Patent Application
20150335629
The present invention relates generally to methods of treating or alleviating a symptom of Fragile X syndrome and related disorders.
Fragile X syndrome (FXS), as implied by its name, is associated with a fragile site expressed as an isochromatid gap in the metaphase chromosome at map position Xq 27.3. Fragile X syndrome is a genetic disorder caused by a mutation in the 5′-untranslated region of the fragile X mental retardation 1 (FMR1) gene, located on the X chromosome. The mutation that causes fragile X syndrome is associated with a CGG repeat in the fragile X mental retardation gene FMR1. In 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, the CGG sequence is repeated from 200 to more than 1,000 times. When a subject has more than about 200 CGG repeats, the fragile X gene becomes hypermethylated, which silences the gene. As a result, fragile X mental retardation protein (FMRP) is not produced and the subject is diagnosed as having fragile X syndrome.
Premutation expansions (55-200 CGG repeats) of the FMR1 gene are frequent in the general population, with estimated prevalences of 1 per 259 females and 1 per 812 males. Carriers of the premutation typically have normal IQ, although emotional problems such as anxiety are common. Older male carriers of the premutation (50 years and older) develop progressive intention tremor and ataxia. These movement disorders are frequently accompanied by progressive cognitive and behavioral difficulties, including memory loss, anxiety, and deficits of executive function, reclusive or irritable behavior, and dementia. This disorder has been designated fragile X-associated tremor/ataxia syndrome (FXTAS). Magnetic resonance imaging in subjects with FXTAS reveals increases in T2-weighted signal intensity in the middle cerebellar peduncles and adjacent cerebellar white matter.
Fragile X syndrome segregates as an X-linked dominant disorder with reduced penetrance. Either sex when carrying the fragile X mutation may exhibit mental deficiency, which is variable in severity. Children and adults with fragile X syndrome have varying degrees of mental retardation or learning disabilities and behavioral and emotional problems, including autistic-like features and tendencies. Young children with fragile X syndrome often have delays in developmental milestones, such as learning how to sit, walk and talk. Affected children may have frequent tantrums, difficulties in paying attention, frequent seizures (e.g., temporal lobe seizures) are often highly anxious, easily overwhelmed, can have sensory hyperarousal disorder, gastrointestinal disorders, and may have speech problems and unusual behaviors, such as hand flapping and hand biting.
Fragile X syndrome can be diagnosed by an established genetic test performed on a sample (e.g., blood sample, buccal sample) from the subject. The test determines whether a mutation or pre-mutation is present in the FMR1 gene of the subject.
Subjects with fragile X syndrome can 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). About 15 to about 20% of subjects with fragile X syndrome meet the full diagnostic criteria for autism. Although mental retardation is a hallmark feature of fragile X syndrome, subjects with fragile X syndrome often display autistic features ranging from shyness, poor eye contact, and social anxiety in mild cases to hand flapping, hand biting and preservative speech in the severely affected. Subjects with fragile X syndrome display other symptoms associated with autism such as attention deficit and hyperactivity, seizures, hypersensitivity to sensory stimuli obsessive-compulsive behavior and altered gastrointestinal function. The FMR1 mutation prevents or greatly decreases expression of a single protein (FMRP). Brain development in the absence of FMRP is thought to give rise to the major symptoms of fragile X syndrome.
In addition to core symptoms, children with fragile X syndrome frequently have serious behavioral disturbances such as irritability, aggression and self-injurious behaviors. In a recent study of males with fragile X syndrome (ages 8-24), self-injurious behavior was reported in 79%, and aggressive behavior in 75%, of subjects during a two month observation period.
Currently available treatment regimens for humans fragile X syndrome include, for example, behavioral modifications and treatment with a range of medications including antidepressant and antipsychotic drugs. Cognitive behavioral therapy has been used to improve language and socialization in fragile X syndrome and autism. In recent years, pharmacological treatment with the atypical antipsychotic risperidone has been commonly employed to augment non-pharmacological approaches in the treatment of individuals with autism. A randomized placebo-controlled trial of risperidone in autistic children demonstrated significant improvement on the irritability subscale of the Aberrant Behavior Checklist and the Clinical Global Impressions-Improvement (McCracken, J. T., et al., N. Engl. J. Med. 347:314-321 (2002)). However, adverse events included weight gain, increased appetite, fatigue, drowsiness, dizziness, and drooling. Social isolation and communication were not improved by administration of risperidone and adverse side effects such as extrapyramidal symptoms and dyskinesias have been associated with risperidone use in autistic children. Since current treatment regimens are frequently not effective or may produce undesirable side-effects with long term use, particularly in the case of antipsychotic drugs, there is a need to develop new treatments.
In various aspects the invention provides methods of treating or alleviating a symptom of Fragile X Syndrome or a related disorder by administering to a subject in need thereof a composition comprising metadoxine. The symptom is for example, impaired learning or impaired sociability. The subject has Fragile X Syndrome or an Autism Spectrum Disorder. The related disorder is an Autism Spectrum Disorder.
In some aspects a total per day dose-of metadoxine of between 100-3000 mg is administered the metadoxine is administered daily, every other day or weekly.
Optionally, the metadoxine is administered in one, two, or three dosage forms per day. In some embodiments the metadoxine is administered in a sustained release oral dosage form, wherein the metadoxine is formulated as a combination of slow release and immediate release forms.
For example, the slow release form provides for sustained release of the 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 proportion of the slow release metadoxine to the immediate release metadoxine is about 65:35.
Unless otherwise defined, 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 pertains. 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 expressly incorporated by reference in their entirety. In cases 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.
The present invention relates to the discovery that metadoxine significantly improves cognitive and social functioning in a valid animal model for the Fragile-X Syndrome.
Specifically, metadoxine significantly improved memory and learning during the contextual fear paradigm in a dose-dependent manner, and the two highest dose levels (150 and 200 mg/kg) fully rescued the Fmr1 KO mice learning and memory deficit to a similar extent of the WT mice levels. Furthermore, a significant improvement in memory in the Fmr1 KO mice treated with 150 mg/kg of metadoxine was found in behavioral tests, such as the T-maze, showing significant improvement in cognitive outcomes. These findings were supplemented by an improved social interaction of KO mice treated with 150 mg/kg of metadoxine. Importantly, improved cognitive executive function, working memory and social interaction following treatment with metadoxine in a valid mouse model of Fragile X correlates with normalization of biochemical markers reflective of neuronal signaling pathways and oxidative stress.
Fragile X syndrome is the most widespread single-gene cause of autism and inherited cause of mental retardation among boys. Anyone with the FMR1 gene mutation can pass it to their children. Approximately 1 in 4,000 males and 1 in 8,000 females have Fragile X syndrome, according to Centers for Disease Control and Prevention (CDC). Not everyone with the mutation will show signs or symptoms of Fragile X, and disabilities will range from mild to severe as well as physical characteristics such as an elongated face, large or protruding ears, large testes (macroorchidism), and behavioral characteristics such as stereotypic movements (e.g. hand-flapping), and social anxiety. Fragile X results from a change or mutation in the Fragile X Mental Retardation 1 (FMR1) gene, which is found on the X chromosome. The gene normally makes a protein called Fragile X Mental Retardation Protein, or FMRP. This protein is important for creating and maintaining connections between cells in the brain and nervous system. The mutation causes the body to make only a little bit or none of the protein, which often causes the symptoms of Fragile X.
Fragile X Syndrome (FXS) often occurs with other conditions such autism spectrum disorders. Autism spectrum disorders (ASDs) are a group of developmental disabilities that can cause significant social, communication and behavior challenges. People with ASDs handle information in their brain differently than other people.
ASDs are “spectrum disorders.” That means ASDs affect each person in different ways, and can range from very mild to severe. People with ASDs share some similar symptoms, such as problems with social interaction. But there are differences in when the symptoms start, how severe they are, and the exact nature of the symptoms. ASDs include Autistic Disorder (also called “classic” autism), Asperger Syndrome and Pervasive Developmental Disorder.
Currently, the Food and Drug Administration (FDA) has not approved any drugs specifically for the treatment of Fragile X or its symptoms. There are medications used off label to treat certain symptoms of Fragile X syndrome, however, results vary greatly by patient and some of these medications carry serious risks, may make symptoms worse at first, or take several weeks to become effective. The present invention provides an unmet need for drugs to treat Fragile X or its symptoms.
Accordingly, the invention provides methods of treating, preventing or alleviating a sign or symptom of Fragile X Syndrome and/or autism spectrum disorders by administering to a subject a composition comprising metadoxine.
In general, the signs and symptoms of Fragile X fall into five categories: intelligence and learning; physical, social and emotional, speech and language and sensory disorders commonly associated or sharing features with Fragile X. include for example. Individuals with Fragile X have impaired intellectual functioning, social anxiety, language difficulties and sensitivity to certain sensations. Treatment with metadoxine improves learning and increases sociability 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 delays, social and communication challenges, and unusual behaviors and interests. Many people with autistic disorder also have intellectual disability. Individuals with Asperger syndrome usually have some milder symptoms of autistic disorder. For example, they may have social challenges and unusual behaviors and interests. Individuals with Pervasive Developmental Disorder (PDD-NOS) People meet some of the criteria for autistic disorder or Asperger syndrome, but not all, may be diagnosed with PDD-NOS. People with PDD-NOS usually have fewer and milder symptoms than those with autistic disorder. The symptoms might cause only social and communication challenges. Treatment with metadoxine improves symptoms of autism.
Metadoxine is an ion-pair between pyrrolidone carboxylate (PCA) and pyridoxine (vitamin B6) with the two compounds linked in a single product by salification. The pairing with PCA synergistically increases the pharmacological activity of pyridoxine (see, e.g., U.S. Pat. No. 4,313,952). Metadoxine is freely soluble in water and in gastric fluid. Oral absorption of the drug is fast with high bioavailability (60-80%). The half-life of metadoxine in human serum is short (40-60 minutes) without appreciable differences 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. Tablets contain 500 mg of metadoxine, microcrystalline cellulose and magnesium stearate. Ampoules contain 300 mg of metadoxine, sodium metabisulfite, EDTA sodium, methyl-p-hydroxybenzoate and water.
In certain embodiments, metadoxine compositions of the invention, e.g., formulated in whole or in part for sustained or controlled release, enable more efficient use of metadoxine in the treatment, prevention and/or alleviation of a sign or symptom of Fragile X syndrome and conditions/disorders related thereto, such as autism spectrum disorders.
In certain of the above described methods of the invention, the metadoxine or acceptable derivative thereof may be formulated for immediate release upon administration to the subject. In certain of the above described methods of the invention, the metadoxine or acceptable derivative thereof may be formulated for sustained and/or controlled release, and may optionally be formulated to have both immediate release and sustained and/or controlled release characteristics upon administration to the subject. 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 described in more detail below.
In certain embodiments, the present invention provides a composition comprising metadoxine or a derivative thereof formulated for sustained and/or controlled release when administered to a subject for improving, treating, preventing and/or alleviating of a sign or symptom of Fragile X syndrome and/or conditions/disorders related thereto, such as autism spectrum disorders.
In certain embodiments, the present invention provides a composition comprising metadoxine or a derivative thereof wherein a portion of the metadoxine or derivative is formulated for sustained and/or controlled release and a portion of the metadoxine or derivative is formulated for immediate release when administered to a subject for improving, treating, preventing and/or alleviating of a sign or symptom of Fragile X syndrome and/or conditions/disorders related thereto, such as autism spectrum disorders.
In certain embodiments, effective serum levels of the active ingredient are achieved within from about 10 to about 20 or 30 or 40 or 50 or 60, 90 minutes, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h following metadoxine or metadoxine derivative administration. In certain embodiments, effective serum levels of the active ingredient in said subject are achieved within from about 5 to about 20 or 30 or 40 or 50 or 60, 90 minutes, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h following metadoxine or metadoxine derivative administration. In certain embodiments, effective serum levels of the active ingredient are achieved within from about 20 to about 20 or 30 or 40 or 50 or 60, 90 minutes, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h following metadoxine or metadoxine derivative administration. In certain embodiments, effective serum levels of the active ingredient are achieved within about 5, 10, 15, 20, 30, 40, 50 or 60, 90 minutes, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h.
The present inventors have developed innovative approaches for the administration of metadoxine or metadoxine derivative based on enteral (via the digestive tract) and/or parenteral (other routes than digestive tract) routes (W02009/004629, the contents of which are incorporated by reference in its entirety). These approaches provide for a rational design of delivery systems with desired properties based on the meticulous selection of the carrier, e.g. appropriate surfactants/co-surfactants composition or micro/nano particles (such as liposomes or nano-liposomes) entrapping the active ingredients, or other additives or excipients, for the delivery system of interest. The enteral delivery systems may be designed for oral administration (tablets, sachets, lozenges, capsules, gelcaps, drops, or other palatable form) or rectal administration (suppository or (mini) enema form). In addition, the delivery system of interest may be in liquid form, for example a drop solution, syrup. Furthermore, the delivery system of interest may be in form of a beverage or food article. Thus, the active ingredient/s used by the invention may be comprised in a beverage, particularly soft drinks like juices, nectars, water, sparkling water and other sparkling drinks, shakes, milk shakes and other milk-based drinks, and the like. Liquid preparations may also be in the form of concentrated syrups, for diluting with water or sparkling water. Alternatively, the active ingredient/s may be comprised in food articles, such as snack bars, health bars, biscuits, cookies, sweets, confectionery products, ice creams, ice lollies, and the like.
Still further, the delivery system may be a food or beverage article comprising a physiologically active pyridoxine derivative, particularly pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine). In certain embodiments, consumption of the food or beverage article of the invention may lead to achievement of serum levels of the active ingredient within from about 10 to about 40-60 minutes following consumption thereof Examples may be sweets, chocolate, candies and candy bars, energy bars, ice creams, pastry products and the like.
The parenteral ways of administration include subcutaneous, transferal (diffusion through the intact skin), transmucosal (diffusion through a mucous membrane), sublingual, buccal (absorbed through cheek near gumline) administration, or administration by inhalation. In certain embodiments, the compositions used by the invention are not administered by invasive modes of treatment (i.e., are non-invasive). In certain embodiments, the metadoxine or metadoxine derivative compositions are not administered by intravenous injection.
In certain embodiments, compositions used by the invention are delivered as a microcrystalline powder or a solution suitable for nebulization; for intravaginal or intrarectal administration, pessaries, suppositories, creams or foams. 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 (absorbed through cheek near gumline) administration, administration by inhalation or ocular administration, e.g., in eye drops.
Administration of metadoxine or metadoxine derivative for medical uses requires safe and efficient delivery systems. The present invention provides delivery systems for safe delivery of a variety of substances due to their special physico-chemical features, particularly direct absorption, by non-invasive means, and consequent avoidance of side effects. The delivery systems significantly enhance efficiency and quality of metadoxine or metadoxine derivative absorption based on its unique physicochemical features, which enables lower concentrations or amounts of active substance to be delivered to a subject in a biologically active form. The delivery systems of the invention provide for the direct access of the active substance to the tissues and thus provide immediate or near-immediate effects of metadoxine or metadoxine derivative to the treated subject. Accordingly, in certain embodiments, the present invention uses a non-invasive pharmaceutical delivery system for the improved administration of a physiologically active pyridoxine, particularly pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine), or a physiologically acceptable derivative thereof, comprising as the active ingredient said physiologically active pyridoxine in a suitable carrier. In certain embodiments, serum levels of the active ingredient are achieved within from about 10 to about 40-60 minutes following administration. In another embodiment, the invention employs a non-invasive pharmaceutical delivery system for the improved administration of a physiologically active pyridoxine derivative, particularly pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine), for use in improvement of cognitive behavior in a subject in need thereof, comprising as the active ingredient said pyridoxine derivative, in a suitable carrier. In certain embodiments, serum levels of said active ingredient are achieved within from about 10 to about 40-60 minutes following administration.
In certain embodiments, the drug delivery systems employed by the invention may be designed for oral, nasal, ocular, rectal, subcutaneous, transferal, transmucosal, sublingual, buccal or inhalation administration. The drug delivery systems may provide the active substance in a controlled release mode. In certain embodiments, the drug delivery systems of the invention may further comprise at least one additional pharmaceutically active agent. The delivery systems used by the invention may generally comprise a buffering agent, an agent that adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art. Supplementary pharmaceutically acceptable active ingredients can also be incorporated into the compositions. The carrier can be 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 solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the 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, lozenges, drops, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Also included are oral rapid-release, time controlled-release, and delayed-release pharmaceutical dosage forms. The active drug components can be administered in a single dosage form or in separate dosage forms to be administered together or independently. The active drug components can be administered in a mixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier”), materials suitably selected with respect to the intended form of administration. Where the delivery system is for oral administration and is in the form of a tablet or capsule or the like, the active drug components can be combined with a non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, modified sugars, modified starches, methylcellulose and its derivatives, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, and other reducing and non-reducing sugars, magnesium stearate, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate and the like. For oral administration in liquid form, the active drug components can be combined with non-toxic pharmaceutically acceptable inert carriers such as ethanol, glycerol, water and the like. When desired or required, suitable binders, lubricants, disintegrating agents and coloring and flavoring agents can also be incorporated into the mixture. Stabilizing agents such as antioxidants, propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7-hydroxycoumarin can also be added to stabilize the dosage forms. Other suitable compounds can include gelatin, sweeteners, natural and synthetic gums such as acacia, tragacanth, or alginates, carboxymethylcellulose, polyethylene, glycol, waxes and the like.
Additional suitable pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. In some embodiments, the pharmaceutically acceptable carrier is magnesium stearate. Additional pharmaceutical excipients commonly accepted and used are found in, for example, Remington's Pharmaceutical Sciences (Gennaro, A., ed., Mack Pub., 1990).
For purposes of parenteral administration, solutions in suitable oil such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. The half-life of metadoxine in human serum is very short. Lu Yuan et al. (Chin. Med. J 2007 120(2) 160-168), showed a mean half life of about 0.8 hour. A way of prolonging serum levels of active moiety is by administering the material in a sustained-release formulation. Because metadoxine is freely soluble in water and in various biological fluids, it is difficult to sustain its release and prolong its absorption time. Therefore, it was unexpected that sustained release could be achieved. A control release dosage form of metadoxine or metadoxine derivative may be based on a predetermined gradual release of the active ingredient in the biological fluids, resulting in a sustained action with small fluctuations of the plasma level over a prolonged period of time.
In certain embodiments, the delivery system used by this invention may be administered in controlled release formulations. In certain embodiments, the method of administration will be determined by the attending physician or other person skilled in the art after an evaluation of the subject's condition and requirements. An embodiment of the method of the present invention is to administer the therapeutic compound described herein in a sustained release form. Any controlled or sustained release method known to those of ordinary skill in the art may be used with the compositions and methods of the invention such as those described in Langer, Science 249(4976):1527-33 (1990). Such method comprises administering a sustained-release composition, a suppository, or a coated implantable medical device so that a therapeutically effective dose of the composition of the invention is continuously delivered to a subject of such a method. Sustained release may also be achieved using a patch designed and formulated for the purpose. The composition of the invention may be delivered via a capsule which allows sustained-release of the agent over a period of time. Controlled or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Sustained release formulae or devices, or any topical formulations, may additionally contain compositions to stabilize the composition or permeate physiological barrier such as skin or mucous membrane. Exemplary additional components may include any physiologically acceptable detergent or solvent such as, for example, dimethylsulfoxide (DMSO).
In all embodiments of the invention, methods and uses of the invention may employ a composition comprising a salt adduct as defined by the invention formulated as a single dose. Said single dose formulation may be an immediate release formulation, a burst formulation, a prolonged release formulation, a sustained release formulation or any other controlled release formulation known to a person skilled in the art.
In other embodiments of the methods and uses of the invention, a composition comprising a salt adduct defined by the invention may be a combined dosage formualtion, wherein different types of formulations are administered to a subject, i.e. any combination of an immediate release formulation, a burst formulation, a prolonged release formulation, a sustained release formulation or any other controlled release formulation known to a person skilled in the art, given either in a single dose or in separate doses given separately, concomitantly or sequentially wherein the gap of time between administration of separate dosages is defined based on the condition and severity of disease or disorder of a subject or the physical condition of said subject.
In some embodiments a composition used by the methods of the invention are formulated as combined dosage forms, wherein at least one dosage from of a suit adduct defined by the invention is in an immediate release form and at least one dosage form of a salt adduct defined by the invention (being the same or different from the salt adduct formulated in the immediate release formulation) is formulated as a controlled (slow and/or sustained) release formulation. In other embodiments the weight ratio of a salt adduct as defined by the invention comprised in said at least one immediate release formulation and at least one controlled release formulation may be 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 employing such combined dosage forms in a method or use of the invention, said at least one immediate release form and at least one controlled release form of a salt adduct defined above, may be administered to a subject separately, concomitantly, sequentially, concurrently, consecutively and so forth. In some embodiments said at least one immediate release form is administered initially. In other embodiments said at least one controlled release formulation is administered initially.
In certain embodiments, the metadoxine or metadoxine derivative in compositions of the invention may be formulated for sustained or controlled release over a period of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be formulated for sustained or controlled release over a period of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be formulated for sustained or controlled release over a period of between about 0.5 or 1 or 2 or 3 or 4 hours and about 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be formulated for sustained or controlled release over a period of between about 5 or 6 or 7 or 8 hours and about 9, 10, 11 or 12 hours.
In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be in immediate, fast of burst release form.
In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be formulated to 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% of the total metadoxine or metadoxine derivative in about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours. In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be formulated to release not less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% of the total metadoxine or metadoxine derivative in about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours.
In certain embodiments, the metadoxine or metadoxine derivative in compositions used by the invention may be in a combination of sustained or slow release and immediate or fast release forms. In certain embodiments, the relative proportion of sustained or slow release metadoxine or metadoxine derivative to immediate or fast release metadoxine or metadoxine derivative is, e.g., 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, a polymeric material is used to sustain or control the release of metadoxine or metadoxine derivative. In certain embodiments, the type of polymeric material and the amount of which is used, has a strong influence on the rate of release of metadoxine or metadoxine derivative from the product 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, fats such as glycerol palmito-stereate, beeswax, glycowax, castorwax, carnaubawax, glycerol monostereate or stearyl alcohol, hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acid derivatives, as well as mixtures of these polymers. Hydrophilic polymers include, but are not limited to, hydrophilic cellulose derivatives such as methyl cellulose, hydroxypropylmethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethylcellulose and hydroxyethyl methyl-cellulose polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polyacrylamide, ethylene vinyl acetate copolymer, polyacrylate, poly-urethane, polyvinylpyrrolidone, polymethylmethacrylate, polyvinyl acetate, polyhydroxyethyl methacrylate, as well as mixtures of these polymers. Furthermore, any mixture of one or more hydrophobic polymer and one or more hydrophilic polymer could optionally be used.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is microcrystalline cellulose such as “AVICEL PH 101” manufactured by FMC BioPolymer's.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is hydroxypropyl methyl-cellulose such as “METHOLOSE”, produced by Shin-Etsu Chemical Co.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is ethyl cellulose such as “ETHOCEL™”, manufactured by The Dow Chemical Company.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is an acrylic polymer such as “EUDRAGIT RS™”, produced by Rohm GmbH.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is a colloidal silicone dioxide such as “AEROSIL™”, manufactured by Degussa.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is a poly(vinyl acetate) such as “KOLLICOAT SR”, manufactured by BASF.
In certain embodiments, a polymeric material to be used in compositions of or used by the invention is an ethyl acetate and vinyl acetate solution such as “DURO-TAK”, manufactured by Delasco Dermatologic Lab & Supply, Inc.
In certain embodiments, the compositions of or used by the invention comprise or consist essentially of about 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg to about 1000, 1500, 2000, 2500 or 3000 mg metadoxine or metadoxine derivative. In certain embodiments, the compositions of or used by the invention comprise or consist essentially of about 5, 100, 500, or 1000 mg to about 2000, 4000, 10,000, 15,000, or 20,000 mg AVICEL PH 101™. In certain embodiments, the compositions of or used by the invention comprise or consist essentially of 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 a polymeric material. In certain embodiments, the polymeric material is METHOLOSE, ETHOCEL E10™ or EUDRAGIT RS™. In certain embodiments, METHOLOSE comprises or consists essentially of between 1 and 90% of the formulation, preferably between 5 and 70%. In certain embodiments, ETHOCEL™ comprises or consists essentially of between 1 and 30% of the formulation, preferably between 2 and 20%. In certain embodiments, EUDRAGIT™ comprises or consists essentially of between 1 and 90% of the formulation, preferably between 5 and 70%.
In certain embodiments, delivery systems of or used by the invention comprise delivery devices. In certain embodiments, the compositions of or used by the invention are delivered by an osmotic process at a controlled rate such as by an osmotic pump. The system may be constructed by coating an osmotically active agent with a rate controlling semipermeable membrane. This membrane may contain an orifice of critical size through which agent is delivered. The dosage form after coming into contact with aqueous fluids, imbibes water at a rate determined by the fluid permeability of the membrane and osmotic pressure of the core formulation. This osmotic imbibition of water results in formation of a saturated solution of active material within the core, which is dispensed at controlled rate from the delivery orifice in the membrane.
In certain embodiments, the compositions of or used by the invention are delivered using biodegradable microparticles. In certain embodiments, the system to prepare microparticles consists of an organic phase comprised of a volatile solvent with dissolved polymer and the material to be encapsulated, emulsified in an aqueous phase. In certain embodiments, the biodegradable polymers that can be used for the microparticle matrix, comprises polylactic acid (PLA) or the copolymer of lactic and glycolic acid (PLAGA). The PLAGA polymer degrades hydrolytically over time to its monomeric components, which are readily removed from the body through natural metabolism
The preparation of or used by the present invention may also contain an absorption enhancer and other optional components. Examples of absorption enhancers include, but are not limited to, cyclodextrins, phospholipids, chitosan, DMSO, Tween, Brij, glycocholate, saponin, fusidate and energy based enhancing absorption equipment.
Optional components present in the dosage forms include, but are not limited to, diluents, binders, lubricants, surfactants, coloring agents, flavors, buffering agents, preservatives, stabilizing agents and the like.
Diluents, also termed “fillers” include, for example, dicalcium phosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, hydrolyzed starches, silicon dioxide, colloidal silica, titanium oxide, alumina, talc, microcrystalline cellulose, and powdered sugar. For administration in liquid form, the diluents include, for example, ethanol, sorbitol, glycerol, water and the like.
Binders are used to impart cohesive qualities to the formulation. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinzed starch), gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia, tragacanth, sodium alginate, celluloses, and Veegum, and synthetic polymers such as polymethacrylates 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.
Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents, with anionic surfactants preferred. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions, associated with cations such as sodium, potassium and ammonium ions. Particularly preferred surfactants include, but are not limited to long alkyl chain sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylhexyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
Stabilizing agents such as antioxidants, include, but are not limited to, propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7-hydroxycoumarin.
If desired, the compositions of or used by the invention may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, preservatives, and the like.
Any of the compositions of or used by the invention may be used alone or in combination with one or more additional therapeutic agents, for the improvement of cognitive behavior. Examples of additional therapeutic agents are: amphetamines, methylphenidate HCl, dexmethylphenidate hydrochloride, atomoxetine, reboxetine, fluoxatine, sertraline, paroxetine, fluoroxamine, citalopram, venlafaxine, bupropion, nefazodone and mirtazapine.
The amount of both the compound and the additional therapeutic agent that may 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 this invention should be formulated so that a dosage of 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 condition and the illness of the patient, and the desired daily dose. In human therapy, the oral daily dose is 10-3000 mg or preferably 100-3000 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 forms, which may be administered in a single daily dose or divided into 2-3 smaller doses for each day in certain cases.
In certain embodiments, the compositions of the present invention may act synergistically in combination with each other and may further act synergistically in the presence of an additional therapeutic agent. Therefore, the amount of compound(s) and additional therapeutic agent(s) in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between 0.1-1 g/kg bodyweight/day of the additional therapeutic agent can be administered.
For convenience, certain terms employed in the specification, examples, and appended embodiments, are collected here. 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.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object 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 by not limited to”
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless 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 refers to administration to a subject of one or more of the compositions of the invention. If it is administered prior to clinical manifestation of the unwanted condition (e.g., clinical or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it contributes to prevention of, i.e., protection of the subject against developing an unwanted condition, whereas if administered after manifestation of an unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or prevent progression of the unwanted condition or side effects there from).
The term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance or substances. The term thus means any substance intended for use in diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. The term “therapeutically effective amount” means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. In certain embodiments, a therapeutically-effective amount of a compound or composition will depend on its therapeutic index, solubility, and the like. For example, certain metadoxine or metadoxine derivatives formulations of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to a selected treatment, as may be determined by the skilled artisan.
The term “effective amount” refers to the amount of a therapeutic reagent that when administered to a subject in an appropriate dose and regimen produces at least one desired result.
A “subject” or “patient” to be treated by a method of the invention may mean 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 Fragile X Syndrome or Autism Spectrum Disorder. In alternative embodiments, the terms “subject” and “healthy subject” and “subject in need” and “patient in need” as used herein exclude subjects under alcohol influence following alcohol consumption of any form, alcoholics (alcohol addicts), and abstinent alcoholics.
As used herein the term “salt adduct” is meant to encompass a salt product of a direct addition of two or more distinct ions, wherein the overall charge of the salt adduct is zero. In certain embodiments, the salt adduct comprises one positively charged moiety having a single positive charge functional group (i.e., the positively charged moiety is charged with +1 net charge) and one negatively charged moiety having a single negative charge functional group (i.e., the negatively charged moiety is charged with −1 net charge). In certain embodiments, the salt adduct comprises one positively charged moiety having two positively charged functional groups, which may be the same or different (i.e., the positively charged moiety is charged with +2 net charge) and two negatively charged moieties, which may be the same or different, and each having a single negative charged functional group (i.e., each negatively charged moiety is charged with −1 net charge). In certain embodiments, the salt adduct comprises two positively charged moieties, which may be the same or different, having each one positively charged functional group (i.e., each positively charged moiety is charged with +1 net charge) and one negatively charged moiety, having two negatively charged functional groups, being the same or different (i.e., the negatively charged moiety is charged with −2 net charge). In certain embodiments, the salt adduct comprises a positively charged moiety charged with +n net charge (originating from one or more positively charged functional groups, which may be the same or different), and a negatively charge moiety having −n (originating from one or more negatively charged functional groups, which may be the same or different) net charge, wherein n is an integer which may be equal to 1, 2, 3, 4, 5 or 6.
As used herein, a “positively charged moiety of a salt adduct” of the invention is the corresponding acid of pyridoxine, or any derivative thereof In certain embodiments, the positive charge of the positively charged moiety stems from the protonated basic nitrogen atom of pyridoxine (as for example in compound (2)) or any derivative thereof (such as for example compounds of formula (I)). In certain embodiments, the positively charged pyridoxine derivative is substituted with a positively charged functional group such as for example —NH3+, —CH2NH3+, NH2R+, —NHR2+ (wherein each R is independently a C1-C6 alkyl), which may, in some embodiments, be present in addition to the positively charged protonated basic aromatic nitrogen atom in the pyridine ring.
It should be understood that moieties of a salt adduct of the invention may contain each at least one chiral center, and thus may exist in, and be isolated as, any stereoisomer thereof including, enantiomers, diastereomers or any mixtures thereof including, but not limited to racemic mixtures. The present invention includes any possible stereoisomer (e.g. enantiomers, diastereomers), any mixtures thereof including, but not limited to, racemic mixtures, of any of the individual moieties of a salt adduct of the invention. Where the herein-described processes for the preparation of each of the moieties of a salt adduct of the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques, such as preparative chromatography. The moieties of a salt adduct of the invention may be each prepared in any mixture of possible stereoisomers thereof, including but not limited to racemic mixtures thereof, or individual stereoisomers (e.g. enantiomers, diastereomers) may be prepared either by enantiospecific synthesis or by chiral chromatographic separation of a racemate. Whenever referring to amino acids, the invention should be understood to encompass natural and non-natural amino acids or any derivative thereof
Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
The term “bio-available” means that at least some amount of a particular compound is present in the systemic circulation. Formal calculations of oral bioavailability are described in terms of an F value (“Fundamentals of Clinical Pharmacokinetics,” John G. Wegner, Drug Intelligence Publications; Hamilton, Ill. 1975). F values are derived from the ratio of the concentration of the parent drug in the systemic circulation (e.g., plasma) following intravenous administration to the concentration of the parent drug in the systemic circulation after administration by a non-intravenous route (e.g., oral). Therefore, oral bioavailability within the scope of the present invention contemplates the ratio or F value of the amount of parent drug detectable in the plasma after oral administration compared to intravenous administration.
The term “treating” or “treatment” refers to mitigating, improving, relieving or alleviating at least on symptom of a condition, disease or disorder in a mammal, such as a human, or the improvement of an ascertainable measurement associated with a condition, disease or disorder. Treatment as used herein also encompasses treatment of healthy individuals.
The term “acceptable derivative” with respect to metadoxine or metadoxine derivatives refers to any salt, conjugate, ester, complex or other chemical derivative of metadoxine or any of the moieties comprising the same, which, upon administration to a subject, is capable of providing (directly or indirectly) metadoxine or a metabolite or functional residue thereof, or measurable metadoxine activity. The term “physiologically compatible metadoxine derivative” may be used interchangeably herein with the term “acceptable derivative” and refers to a functional, active, pharmaceutically acceptable derivative of metadoxine.
The term “excipient” refers to an inactive substance used as a carrier for the active ingredient in a formulation.
The term “controlled release” refers to any formulation which delivers an agent at a controlled rate for an extended time and is designed to achieve a desired agent level profile.
The term “sustained release” is used in its conventional sense to refer to a formulation that provides for gradual release of an active material over an extended period of time, which in certain embodiments may also further result in substantially constant blood levels over an extended time period, i.e., controlled release.
The term “immediate release” is used in its conventional sense to refer to a formulation that provides for non delayed or controlled release of an active material upon administration.
The term “half-life” of a substance is the time it takes for a substance to lose half of its pharmacologic, physiologic, or other activity. Biological half-life is an important pharmacokinetic parameter and is usually denoted by the abbreviation tin.
The term “non-invasive” refers to modes of treatment which do not puncture the skin.
The term “non-chronic administration” may be used interchangeably herein with the term “acute administration” and refers to giving a measured or non-measured quantity or portion of a medication to a subject on a non-regular basis. Non-chronic administration may be a single dose treatment or a multiple dose treatment, and may optionally be given over time. Typically but not always, a non-chronic administration is given to treat or prevent a non-chronic condition. Certain chronic conditions may also benefit from non-chronic administration of a metadoxine or metadoxine derivatives composition described herein.
The term “chronic administration” refers to giving a measured quantity of a medication on a regular basis to a subject. In some embodiments, chronic administration is to treat or prevent one or more chronic conditions, problems or diseases. Chronic diseases have one or more of the following characteristics: they are permanent, leave residual disability, are caused by nonreversible pathological alteration, require special training of the patient for rehabilitation, or may be expected to require a long period of supervision, observation, or care.
The term “single dose treatment” refers to giving a measured quantity of a medication to be taken at one time. It is given to treat non-chronic conditions on an irregular basis, depending on personal need.
The term “tmax” refers to the time to peak concentration. Calculation of time at which maximum concentration occurs after a single dose administration is performed according to the formula:
Where λα and λz are the apparent absorption and elimination rate constants, respectively.
Mice were injected once daily for one week prior and during behavioural testing. All mice were intraperitonealy injected with 150 mg/kg/day of Metadoxine (referred as M in the figures) and vehicle (referred as V in the figures).
The experiments presented herein demonstrate that Metadoxine significantly improved memory and learning during the contextual fear paradigm. A significant improvement in memory was also detected in the fmr1 KO mice treated with Metadoxine in several different tests such as Y-maze and T-maze showing significant improvement in cognitive functions. Metadoxine treatment also showed positive effect in some of the social interaction paradigms.
1A. Contextual Fear Conditioning: Test of Memory and Learning
Contextual fear conditioning is the most basic of the conditioning procedures. It involves taking an animal and placing it in a novel environment, providing an aversive stimulus, and then removing it. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the aversive stimulus. Freezing is a species-specific response to fear, which has been defined as “absence of movement except for respiration”. This may last for seconds to minutes depending on the strength of the aversive stimulus, the number of presentations, and the degree of learning achieved by the subject.
Contextual fear conditioning test is used to examine both hippocampus-dependent memory and amygdala-dependent emotional memory and learning.
As shown in
1B. Contextual Fear Conditioning: Test of Memory and Learning
Fmr1 KO mice treated with Metadoxine (KO-M) at concentrations ranging from 100 to 200 mg/kg exhibited a significant dose-dependent improvement in learning and memory when compared to the vehicle-treated Fmr1 KO group (KO-V). However, a significant difference (p<0.01) was found only between the WT Metadoxine-treated group (WT-M) and the Fmr1 KO mice treated with 100 mg/kg of Metadoxine, suggesting that despite a significant improvement in the Fmr1 KO group receiving 100 mg/kg Metadoxine, only 150 and 200 mg/kg dose levels of Metadoxine fully rescued the Fmr1 KO mice learning deficit. (
2. Social Interaction
A. Social Approach
Mice are a social species, which engage in easily scored social behaviors including approaching, following, sniffing, all grooming, aggressive encounters, sexual interactions, and parental behaviors, nesting and sleeping in a group huddle. Social recognition and social memory in mice are evaluated by amount of time spent sniffing a novel mouse upon repeated exposures, to induce familiarity, and reinstatement of high levels of sniffing when a novel stimulus animal is introduced.
As shown in
B. Sociability Partition Test
This test helps assessing behavioral responses to another individual placed in the neighboring sector of the cage, divided in half by a transparent partition with holes. The animal behavior response is expected to differ depending on the physiological and psychological state of an individual and its social experience. The “partition” test can be informative and productive in experiments designed to study the neurochemical and neurophysiological mechanisms of sociability, olfaction and aggressive behavior.
In the first 20-30 s, the tested mice moved about but avoided the partner in the cage. Then, avoiding behaviors disappeared and social interactions became frequent, including aggression
C. Sociability and Social Novelty Preference Test
Briefly, mice are placed in a rectangular apparatus (36×20×20 cm) divided into 3 chambers (left and right chambers 13.5×20×20 cm; center chamber 9×20×20 cm) by transparent partitions with small circular openings allowing easy access to all compartments. The test is composed of 3 sequential 10 minute trials; trial 1: habituation (the mouse is allow to explore the 3 chambers), trial 2: sociability (an unfamiliar mouse is placed into a mesh wire cage in either the left or right chambers and exploration by the test mouse of the 3 chambers and recorded for a further 10 minutes), trial 3: social novelty preference (a novel mouse is placed into a mesh wire cage in the chamber opposite the (now familiar) mouse from the previous stage. Exploration of the 3 chambers by the test mouse is again recorded for 10 minutes. Stimulus mice used in this test were of the C57 strain. Each chamber was cleaned and lined with fresh bedding between trials.
3. Y Maze Alternation. Test of Learning and Preservation
The Y maze is a spatial reference memory task in which the animal has to learn which of two arms is baited with a food reward. In the Y-maze paradigm used in this study, mice had to learn which of the two arms forming the Y was baited with food. The day prior to the start of the training, mice were allowed to freely explore the maze for 5 min. Next, they received two trials, one in which the food was located in the left arm and one in which the food was positioned in the right arm. This procedure prevented the development of a preference for one of the arms.
As shown in
As shown in
4. Y Paddling Maze. Test of Learning and Memory
A left-right discrimination paradigm in the Y maze, instead of the more often applied Morris water maze, was chosen for this experiment. The water maze paradigm involves handling and swim stress and causes a considerable elevation of plasma corticosterone levels. Because in this case the primarily interest was the cognitive activity, experiment was designed in such a way that stress levels are minimized. Although the Y-maze learning can also be stressful to the mice because of novelty, we reduced stress associated with this task by habituating the mice to the maze and by letting them voluntarily enter the apparatus. The Y paddling maze task combines elements of two hippocampal-dependent tasks: the paddling pool spatial cognition test and the appetitive Y-maze.
Briefly, a clear perspex Y-maze is filled with 2 cm of water at 20° C., sufficient to motivate mice to leave the maze by paddling to an exit tube at the distal end of one arm. The mouse exits to a burrowing tube in which it is returned to its home cage. The maze is placed in the middle of a room surrounded by prominent visual cues.
As demonstrated in
5. Rewarded T Maze Alternation. Test of Working Memory
The T-maze is an elevated or enclosed apparatus in the form of a T placed horizontally. Animals are started from the base of the T and allowed to choose one of the goal arms abutting the other end of the stem. If two trials are given in quick succession, on the second trial the rodent tends to choose the arm not visited before, reflecting memory of the first choice. This is called ‘spontaneous alternation’. This tendency can be reinforced by making the animal hungry and rewarding it with a preferred food if it alternates. The spontaneous rewarded alternation its very sensitive to dysfunction of the hippocampus, but other brain structures are also involved.
After a 4-d habituation period on the T-maze, mice were trained to alternate arm choices to receive sweet condensed milk as reward. T maze learning task is used to detect problems in spatial working memory skills.
As shown in
6. Successive Alleys. Test of Hyperactivity
The Fmr1KO-Vehicle mice displayed a significantly shorter latency to enter the first open alley (p<0.001) and spent significantly more time in the open alleys (p<0.001) than wild types, indicating a significantly higher levels of hyperactivity than their WT-V controls. They also made more crossings between alleys (Graphs bellow represent an Alley entry, moving from 1 or safer to 4 or not safe). Fmr1 KO mice treated with Metadoxine showed a significant reduction in open arm entries (p<0.001) as well as time spend in the center (p<0.005), indicating a reduction in hyperactivity. See,
Mice were sacrificed on the day of the last behavior tests, and the tested mice brains were removed and stored at −80 C for further studies.
Statistics
Multivariate analysis of variance was employed to assess group differences across data. Repeated measures ANOVA were performed to analyze data. Statistically significant effects in each ANOVA were followed with post hoc comparisons, using the Newman-Keuls test. A p-value of less than 0.05 was considered significant.
After treatment with Metadoxine (150 mg/kg), the brain tissues of WT-V, WT-M, KO-M and KO-M mice were harvested and analyzed for biomarkers reflecting neuronal signaling pathways known to be involved in the pathophysiology of Fragile X syndrome. Specifically, FXS neuronal phenotype is thought to be mediated via induction of RAS-MEK-ERK and PI3K-Akt-mToR pathways. Reduced cAMP induction and PKA signaling have been also linked to FXS.
The activation of ERK1/2 and AKT, as indexed by phosphorylated ERK1/2 (pERK1/2) and phosphorylated AKT (pAKT), respectively, were increased in KO-V mice as compared to WT-V, while increased pERK and pAKT levels were normalized in KO-M (150 mg/kg) mice, as indicated by a significant reduction of pERK and pAKT levels as compared to KO-V (p<0.01 and p<0.05, respectively). However, we did not see any improvement in cAMP levels nor PKA activity in KO-M mice as compared to KO-V. (
There is a growing body of evidence that oxidative stress and subsequently oxidant-mediated neuronal damage play a role in the pathophysiology of FXS. We therefore examined the effect of Metadoxine on a key component in oxidative stress, the anti-oxidant Glutathione S-Transferase (GST) protein levels in WT-V, WT-M, KO-V and KO-M animals.
The data shows a decrease in GST protein levels in KO-V mice as compared to WT-V group, while KO-M mice exhibited significant increased GST levels (p<0.01) when compared to KO-V mice, indicating that treatment of KO mice with Metadoxine (150 mg/kg) reduces the oxidative damage induced in KO-V mice. (
This application is a continuation of U.S. Ser. No. 14/038,258, filed on Sep. 26, 2013, which claims priority to and benefit of provisional applications U.S. Ser. No. 61/875,384, filed on Sep. 9, 2013. The specification of each above-listed application is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61875384 | Sep 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14038258 | Sep 2013 | US |
| Child | 14819013 | US |
