 150 mg / kg metadoxine once daily for 7 days on contextual fear conditioning in N = 10 WT and Fmrl knockout mice groups was investigated. Vehicle-treated Fmrl knockout mice showed a learning deficit in a contextual fear conditioning paradigm that is reflected in decreased immobility response during the test session (Figure 1, Panel A (p <0.0001)). Metadoxine administration reversed the learning deficit effect in Fmrl knockout mice, and this reversal was partial because metadoxine treated mice were different from metadoxine treated WT mice (p <0.05). This study was repeated to examine the dose-dependent effects of intraperitoneal administration of vehicle, 100 or 200 mg / kg metadoxine once daily for 7 days on contextual fear conditioning in N = 10 WT and Fmrl knockout mice groups (Figure 1). Panels B and C). In this study, vehicle-treated Fmr1 knockout mice showed learning deficits compared to vehicle-treated WT mice (p <0.0001), which was a reproduction of the first study. 100 mg / kg metadoxine had reversed defects in Fmrl knockout mice (P <0.05), but metadoxine treated Fmrl knockout mice were different from metadoxine treated wild type mice (p <0.0001). ) This was a partial reversal. The learning deficit seen in Fmrl knockout mice was 200 mg / kg i. p. It was completely reversed after treatment with metadoxine (treated Fmr1 mice were different from vehicle-treated Fmr1 knockout mice (P <0.0001) but not metadoxine-treated WT mice). Metadoxine treatment had no effect on WT mice in either test (Figure 1, Panels AC).

Social access : Vehicle-treated Fmrl knockout mice showed low social access as indicated by scent sniffing (Figure 2 (p <0.0001)). Intraperitoneal treatment with 150 mg / kg metadoxine once daily for 7 days increased the social access of Fmr1 knockout mice (p <0.0001 compared to vehicle-treated Fmr1 knockout mice). Fmr1 knockout mice treated with metadoxine were different from metadoxine-treated WT mice, although they tended to approach the effects of WT mice (p <0.05). Metadoxine treatment had no effect on WT mice.

Y-maze spontaneous alternation behavior : The effect of treatment with vehicle or 150 mg / kg metadoxine once a day for seven days on spontaneous alternation behavior in N = 10 WT or Fmrl knockout mice groups is shown in FIG. Vehicle treated Fmrl knockout mice showed lower spontaneous alternation behavior than vehicle treated W mice (p <0.0001). Metadoxine treatment increased spontaneous alternation behavior compared to vehicle treatment in Fmrl knockout mice (p <0.0001), whereas metadoxine treated Fmrl knockout mice showed a deficiency compared to metadoxine treated WT mice (p <0). .01). Thus, metadoxine resulted in partial reversal of the defect found in Fmrl knockout mice.

Y-maze reference memory task : FIG. 3 panel B shows the effect of treatment with vehicle or 150 mg / kg metadoxine once daily for 7 days on rewarded reference memory learning in N = 10 WT or Fmrl knockout mice. Vehicle treated Fmr1 knockout mice had a lower appropriate arm selection than vehicle treated WT mice (p <0.0001). Metadoxine-treated Fmr1 knockout mice were not different from metadoxine-treated WT mice, so metadoxine treatment reduced this deficiency compared to vehicle-treated Fmr1 knockout mice (p <0.0001). Metadoxine treatment had no effect on WT mice.

Y Water Maze Left-Right Discrimination : The effect of treatment with vehicle or 150 mg / kg metadoxine once daily for 7 days on aversive motivational spatial discrimination learning in N = 10 WT or Fmrl knockout mice groups is shown in FIG. . Vehicle treated Fmrl knockout mice showed a higher number of inappropriate arm selections than vehicle treated WT mice. This deficiency was alleviated by metadoxine treatment.

T-maze rewarded alternation behavior task : FIG. 4 shows the effect of treatment with vehicle or 150 mg / kg metadoxine once a day for 7 days on reward alternation behavior working memory in a group of N = 10 WT or Fmr1 knockout mice. Vehicle treated Fmrl knockout mice showed a longer latency to reach the correct arm compared to vehicle treated WT mice (p <0.0001). Metadoxine treatment alleviates this deficiency compared to vehicle treatment in Fmrl knockout mice (p <0.0001), since metadoxine treated Fmrl knockout mice responded slower than WT mice (p <0.0001). The reversal was partial.

Continuous alley : The effect of treatment with vehicle or 150 mg / kg metadoxine once a day for 7 days on behavior in a continuous alley task in a group of N = 10 WT or Fmrl knockout mice is shown in FIG. 5 and further described below. .

  The continuous path test effectively measured anxiety (latency 1 approach latency) and overactivity (paths 2-4). Progression from alley 1 to continuous alleys 2, 3, and 4 involves exposure to an increasingly brighter environment with increasingly lower walls and narrower, more open open arms. . Time spent in the open arm and entry into the open arm is an indicator of anxiety; conversely, increased time spent in the more open arm represented overactivity. These factors enabled high-sensitivity tests that collectively handle a range of anxiety-like behaviors with excessive activity.

Path 1 : Fmr1 knockout mice showed greater anxiety than WT mice (p <0.001). Since complete normalization was seen, Fdrl1 knockout mice treated with metadoxine showed improved anxiety compared to vehicle treated Fmr1 knockout mice (p <0.001). There was no difference between metadoxine-treated Fmrl knockout and metadoxine-treated WT mice. Metadoxine treatment had no effect on WT mice.

Path 2 : WT mice showed lower activity in path 2 when compared to Fmr1 knockout mice (p <0.0001). Metadoxine treatment reduced hyperactivity in Fmr1 knockout mice (p <0.001), but metadoxine-treated Fmr1 knockout and WT mice were different (p <0.001), and this reversal of hyperactivity was partially It was something. Metadoxine treatment had no effect on WT mice.

Path 3 : Fmr1 knockout mice showed overactivity compared to WT mice (p <0.0001). This hyperactivity was not reversed by metadoxine since metadoxine-treated Fmr1 knockout mice were not different from vehicle-treated Fmr1 knockout mice. Metadoxine treatment had no effect on WT mice.

Path 4 : Fmr1 knockout mice showed overactivity compared to WT mice (p <0.01). Metadoxine treatment reversed this overactivity, as metadoxine-treated Fmr1 knockout mice showed lower activity than vehicle-treated Fmr1 knockout mice (p <0.01). This effect represented a normalization because metadoxine-treated Fmrl knockout mice were not different from metadoxine-treated WT mice. Metadoxine treatment had no effect on WT mice.

  Overall, while not wishing to be bound by any particular theory, it is believed that continuous alley testing has shown that Fmrl knockout mice exhibit increased anxiety and hyperactivity compared to WT mice. Metadoxine treatment alleviated this anxiety and hyperactivity in Fmrl knockout mice, while WT mice remained unaffected.

Biochemical analysis
ERK and Akt phosphorylation : Intraperitoneal treatment of either vehicle or 150 mg / kg metadoxine once daily for 7 days against total brain phosphorylation of brain ERK or Akt in N = 5 Fmrl knockout or WT mice. The effect is shown in FIG. The phosphorylation level was evaluated as the ratio of phosphorylation to total ERK. An increase in this ratio indicated ERK activation. ERK phosphorylation was increased in vehicle-treated Fmrl knockout mice compared to vehicle control (p <0.001), but this effect reproduced the abnormal activation of ERK seen in human subjects with fragile X syndrome (Wang et al., 2012). This effect was reduced by metadoxine treatment (p <0.01) since no difference was seen when compared to metadoxine treated WT mice. Metadoxine had no effect on ERK phosphorylation in WT mice or total ERK levels in any mouse. The ratio of phosphorylated Akt to total AKT was also increased in vehicle treated Fmrl knockout mice compared to vehicle treated WT mice (p <0.0001). Metadoxine treatment reduced the relative levels of phosphorylated Akt in Fmr1 knockout mice, as Fmr1 knockout mice were not different from controls (p <0.01). Metadoxine treatment had no effect on WT mice or on total Akt levels in any mouse.

Example 3: Evaluation of metadoxine in the Fmr1 knockout fragile X mouse model (Test 2)
Behavioral effects of metadoxine in 6-month-old Fmr1 knockout mice
Contextual fear conditioning : The first study investigated the effect of intraperitoneal administration of vehicle or 150 mg / kg metadoxine once daily for 7 days on contextual fear conditioning in a group of 6 months old WT and Fmrl knockout mice with N = 10 It was. Vehicle-treated Fmrl knockout mice (KO-V) showed a learning deficit in the contextual fear conditioning paradigm compared to vehicle-treated WT mice (WT-V), reflected in decreased immobility response during the test session (FIG. 7 (p <0.0001)). Metadoxine administration reversed the learning deficit effect in Fmrl knockout mice (p <0.0001 KO-M-150 vs. KO-V). This was a complete reversal because metadoxine-treated KO mice were not different from metadoxine-treated WT mice.

Social access and social memory : Social access data (first trial 1) is shown in FIG. 8 panel A (number of sniffing) and panel C (smelling duration). Odor sniffing social memory data (24 hours after trial 2, trial 1) are shown in FIG. 8 panel B (number of sniffing) and panel D (duration of smell sniffing). These results are further discussed below.

  In trial 1, Fmr1 knockout mice had more olfactory sniffing (p <0.0001) than WT mice (see FIG. 8 panel A), and odor sniffing lasted less (p <0. 0001) (FIG. 8 panel C). These social interaction deficiencies are consistent with those reported by other investigators in Fmrl knockout mice (Thomas et al., 2011). Metadoxine treatment did not differ from metadoxine-treated WT mice in terms of the number of scent sniffing, both in terms of number of face-to-face and odor sniffing, so metadoxine treatment reversed the abnormalities in Fmrl knockout mice. (P <0.0001 KO-M-150 vs. KO-V, respectively). Although relief was shown in terms of scent duration measurements, Fmrl knockout mice remained different compared to metadoxine-treated WT mice (p <0.05), so this effect was partially It was a thing. Metadoxine had no effect on WT mice. These data indicated that abnormal social access behavior in Fmrl knockout mice was rescued by metadoxine.

  In trial 2, Fmr1 knockout mice showed both increased scent sniffing and increased scent sniffing time compared to wild type mice (p <0.0001 for each measurement, FIG. 8 panel, respectively). B and D). This reflected a failure to acclimatize, and thus a lack of social memory. Metadoxine treatment alleviated these differences (p <0.0001 KO-M-150 vs. KO-V). This reversal in the number of scent sniffing was partial, as the difference between metadoxine treated Fmrl knockout mice and metadoxine treated WT mice was maintained (p <0.05). This reversal by metadoxine was complete with respect to scent duration, as no difference was seen between metadoxine-treated Fmrl knockout and metadoxine-treated WT mice. Metadoxine treatment had no effect on WT mice. These data indicated that metadoxine reduced social memory impairment in Fmrl knockout mice. This reduction in social memory deficit is shown below by calculation of the social memory ratio (described in Example 1).

  The social memory ratio was defined as the duration of smell sniffing: trial 2 / trial 1 + 2. Accordingly, an example without storage is, for example, 20 / (20 + 20) = 0.5, and an example with storage is, for example, 10 / (20 + 10) = <0.5.

The calculated social memory ratio was as follows.
WT-V Trial 2 / Trial 1 + Trial 2: 12.4 / 12.4 + 26.8 = 0.3, <0.5 Memory KO-V Trial 2 / Trial 1 + Trial 2: 325/325 + 24.1 = 0. 9. WT-M Trial 2 / Trial 1 + Trial 2: 12.5 / 38.5 + 12.5 = 0.2, <0.5 Memory KO-M Trial 2 / Trial 1 + Trial 2: 12.7 / 28.4 + 12.7 = 0.3, <0.5 with memory

Biochemical effects of metadoxine in 6 month old Fmr1 knockout mice Effect of ip treatment with either vehicle or 150 mg / kg metadoxine once daily for 7 days on N = 10 Fmr1 knockout or whole brain pERK in WT mice (Figure 9 panel A) and pAkt of the brain after the behavior test (FIG. 9, panel B) are shown in FIG. Specifically, FIG. 9 panel A shows increased brain levels of pAkt in Fmrl knockout mice as compared to WT mice as seen in previous studies (P <0.0001). Metadoxine treatment reversed this increase in brain pAkt (p <0.0001 KO-M-150 vs. KO-V) since metadoxine-treated Fmrl knockout mice were not different from metadoxine-treated WT mice. FIG. 9 panel B shows increased brain levels of pERK in Fmrl knockout mice compared to WT mice as seen in previous studies (p <0.0001 KO-M-150 vs. KO-V). This increase was reversed by metadoxine treatment because metadoxine-treated Fmrl knockout mice were not different from metadoxine-treated WT mice (p <0.0001).

Effect of metadoxine after intraperitoneal or oral administration on the behavior of 2-month-old mice FIG. 10 shows a 150 mg / kg ip dose once daily for 7 days for 2-month-old Fmrl knockout and contextual fear conditioning in WT mice. The effects of metadoxine administration at 150 and 300 mg / kg oral doses are shown. Specifically, FIG. 10 panel A shows contextual fear conditioning data obtained from Fmr1 knockout and WT mice after vehicle ip and oral treatment. There was no difference regarding the route of vehicle administration. Fmrl knockout mice showed a reduction in immobile response behavior compared to WT mice after vehicle treatment via ip and oral routes (p <0.0001 in each case). FIG. 10 panel B shows the effect of metadoxine treatment by both routes of administration in WT mice. No effect was seen. FIG. 10 panel C shows that ip 150 mg / kg and oral 150 and 300 mg / kg metadoxine treatment in Fmrl knockout mice reversed the reduction in immobility response behavior seen in Fmrl knockout mice (p <0.01, p < 0.0001, and p <0.0001, KO-M-ip, KO-M-po150, and KO-M-po 300 versus KO-V-ip and KO-V po, respectively). The administration effect of 150 mg po metadoxine was not different from the administration effect of 300 mg / kg po metadoxine. The effects of 150 and 300 mg / kg oral metadoxine in Fmrl knockout mice were not different from the effects of 150 mg / kg ip metadoxine. In each case, this reversal was complete because metadoxine-treated Fmrl knockout mice were not different from metadoxine-treated WT mice.

  FIG. 11 shows the effect of administration of metadoxine at 150 mg / kg ip or 150 and 300 mg / kg oral doses once daily for 7 days on social access and social memory in Fmrl knockout and WT mice. Specifically, FIG. 11 panel A shows the effect of vehicle or 150 mg / kg ip or 150 and 300 mg / kg oral metadoxine on social access behavior in Fmrl knockout or WT mice. After ip or oral treatment with vehicle, the duration of scent behavior in Fmrl knockout mice was reduced compared to WT mice (p <0.0001 in each case). Metadoxine treatment had no effect on WT mice at any dose. However, 150 mg / kg ip, 150 mg / kg, and 300 mg / kg oral metadoxine treatment resulted in reversal of the deficits in social access seen in Fmrl knockout mice (p <0.0001 KO-M-po150 respectively). And KO-M-po300 vs. KO-V po). This effect of oral metadoxine was not dose dependent between 150-300 mg / kg. This reversal was complete because metadoxine treated Fmrl knockout mice were not different from metadoxine treated WT mice. The effect of 150 mg / kg ip metadoxine in Fmrl knockout mice was not different from the effect of 150 mg / kg oral or 300 mg / kg oral metadoxine. FIG. 11 panel B shows the effect of 150 mg / kg ip or 150 and 300 mg / kg oral vehicle or metadoxine on social memory in Fmrl knockout or WT mice. After ip or vehicle treatment, the duration of scent behavior in Fmrl knockout mice was increased compared to WT mice (p <0.0001 in each case). Metadoxine treatment had no effect on WT mice at any dose. However, 150 mg / kg ip, 150 mg / kg oral, and 300 mg / kg oral metadoxine treatment resulted in reversal of the deficits in social access seen in Fmrl knockout mice (p <0.0001, p <0. 05, and p <0.01 KO-M-ip150, KO-M-po150, and KO-M-po 300 vs. KO-V-ip and KO-V po, respectively). This reversal was complete because metadoxine treated Fmrl knockout mice were not different from metadoxine treated WT mice. The effect of 150 mg / kg ip metadoxine in Fmrl knockout mice was not different from the effect of 150 mg / kg oral or 300 mg / kg oral metadoxine. In addition, the dose-dependent effects of these oral metadoxine treatments were not observed between 150 mg / kg and 300 mg / kg.

Effect of metadoxine on biochemical markers after intraperitoneal or oral administration in 2-month-old mice
Peripheral lymphocytes : FIG. 12 shows one day of 7 days against lymphocytes pAkt (FIG. 12 panel A) and pERK (FIG. 12 panel B) as determined by flow cytometry in 2 month old Fmrl knockouts and WT mice. The effects of administration of metadoxine at a single dose of 150 mg / kg ip or 150 mg / kg and 300 mg / kg are shown. Specifically, FIG. 12 panel A shows that vehicle-treated Fmrl knockout mice exhibited increased phosphorylation of lymphocyte Akt compared to WT mice that received comparable vehicle treatment (ip and oral administration). P <0.0001) for both. Akt levels were not different between metadoxine-treated Fmrl knock-out mice and WT mice that received the same treatment, so metadoxine at 150 mg / kg ip or 150 mg / kg or 300 mg / kg oral dose once daily for 7 days Treatment normalized the overactivated Akt. FIG. 12 panel B shows that vehicle-treated Fmrl knockout mice showed increased phosphorylation of lymphocyte ERK compared to WT mice that received comparable vehicle treatment (p <0 for both ip and oral administration). .0001). pERK levels were not different between metadoxine-treated Fmrl knockout mice and WT mice that received the same treatment, so metadoxine at 150 mg / kg ip or 150 mg / kg or 300 mg / kg oral dose once daily for 7 days Treatment normalized over-activated ERK.

Brain Region : FIG. 13 shows the effect of 150 mg / kg metadoxine administration for 7 days on hippocampal, prefrontal cortex, and striatal pERK levels. pERK levels were increased in Fmr1 knockout mice compared to WT mice in all three brain regions (p <0.0001 in all cases). pERK levels were decreased in metadoxine-treated Fmr1 knockout mice compared to vehicle-treated Fmr1 knockout mice (p <0.0001 in all cases). There is no difference in hippocampus and striatum between the KO-M and WT-M groups, indicating a complete reversal of ERK activation. The effect on the prefrontal cortex was partial and the KO-V and KO-M groups remained different (p <0.05). Metadoxine had no effect on WT mice.

  FIG. 14 shows the effect of 7 days of 150 mg / kg metadoxine administration on pAkt levels in the hippocampus, prefrontal cortex and striatum. pAkt levels were increased in Fmr1 knockout mice compared to WT mice in all three brain regions (p <0.0001 in all cases). pAkt levels were reduced in metadoxine-treated Fmr1 knockout mice compared to vehicle-treated Fmr1 knockout mice in all three brain regions (p <0.0001 in all cases). In all cases, there is no difference between the KO-M and WT-M groups, indicating a complete reversal of Akt activation. Metadoxine had no effect on WT mice. Decreased levels of phosphorylated ERK and Akt in the brain and blood correlate with improved behavioral outcomes in Fmrl knockout mice, suggesting that phosphorylation levels are biomarkers of metadoxine treatment response.

Effect of metadoxine on dendritic filopodia density and primary hippocampal neuron maturation from Fmrl knockout mice in vitro FIG. 15 (panels AC) shows the effect of treatment with 300 μM metadoxine for 5 hours. The dendrites were divided into 10 segments of 10 μm based on the distance from the neuronal cell body (from proximal to distal from left to right). The process spine density was increased in neurons derived from Fmrl knockout mice in segment 3 compared to neurons derived from WT mice. Specifically, FIG. 15 panel A shows the filopodia density of neurons. Primary hippocampal neurons from Fmr1 knockout mice showed an increase in the density of filopodia (p <0.001). Treatment with 300 μM metadoxine alleviated an abnormal increase in neuronal filopodia density in Fmrl knockout mice (p <0.001). Neurons derived from Fmr1 knockout mice have immature features, are longer (FIG. 15 panel B (p <0.01)) and narrower (FIG. 15 panel C (p <0.01)). Showed legs. Metadoxine treatment reversed this increase in the length of the fistula (FIG. 15 panel B (p <0.01)) and reversed the increase in width (FIG. 15 panel C (p <0.001)).

Effect of metadoxine on de novo hippocampal protein synthesis in Fmr1 knockout mice in vitro FIG. 16 shows the effect of treatment with either vehicle or 300 μM metadoxine on basal de novo protein synthesis in 400 μM hippocampal sections from Fmr1 knockout or WT mice. . Protein synthesis was higher in the hippocampus from vehicle-treated Fmrl knockout mice than in the vehicle-treated WT control hippocampus (p <0.0001). Metadoxine treatment reduced the rate of protein synthesis in the hippocampus of Fmrl knockout mice. This effect was partial because the hippocampus from Fmrl knockout mice maintained a higher protein synthesis rate than the hippocampus from metadoxine-treated WT mice (p <0.001).

Claims (10)

  A method of treating or alleviating symptoms of fragile X syndrome or related disorders, comprising administering to a subject in need thereof a composition comprising metadoxine.   2. The method of claim 1, comprising administering a total daily dose of metadoxine between 100 and 3000 mg.   The method of claim 1, wherein the metadoxine is administered daily, every other day, or weekly.   The method of claim 1, wherein the metadoxine is administered in one, two, or three dosage forms per day.   2. The method of claim 1, wherein the metadoxine is administered in a sustained release oral dosage form and the metadoxine is formulated as a combination of a sustained release form and an immediate release form. (A) the sustained release form provides a sustained release of said metadoxine for at least 8 hours; and
(B) the relative ratio of sustained release metadoxine and immediate release metadoxine is between about 60:40 and 80:20;
The method of claim 5.   7. The method of claim 6, wherein the relative ratio of the sustained release metadoxine and the immediate release metadoxine is about 65:35.   The method of claim 1, wherein the symptom is a learning disorder or a social behavior disorder.   The method of claim 1, wherein the subject has fragile X syndrome or autism spectrum disorder.   The method of claim 1, wherein the associated disorder is an autism spectrum disorder.

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