CN105517546A - Methods of treating fragile X syndrome and related disorders - Google Patents

Abstract

The present invention provides methods of alleviating a sign or a symptom of Fragile X Syndrome and relates disorders such as autism spectrum disorders.

Description

The method for the treatment of fragile X mental retardation and relevant disease

Related application

This application claims the provisional application USSN61/875 in JIUYUE in 2013 submission on the 9th, 384, in the USSN14/038258 of JIUYUE in 2013 submission on the 26th with in the provisional application USSN61/991 of submission on May 9th, 2014, the priority of 351 and rights and interests, its content is incorporated herein by reference in their entirety at this respectively.

Technical field

The present invention relates generally to the method for the symptom treating or alleviate fragile X mental retardation and relevant disease.

Background technology

As its name implies, fragile X mental retardation (FragileXSyndrome, FXS) is relevant to the fragile site being expressed as isochrome crack in metaphase chromosome at Map Location Xq27.3 place.Fragile X mental retardation is a kind of hereditary, and it is caused by the sudden change of 5 '-untranslated region of fragile X mental retardation 1 (fragileXmentalretardation1, the FMR1) gene be positioned on X chromosome.The sudden change of FXS is caused to repeat relevant to the CGG in fragile X mental retardation gene FMR1.In most healthy individuals, CGG repeat total quantity for being less than 10 to 40, average out to about 29.In fragile X mental retardation, CGG sequence repeats 200 times to more than 1,000 time.When object have exceed about 200 CGG repeat time, fragile X gene becomes supermethylation, and this makes this gene silencing.As a result, do not produce or produce fragile X mental retardation egg from (fragileXmentalretardationprotein, FMRP) with the level reduced, and this Object table reveals the performance of FXS.

The premutation amplification (55 to 200 CGG repeat) of FMR1 gene is comparatively frequent in ordinary group, wherein estimates that prevalence rate is women 1/259 and male 1/812.The carrier of premutation has normal IQ usually, but emotional problems such as anxiety is comparatively common.There is Progressive symmetric erythrokeratodermia intentional tremor and ataxia in old male's premutation carrier (50 years old and more than).These dyskinesia are and behavior difficulty cognitive along with Progressive symmetric erythrokeratodermia continually, comprises the loss of memory, anxiety and n-back test defect, stays alone or irritability behavior, and dementia.This disease has regarded as fragile X dependency tremor/ataxia syndrome (fragileX-associatedtremor/ataxiasyndrome, FXTAS).The T2 weighted signal strength that the nuclear magnetic resonance of the object suffering from FXTAS discloses in middle cerebellar peduncle and contiguous cerebellar white matter is strengthened.

The chain dominant disease of X that FXS reduces as penetrance and being separated.Any sex of carrying fragile X sudden change all can show the dysnoesia of the different order of severity.Suffer from the child of FXS and the be grown up dysnoesia or learning disorder and behavior and emotional problem that have in various degree, comprise self-closing sample characteristic sum tendency.The child suffering from FXS has the delay of growth critical event (such as learning how to sit, walk and speak) usually.Affected child can have that frequent angry, attention is difficult to concentrate, frequent epilepsy (such as, temporal lobe epilepsy shows effect), usually HA, be easy to be at a loss, the disorder of sensualness hyperarousal, gastrointestinal disturbance can be had, and the problem of speaking and Deviant Behavior can be had, such as, wave hands and sting hands.

FXS diagnoses by the heredity test carrying out setting up to the sample (such as, blood sample, buccal sample) from object.Described test determines whether there is sudden change or premutation in the FMR1 gene of this object based on the quantity that CGG repeats.

The object suffering from FXS also can have infantile autism.To suffer from all children of infantile autism sudden change and the X syndrome that enbrittles (FXS) that about 5% has FMR1 gene after diagnosing.Autism spectrum disorder (autismspectrumdisorder, ASD) sees about 30% male and 20% suffering from FXS to be suffered from the women of FXS, and other 30%FXS individuality shows infantile autism symptom but do not diagnose and has ASD.Although dysnoesia is the symbolic characteristic of FXS, the object suffering from FXS usually shows infantile autism feature, in range from mild case shy, lack eye contact and Social Anxiety to severe by waving hands, sting hands and perseveration in invasion and attack person.The Object table suffering from FXS reveals other symptoms relevant to infantile autism, and such as attention deficiency is with how dynamic, epilepsy, to sensory stimuli allergy, compelling sex behavior and gastrointestinal function change.FMR1 sudden change stops or significantly reduces the expression of single protein (FMRP).Not think to there is the brain development in FMRP situation and cause the cardinal symptom of FXS.

Except core symptom, the child suffering from FXS also has serious conduct disorder continually, such as irritability, attack and autolesionism etc.To suffering from the nearest research of male (8 to 24 years old) of FXS, being reported in the bimestrial observation period, having autolesionism in the object of 79% and have aggressive behavior in the object of 75%.

At present, the available treatment scheme for the people suffering from FXS comprise such as behavior modification and with multi-medicament (without FDA approval be used for the treatment of FXS) treat, described medicine comprises antidepressant and antipsychotic drug.Cognitive behavioral therapy has been used to improve language and social activity in the individuality suffering from FXS and infantile autism.In recent years, in the treatment of individuality suffering from infantile autism, generally utilized the pharmacological treatment of use atypical antipsychotic agents risperidone to strengthen non-pharmacological methods.The randomized placebo controlled trial of carrying out risperidone in autism children proves that the irritability subscale that clinical global impression improves (ClinicalGlobalImpressions-Improvement) and Deviant Behavior scale (AberrantBehaviorChecklist) significantly improves (McCracken, J.T., Deng, N.Engl.J.Med.347:314-321 (2002)).But adverse events comprises body weight increase, appetite increases, tired, drowsiness, dizzy and sialorrhea.Do not improve social isolation by using risperidone and exchange, and adverse side effect (such as extrapyramidal symptom and the dyskinesia) uses relevant with the risperidone in autism children.Lost efficacy continually due to current therapeutic scheme or less desirable side effect (particularly at antipsychotic drug) can be produced when life-time service, and therefore needing to develop new treatment.

Summary of the invention

In many aspects, the invention provides the method for the symptom for the treatment of or alleviation fragile X mental retardation or relevant disease, it is undertaken by using to the individuality having this to need the compositions comprising metadoxine (metadoxine).Described symptom is for such as learning impaired or social competence is impaired.Described object suffers from fragile X mental retardation or autism spectrum disorder.Described relevant disease is autism spectrum disorder.

In some respects, use the metadoxine of every TDD of 100mg to 3000mg, and every day, every other day or weekly use metadoxine.

Optionally, metadoxine is used with 1,2 or 3 dosage forms every day.In some embodiments, metadoxine is used with sustained release oral, and wherein metadoxine is configured to the combination of slow releasing form and releasing pattern immediately.

Such as, slow releasing form provides the sustained release of at least 8 hours of metadoxine.Slow releasing metadoxine is about 60: 40 to 80: 20 with the relative scale discharging metadoxine immediately.Preferably, slow releasing metadoxine is about 65: 35 with the relative scale discharging metadoxine immediately.

Unless otherwise defined, otherwise all technology used herein and scientific terminology have understood identical implication usual with those skilled in the art.Although can use in the embodiment of this invention and those similar or methods of being equal to described and material herein, still suitable method and material are described below.All open, patent applications mentioned in this article, patent and other lists of references are incorporated to its entirety all clearly by reference.If any conflict, will be as the criterion with this description (comprising definition).In addition, described herein material, method and embodiment are only exemplary and not intended to be limiting.

Other features and advantages of the present invention will by the following detailed description with claims and become obvious and be encompassed in wherein.

Accompanying drawing is sketched

Fig. 1 shows and knocks out in (KO) or wild type (WT) mice at 2 monthly age Fmr1 the intraperitoneal once a day (ip) carrying out 7 days supporting agents (V) or metadoxine (M) (100,150 or 200mg/kg) and use effect to scene fear conditioning.Especially, the effect that A shows supporting agent or 150mg/kg metadoxine is schemed.Figure B shows the effect of supporting agent or 100mg/kg metadoxine.Figure C shows the effect of supporting agent or 200mg/kg metadoxine.Shown data are meansigma methods ± average standard error (sem), N=10 mice/group. ^*p < 0.05, ^{* * *}p < 0.0001 and NS=is not remarkable.

Fig. 2 shows and knocks out at 2 monthly age Fmr1 the intraperitoneal once a day carrying out 7 days supporting agents (V) or 150mg/kg metadoxine (M) in (KO) or wild type (WT) mice and use the effect of social activity close to behavior.Shown data are meansigma methods ± sem, N=10 mice/group. ^*p < 0.05 He ^{* * *}p < 0.0001.

Fig. 3 shows and knocks out at 2 monthly age Fmr1 the intraperitoneal once a day carrying out 7 days supporting agents (V) or 150mg/kg metadoxine (M) in (KO) or wild type (WT) mice and use effect to Y shape labyrinth spontaneous alternately (Y-mazespontaneousalternation) (figure A), Y shape labyrinth incentive alternately (Y-mazerewardedalternation) (figure B) or Y shape labyrinth water maze spatial discrimination (Y-mazewatermazespatialdiscrimination) (figure C).Shown data are meansigma methods ± sem, N=10 mice/group. ^{* *}p < 0.001, ^{* * *}p < 0.0001 and NS=is not remarkable.

Fig. 4 shows and knocks out at 2 monthly age Fmr1 the intraperitoneal once a day carrying out 7 days supporting agents (V) or 150mg/kg metadoxine (M) in (KO) or wild type (WT) mice and use the incentive effect replaced in T-shaped labyrinth.Shown data are meansigma methods ± sem, N=10 mice/group. ^＊＊＊＊p＜0.0001。

Fig. 5 show N=10 wild type (WT) or Fmr1 knock out in the group of (KO) 2 monthly age mice carry out 7 days supporting agents (V) or 150mg/kg metadoxine (M) process once a day to the effect of the behavior in continuous passage task (successivealleytask).The continuous passage of equipment present further cause anxiety environment to study mice.Therefore, move down from passage and be evaluated as anxiety.In addition, also can quantize mass activity level in the device.

Fig. 6 shows and knocks out at 2 monthly age Fmr1 the effect that the intraperitoneal once a day carrying out 7 days supporting agents (V) or 150mg/kg metadoxine (M) in (KO) or wild type (WT) mice uses the overall brain phosphorylation level to ERK (instruction ERK is active) (figure A) and Akt (instruction Akt is active) (figure B).Shown data are meansigma methods ± sem, N=5 mice/group. ^*p < 0.01, ^{* *}p < 0.001, ^{* * *}p < 0.0001 and NS=is not remarkable.

Fig. 7 shows and to knock out in (KO) or wild type (WT) mice ip once a day at 6 monthly age Fmr1 and use supporting agent (V) or 150mg/kg metadoxine (M) effect to scene fear conditioning in 7 days.Shown data are meansigma methods ± sem, N=10 mice/group. ^{* * *}p < 0.0001 and ns=is not remarkable.

Fig. 8 shows as visited measured by the persistent period by smelling to visit bout number of times or smell, and to knock out in (KO) or wild type (WT) mice ip once a day use supporting agent (v) or 150mg/kg metadoxine (M) 7 days to the effect of social activity close to (figure A and C) and social memory (scheming B and D) behavior at 6 monthly age Fmr1.Shown data are meansigma methods ± sem, N=10 mice/group. ^*p < 0.05, ^{* * *}p < 0.0001 and ns=is not remarkable.

Fig. 9 shows and to knock out in (KO) or wild type (WT) mice ip once a day at 6 monthly age Fmr1 and use supporting agent (V) or 150mg/kg metadoxine (M) effect to the overall brain phosphorylation level of ERK (figure A) and Akt (scheming B) in 7 days.Shown data are meansigma methods ± sem, N=10 mice/group. ^*p < 0.05, ^*p < 0.01, ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 10 shows and to knock out in (KO) or wild type (WT) mice ip or oral administration (PO) supporting agent (V) or 150mg/kg and 300mg/kg metadoxine (M) effect to scene fear conditioning in 7 days once a day at 2 monthly age Fmr1.Shown data are meansigma methods ± sem, N=10 mice/group.Particularly, scheme A show Fmr1 knock out with wild-type mice in use ip or the per os process of supporting agent.Figure B shows in wild-type mice the ip and per os process that use metadoxine.Figure C shows the ip and per os process that use metadoxine in Fmr1 knock-out mice. ^*p < 0.01, ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 11 shows and to knock out in (KO) or wild type (WT) mice ip once a day or oral administration (PO) supporting agent (V) or 150mg/kg or 300mg/kg metadoxine (M) 7 days to the effect of social activity close to (figure A) and social memory (scheming B) at 2 monthly age Fmr1.Shown data are meansigma methods ± sem, N=10 mice/group. ^*p < 0.01, ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 12 show as use flow cytometry assessed, to knock out in (KO) and wild type (WT) mice ip or oral administration (PO) supporting agent (V) or 150 or 300mg/kg metadoxine (M) effect to lymphocyte biomarker in 7 days once a day at 2 monthly age Fmr1.Shown in biomarker be that Fmr1 knocks out or pAkt in wild-type mice (figure A) and pERK (scheming B).Shown data are meansigma methods ± sem, N=10 mice/group. ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 13 shows ip once a day and uses supporting agent (V) or 150mg/kg metadoxine (M) knock out the pERK level in (KO) mouse brain region for 7 days effect to 2 monthly age wild types (WT) and Fmr1.Institute's analyzed area is that Fmr1 knocks out or Hippocampus in wild-type mice (figure A), prefrontal cortex (figure B) and striatum (scheming C).Shown data are meansigma methods ± sem, N=10 mice/group. ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 14 shows ip once a day and uses supporting agent (V) or 150mg/kg metadoxine (M) knock out the pAkt level in (KO) mouse brain region for 7 days effect to 2 monthly age wild types (WT) and Fmr1.Institute's analyzed area is that Fmr1 knocks out or Hippocampus in wild-type mice (figure A), prefrontal cortex (figure B) and striatum (scheming C).Shown data are meansigma methods ± sem, N=10 mice/group. ^{* * *}p < 0.0001 and ns=is not remarkable.

Figure 15 shows and carries out 5 hours extracorporeal treatment to the effect of the filopodium density knocked out from Fmr1 in the hippocampal neuron culture of (KO) or wild type (WT) mice (figure A), length (figure B) and width (figure C) with supporting agent (V) or 300 μMs of metadoxines (M).Shown data are meansigma methods ± sem (wild type, N=20 neuron; Fmr1 knock-out mice, N=20 neuron). ^*p < 0.01, ^{* *}p < 0.001 and ns=is not remarkable.

Figure 16 shows and carries out the effect of extracorporeal treatment to the protein basis de novo synthesis knocked out from Fmr1 in 400 μMs of hippocampal slices of (KO) or wild type (WT) mice with supporting agent (V) or 300 μMs of metadoxines (M).Shown data are meansigma methods ± sem, N=6 section/group. ^*p < 0.001 and ^{* * *}p < 0.0001.

Detailed Description Of The Invention

The present invention relates to following discovery: metadoxine significantly improves cognitive and social functions in effective animal model of fragile X mental retardation.

Especially, metadoxine is with dosage-dependent manner Improving memory and study significantly during the frightened pattern of scene, and the learning and memory defect of Fmr1KO mice is fully remedied the similar degree to WT mice level by two maximum dose level levels (150mg/kg and 200mg/kg).In addition, find significantly to improve in memory through the Fmr1KO mice of 150mg/kg metadoxine process in performance testing (such as T-shaped labyrinth), show that cognitive result is significantly improved.Social activity interaction improvement through the KO mice of 150mg/kg metadoxine process supplements these and finds.Importantly, in effective mice fragile X model, cognitive n-back test, working memory are relevant with the social normalization improved with the biochemical marker reflecting neuron signal pathway and oxidative stress of interacting after with metadoxine process.

Fragile X mental retardation is the most general single-gene reason of infantile autism in boy and the heritability reason of mental retardation.Anyone with FMR1 gene mutation can be entailed its children.According to CDC (CenterforDiseaseControlandPrevention, CDC), about 1/4,000 male and 1/8,000 women suffers from fragile X mental retardation.Not there is this sudden change everyone all will show the S or S of fragile X, and obstacle range from mild is to severe, and physical trait such as facial elongated, large ear or protruding ear, greatly testis (huge testis disease), and behavior characteristics, such as stereotyped movement (such as waving hands) and Social Anxiety.Fragile X is caused by the change in fragile X mental retardation 1 (FMR1) gene that X chromosome exists or sudden change.Described gene generally produces the protein being called fragile X mental retardation protein or FMRP.This protein is significant to the contact produced and maintain in brain and nervous system between cell.Described sudden change causes body only produce a bit or do not produce this protein, and this usually causes the symptom of fragile X.

The generation of fragile X mental retardation (FXS) is usually such as, along with other diseases, autism spectrum disorder.Autism spectrum disorder (ASD) is one group of dysplasia that can cause remarkable social activity, interchange and behavior difficulty.The people suffering from ASD in its brain to be different from other people mode process information.

ASD is " pedigree obstacle ".This means that ASD acts on everyone in a different manner and scope can from very slight to severe.The people suffering from ASD has some similar symptoms, such as social interactional problem.But when symptom starts, the definite character of its order of severity and symptom there are differences.ASD comprises autistic disorder's (also referred to as " typical case " infantile autism), A Sibogeer syndrome (AspergerSyndrome) and pervasive developmental disorders (PervasiveDevelopmentalDisorder).

At present, Food and Drug Administration (FoodandDrugAdministration, FDA) not yet ratifies any medicine being used for the treatment of fragile X or its symptom especially.Exist and indicate the outer medicine using some symptom treat fragile X mental retardation, but result because of patient very big difference and have some to carry serious risk, severity of symptoms can be made at the very start or need a few Zhou Caineng onset in these medicines.The invention provides the outstanding demand to the medicine for the treatment of fragile X or its symptom.

Correspondingly, the invention provides treatment, prevention or alleviate the method for S or S of fragile X mental retardation and/or autism spectrum disorder, it is undertaken by using the compositions comprising metadoxine to object.

In general, the S&S of fragile X is divided into five classes: intelligence and study; Health, social and emotion, speaks and language and sensory disturbance, and it is usually relevant to fragile X or have common characteristic with fragile X.Comprise such as, the individuality suffering from fragile X has impaired intellectual function, Social Anxiety, language problem and responsive to some sensation.The treatment of metadoxine is used in the object suffering from fragile X mental retardation, to improve study and improve social competence.

Autism spectrum disorder is usually relevant to the individuality suffering from fragile X mental retardation.The S&S of infantile autism comprises remarkable delayed speech, social and communication difficult, and the behavior of exception and interest.The a lot of people suffering from infantile autism disease also have dysnoesia.Suffer from some mild that the syndromic individuality of A Sibogeer has autistic disorder usually.Such as, they can have social difficulty and abnormal behavior and interest.Suffer from the individuality of pervasive developmental disorders (PDD-NOS): meet infantile autism obstacle or the more syndromic standards of A Sibogeer and and the people of not all standard is diagnosable for suffering from PDD-NOS.With suffer from autistic disorder those compared with, the people suffering from PDD-NOS has less and slight symptom usually.Described symptom can only cause social activity and communication difficult.Carry out with metadoxine treating the symptom improving infantile autism.

Metadoxine is the ion pair of 2-pyrrolidone-5-carboxylic acid's ester (pyrrolidonecarboxylate, PCA) and pyridoxol (vitamin B6), and wherein these two kinds of compounds are connected in single product by salification.With the pairing of PCA improve synergistically pyridoxol pharmacologically active (see, such as United States Patent (USP) 4,313,952).Metadoxine can freely dissolve in water and gastric juice.This medicine can absorb and has high bioavailability (60% to 80%) by per os fast.The half-life of metadoxine in human serum shorter (40 to 60 minutes), and between per os and intravenous are used and no significant difference (Addolorato etc., the same; LuYuan etc., Chin.Med.12007120 (2) 160-168).

In some countries, metadoxine is commercially available as prescription drug using the form of 500mg tablet and 300mg injection.Tablet comprises 500mg metadoxine, microcrystalline Cellulose and magnesium stearate.Ampoule comprises 300mg metadoxine, sodium pyrosulfite, EDETATE SODIUM, methyl parahydroxybenzoate and water.

In certain embodiments, metadoxine compositions of the present invention (such as, being mixed with the metadoxine compositions for lasting or controlled release in whole or in part) can make metadoxine more efficiently be used for the treatment of, prevent and/or alleviate the S or S of fragile X mental retardation and associated conditions/disease (such as autism spectrum disorder) thereof.

In some method above-mentioned of the present invention, metadoxine or its acceptable derivates can be mixed with and discharge immediately after being applied to object.In some method above-mentioned of the present invention, metadoxine or its acceptable derivates are joined can be made into and are continued and/or controlled release after being applied to object, and are optionally mixed with to have after being applied to object simultaneously and discharge immediately and continue and/or controlled-release character.In certain embodiments, metadoxine or its pharmacologically acceptable derivates be formulated for non-chronic administration.The metadoxine preparation that can be used for the inventive method is hereafter describing in more detail.

In certain embodiments, the invention provides to comprise to be mixed with and to continue when being applied to object and/or the compositions of metadoxine or derivatives thereof of controlled release, its for improving, treatment, prevention and/or alleviate the S or S of fragile X mental retardation and/or its associated conditions/disease (such as autism spectrum disorder).

In certain embodiments, the invention provides the such compositions comprising metadoxine or derivatives thereof, wherein the metadoxine of a part or derivant are mixed with and continue when being applied to object and/or controlled release and the metadoxine of a part or derivant are mixed with for discharging immediately, described compositions is used for improving, treatment, prevention and/or alleviation fragile X mental retardation and/or its associated conditions/disease (such as autism spectrum disorder) S or S.

In certain embodiments, after using metadoxine or metadoxine derivant, in 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, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, reach effective serum levels of active component.In certain embodiments, after using metadoxine or metadoxine derivant, in described object, in about 5 minutes to about 20 minutes or 30 minutes or 40 minutes or 50 minutes or 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, reach effective serum levels of active component.In certain embodiments, after using metadoxine or metadoxine derivant, in 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, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, reach effective serum levels of active component.In certain embodiments, in 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, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, reach effective serum levels of active component.

The present inventor has developed the innovative approach (WO2009/004629, its content by reference entirety is incorporated to) using metadoxine or metadoxine derivant based on enteral (through digestive tract) and/or parenteral (other approach except digestive tract) approach.These methods are based on the appropriate design careful selection of the carrier for object delivery system being provided to the delivery system with desired characteristic, the surfactants/cosurfactants compositions that described carrier is such as suitable or catch the micrometer/nanometer granule (such as liposome or nanometer liposome) of active component, or other additives or excipient.Intestinal delivery system can be designed for oral administration (tablet, bag agent (sachet), lozenge, capsule, soft gelatin capsule (gelcap), drop or other can accept form) or per rectum use (suppository or (miniature) enema forms).In addition, described object delivery system can be liquid form, such as dropping liquid agent, syrup.In addition, described object delivery system can be beverage or food form.Therefore, active component used in the present invention can comprise in the beverage, particularly soft drink such as fruit juice, nectar, water, soaked (sparklingwater) and other effervescent beverages, bland (shake), milk shake and other beverages etc. based on emulsion.Liquid preparation can also be the form of heavy syrup agent, soakedly to dilute with water or rise.Or active component can be included in food, such as snack bar, health bar, cookies, cookie, sweet food, pan work, ice cream, ice lolly etc.

Moreover described delivery system can be the Foods or drinks comprising physiologically active derivatives of pyridoxine (particularly pyridoxol L, 2-Pyrrolidone-5 carboxylate (metadoxine)).In certain embodiments, edible Foods or drinks goods of the present invention can cause the serum levels reaching active component after it is edible in about 10 minutes to about 40-60 minute.Example can be sweet food, chocolate, confection and candy bar, energy bar, ice cream, cake product etc.

Parenteral administration mode is comprised subcutaneous, transdermal (skin diffusion by complete), thoroughly mucosa (being spread by mucosa), Sublingual, uses through cheek (buccal absorption by near gingiva line), or is used by suction.In certain embodiments, compositions used in the present invention is not used (that is, right and wrong are invasive) by invasive therapeutic modality.In certain embodiments, metadoxine or metadoxine derivative composition are not used by intravenous injection.

In certain embodiments, compositions used in the present invention is sent as the microcrystalline powder of applicable atomization or solution; Intravaginal or internal rectum are used, sends as vaginal suppository, suppository, cream or foam.Preferred preparation is the preparation for oral administration.Another preferred formulation is used for local application.Another preferred formulation is used for saturating mucosal administration, sublingual administration, uses (buccal absorption by near gingiva line) through cheek, is used or use (such as, in eye drop) through eye by suction.

Metadoxine is used or metadoxine derivant needs safe and efficient delivery system for medical application.The invention provides such delivery system, it sends many kinds of substance due to its special physicochemical characteristic (particularly directly absorbing) safely by non-invasive manner, thus avoids side effect.Described delivery system strengthens based on the physicochemical characteristic of its uniqueness the efficiency and quality that metadoxine or metadoxine derivant absorb significantly, and this makes it possible to the active substance of biologically active form to Object delivery low concentration or amount.Delivery system of the present invention makes active substance directly arrive tissue, and thus to treated object provide metadoxine or metadoxine derivant immediately or close to effect immediately.Therefore, in certain embodiments, the present invention uses for improving physiologically active pyridoxol (particularly pyridoxol L, 2-Pyrrolidone-5 carboxylate (metadoxine)) or the non-invasive drug delivery system used of the upper acceptable derivates of its physiology, it comprises the described physiologically active pyridoxol in suitable carrier as active component.In certain embodiments, the serum levels of described active component is reached after application in about 10 minutes to about 40-60 minute.In another embodiment, the present invention adopts for improving physiologically active derivatives of pyridoxine (particularly pyridoxol L, 2-Pyrrolidone-5 carboxylate (metadoxine)) the non-invasive drug delivery system used, it for improving cognitive behavior in the object having this to need, and described system comprises the described derivatives of pyridoxine in suitable carrier as active component.In certain embodiments, the serum levels of described active component is reached after application in about 10 minutes to about 40-60 minute.

In certain embodiments, drug delivery system of the present invention can be designed for per os, per nasal, through eye, per rectum, subcutaneous, transfer, thoroughly mucosa, Sublingual, use through cheek or suction.Described drug delivery system can provide active substance with controlled release profile.In certain embodiments, drug delivery system of the present invention also can comprise the other pharmaceutically active agents of at least one.Delivery system used in the present invention generally can comprise buffer agent (namely regulating the reagent of its Osmolalities) and optionally comprise one or more of pharmaceutically suitable carrier as known in the art, excipient and/or additive.Also the pharmaceutically acceptable active component supplemented can be mixed in described compositions.Described carrier can be the solvent or the disperse medium that contain such as water, ethanol, polyhydric alcohol (such as glycerol, propylene glycol and liquid macrogol etc.), its suitable mixture and vegetable oil.Can such as by using coating (as lecithin), maintaining suitable mobility by the particle size (in the case of a dispersion) needed for maintenance and by use surfactant." pharmaceutically suitable carrier " used herein comprises any and all solvents, disperse medium, coating, antibacterial agent and antifungal etc.This type of medium and reagent needles to the use of pharmaceutically active substance known in the art.Unless due to any conventional media or reagent incompatible with described active component, otherwise can consider to use it in therapeutic composition.Can consider, active agent delivery can be carried out by any pharmaceutically acceptable approach and with any pharmaceutically acceptable dosage form.Oral form includes but not limited to tablet, capsule, pill, bag agent, lozenge, drop, powder, granule, elixir, tincture, suspensoid, syrup and Emulsion.Also comprise that per os discharges rapidly, the pharmaceutical dosage form of release and delayed release when controlling.Active medicine component can be used with single dosage form or with independent dosage form simultaneously or individual application.Active medicine component can with suitable pharmaceutical diluents, excipient or carrier (being referred to as herein " carrier "), the mixture of material suitably selected for target administration form in use.When described delivery system is used for oral administration and is the form of tablet or capsule etc., active medicine component and nontoxic pharmaceutical acceptable inert carriers can be combined, described inert carrier such as lactose, starch, sucrose, glucose, modified sugars, modified starch, methylcellulose and derivant thereof, calcium hydrogen phosphate, calcium sulfate, mannitol, sorbitol and other reducing sugar and non-reducing sugar, magnesium stearate, stearic acid, sodium stearyl fumarate, Tridocosanoin, calcium stearate etc.For the oral administration of liquid form, can by combinations such as active medicine component and nontoxic pharmaceutical acceptable inert carriers such as ethanol, glycerol, water.When expectation or when needing, also suitable adhesive, lubricant, disintegrating agent and coloring agent and flavoring agent can be mixed in mixture.Also can add stabilizing agent such as antioxidant, propyl gallate, sodium ascorbate, citric acid, pyrosulfurous acid calcium, hydroquinone and umbelliferone with stabilizer type.Other suitable compounds can comprise gelatin, sweeting agent, natural and rubber polymer (such as arabic gum, Tragacanth or alginate), carboxymethyl cellulose, Polyethylene Glycol, wax etc.

Other the suitable pharmaceutically suitable carrier that can be used in these pharmaceutical compositions include but not limited to: ion-exchanger, aluminium oxide, aluminium stearate, magnesium stearate, lecithin, serum albumin (such as human serum albumin), buffer substance (such as phosphate), glycine, sorbic acid, potassium sorbate, the partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte (such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, silica sol, magnesium trisilicate), polyvinylpyrrolidone, based on cellulosic material, Polyethylene Glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, Polyethylene Glycol and lanoline.In some embodiments, described pharmaceutically suitable carrier is magnesium stearate.Generally accept and use other drug excipients see in such as Remington ' sPharmaceuticalSciences (Gennaro, A. edit, MackPub., 1990).

For the object of parenteral administration, the solution in suitable oil (such as Oleum sesami or Oleum Arachidis hypogaeae semen) or aqueous propylene glycol can be used in, and the sterile aqueous solutions of corresponding water soluble salt.If necessary, such aqueous pharmaceutical suitably can be cushioned, and first make liquid diluent isotonic with enough saline or glucose.These aqueous solutions are particularly useful for the object of intravenous, intramuscular, subcutaneous and peritoneal injection.In this respect, the sterile aqueous media used all easily obtains by standard technique well known to those skilled in the art.Use the method for the various pharmaceutical composition of a certain amount of active fraction preparation to be known to those skilled in the art, or will be obvious according to present disclosure.The half-life of metadoxine in human serum is very short.LuYuan etc. (Chin.Med.J2007120 (2) 160-168) show mean half-life and are about 0.8 hour.The mode extending active part serum levels is undertaken by the material be applied in extended release preparation.Because metadoxine can freely be dissolved in water and multiple biofluid, be therefore difficult to maintain its release and extend its soak time.Therefore, unexpectedly sustained release is realized.The Co ntrolled release dosage form of metadoxine or metadoxine derivant can discharge gradually based on active component making a reservation in biofluid, thus produces the continuous action of blood plasma level fuctuation within a narrow range in for a long time.

In certain embodiments, delivery system used in the present invention can be used with controlled release formulation.In certain embodiments, application process is determined by attending doctor or others skilled in the art after evaluating the situation of object and requirement.An embodiment of the inventive method uses described therapeutic compound herein with sustained release form.Any controlled or sustained release method known to persons of ordinary skill in the art all can use together with the compositions and methods of the invention, such as, described in Langer, Science249 (4976): 1527-33 (1990) those.Such method comprises uses sustained-release composition, suppository or the implantable medical device through bag quilt, thus the present composition of the continuous delivery treatments effective dose of object to the method.Also can use and to design and the patch (patch) be mixed with for described object realizes sustained release.Compositions of the present invention is sent by capsule, and it allows sustained release in medicament a period of time.Controlled release or sustained-release composition are included in the preparation in lipotropy storehouse (such as, fatty acid, wax, oil).The present invention also comprises the particulate composition (such as the husky amine of poloxamer or pool Lip river) through polymer coating.Sustained-release formulations or device, or any topical preparation additionally can comprise the component stablizing described compositions or infiltration physiologic barrier (such as skin or mucosa).Exemplary annexing ingredient can comprise the upper acceptable detergent of any physiology or solvent such as, as dimethyl sulfoxide (DMSO).

In all embodiments of the present invention, method of the present invention and purposes all can adopt the compositions comprising salt adduct being as defined by the present invention formulated as single dose.Described single-dose preparations can be immediate release formulation, explosion type (burst) preparation, prolongation release reagent, extended release preparation or any other controlled release formulation well known by persons skilled in the art.

In other embodiments of the inventive method and purposes, the compositions comprising the salt adduct that the present invention limits can be wherein use the unitized dose preparation of dissimilar preparation to object, i.e. immediate release formulation, explosion type preparation, extend release reagent, the combination in any of sustained-release agents or any other controlled release formulation well known by persons skilled in the art, it gives with single dose or with individually dosed independence, with or give successively, wherein use individually dosed between interval determine based on object disease or the disease of obstacle and the health of the order of severity and described object.

In some embodiments, the compositions that the inventive method uses is formulated as combination dosage forms, the at least one dosage form of salt adduct that wherein the present invention limits be releasing pattern immediately, and at least one dosage form of salt adduct (identical or different with the salt adduct be formulated in immediate release formulation) that the present invention limits is formulated as controlled (slowly and/or lasting) delivery formulations.In other embodiments, the weight ratio of salt adduct in described at least one immediate release formulation and at least one controlled release formulation that the present invention limits can 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 adopting such combination dosage forms in method of the present invention or purposes, can by the described at least one of the salt adduct limited above releasing pattern and at least one controlled release form is independent, adjoint, successively, simultaneously, in succession etc. be applied to object immediately.In some embodiments, described at least one immediately releasing pattern use at first.In other embodiments, described at least one controlled release formulation is used at first.

In certain embodiments, the metadoxine in the present composition or metadoxine derivant can be mixed with for continuing or controlled release within the time of at least 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be mixed with for continuing or controlled release within the time of about 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be mixed with for littlely continuing or controlled release within the time of about 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours in about 0.5 hour or 1 hour or 2 hours or 3 hours or 4.In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be mixed with for littlely continuing or controlled release within the time of about 9 hours, 10 hours, 11 hours or 12 hours in about 5 hours or 6 hours or 7 hours or 8.

In certain embodiments, this explanation use metadoxine in compositions or metadoxine derivant can for discharging immediately, release or outburst releasing pattern fast.

In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be mixed with to discharge in about 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours up to whole metadoxine or metadoxine derivant 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%.In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be mixed with release in about 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours and be no 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 whole metadoxine or metadoxine derivant.

In certain embodiments, the metadoxine in compositions used herein or metadoxine derivant can be continue or slow releasing form with immediately or the combination of fast release form.In certain embodiments, continue or slow releasing metadoxine or metadoxine derivant with immediately or the relative scale discharging metadoxine or metadoxine derivant be fast such as 1: 99,5: 95,10: 90,15: 85,20: 80,25: 75,30: 70,35: 65,40: 60,45: 55,50: 50,55: 45,60: 40,65: 35,70: 30,75: 25,80: 20,85: 15,90: 10,95: 5 or 99: 1.

In certain embodiments, polymeric material is used to continue or control the release of metadoxine or metadoxine derivant.In certain embodiments, the type of polymeric material and consumption have strong effect to the speed that metadoxine or metadoxine derivant discharge from product of the present invention.The example of polymer comprises both hydrophobicity and hydrophilic polymer.The example of hydrophobic polymer includes but not limited to ethyl cellulose and other cellulose derivatives, fat (such as glyceryl palmitostearate, Cera Flava, sugared wax (glycowax), castor wax, palm wax, glyceryl monostearate or stearyl alcohol), hydrophobic polypropylene amide derivatives and hydrophobic methyl acrylic acid derivative, and the mixture of these polymer.Hydrophilic polymer includes but are not limited to hydrophilic cellulose derivant such as methylcellulose, hydroxypropyl emthylcellulose, hydroxyethyl-cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose and hydroxyethylmethyl-cellulose polyvinyl alcohol), polyethylene, polypropylene, polystyrene, polyacrylamide, vinyl-vinyl acetate copolymer, polyacrylate, polyurethane, polyvinylpyrrolidone, polymethyl methacrylate, polyvinyl acetate, poly hydroxy ethyl acrylate, and the mixture of these polymer.In addition, any mixture of one or more of hydrophobic polymer and one or more of hydrophilic polymer is optionally used.

In certain embodiments, in the present composition or polymeric material used in the present invention be microcrystalline Cellulose, " AVICELPH101 " that such as produced by FMCBioPolymer.

In certain embodiments, in the present composition or polymeric material used in the present invention be hydroxypropyl emthylcellulose, " METHOLOSE " that such as produced by Shin-EtsuChemicalCo.

In certain embodiments, in the present composition or polymeric material used in the present invention be ethyl cellulose, " the ETHOCEL such as produced by DowChemicalCompany ^tM".

In certain embodiments, in the present composition or polymeric material used in the present invention be acrylic polymer, " the EUDRAGITRS such as produced by RohmGmbH ^tM".

In certain embodiments, in the present composition or polymeric material used in the present invention be silicon dioxide colloid, " the AEROSIL such as produced by Degussa ^tM".

In certain embodiments, in the present composition or polymeric material used in the present invention be polyvinyl acetate, " KOLLICOATSR " that such as produced by BASF.

In certain embodiments, for in the present composition or polymeric material used in the present invention be ethyl acetate and vinyl acetate solution, " DURO-TAK " that such as produced by DelascoDermatologicLab & Supply, Inc..

In certain embodiments, the present composition or compositions used in the present invention comprise about 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg or 900mg to about 1000mg, 1500mg, 2000mg, 2500mg or 3000mg metadoxine or metadoxine derivant, or substantially consisting of.In certain embodiments, the present composition or compositions used in the present invention comprise about 5mg, 100mg, 500mg or 1000mg to about 2000mg, 4000mg, 10,000mg, 15,000mg or 20,000mgAVICELPH101 ^tM, or substantially consisting of.In certain embodiments, the present composition or compositions used in the present invention comprise about 25mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg or 600mg extremely about 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 5000mg, 10,000mg, 15,000mg or 20000mg polymeric material, or substantially consisting of.In certain embodiments, described polymeric material is METHOLOSE, ETHOCELE10 ^tMor EUDRAGITRS ^tM.In certain embodiments, METHOLOSE accounts for or substantially accounts for 1% to 90% of preparation, and preferably 5% to 70%.In certain embodiments, ETHOCEL ^tMaccount for or substantially account for 1% to 30% of preparation, preferably 2% to 20%.In certain embodiments, EUDRAGIT ^tMaccount for or substantially account for 1% to 90% of preparation, preferably 5% to 70%.

In certain embodiments, delivery system of the present invention or delivery system used in the present invention comprise delivery apparatus.In certain embodiments, the present composition or compositions used in the present invention is sent by permeating method (such as by osmotic pumps) with controlled speed.Described system can by being built by osmotically active agent with the fast semipermeable membrane bag of control.This film can comprise the hole of the critical dimension of being sent medicament by it.This dosage form sucks water with the speed determined by the fluid permeability of film and the osmotic pressure of core formulation after contacting with aqueous fluids.The infiltration of this water sucks and causes the saturated solution forming active material in the core, and it distributes from the delivery orifice film with controllable rate.

In certain embodiments, use degradable microgranule to send the present composition or compositions used in the present invention.In certain embodiments, the system preparing microgranule is made up of following: be emulsifiable in the organic facies be made up of the volatile solvent with dissolve polymer and encapsulated material in aqueous phase.In certain embodiments, the degradable polymer that can be used for matrix of microparticles comprises the copolymer (PLAGA) of polylactic acid (PLA) or lactic acid and glycolic.PLAGA polymer is degraded to monomer whose component with hydrolysis method in time, and described monomer component is easily removed from body by natural metabolism.

Invention formulation or preparation used in the present invention also can comprise absorption enhancer and other optional components.The example of absorption enhancer includes but not limited to cyclodextrin, phospholipid, chitosan, DMSO, tween, Brij, glycocholate, saponin, fusidate and the influx and translocation equipment based on energy.

The optional components be present in dosage form includes but not limited to diluent, adhesive, lubricant, surfactant, coloring agent, flavoring agent, buffer agent, antiseptic, stabilizing agent etc.

Diluent (also referred to as " filler ") comprises such as: dicalcium phosphate dihydrate, calcium sulfate, lactose, cellulose, Kaolin, mannitol, sodium chloride, dried starch, hydrolyzed starch, silicon dioxide, silica sol, titanium dioxide, aluminium oxide, Talcum, microcrystalline Cellulose and Icing Sugar.For using in liquid form, diluent comprises such as ethanol, sorbitol, glycerol, water etc.

Adhesive is for giving preparation to stick together performance.Suitable binder materials includes but not limited to starch (comprising corn starch and pregelatinized starch), gelatin, sugar (comprising sucrose, glucose, dextrose, lactose and sorbitol), Polyethylene Glycol, wax, natural and rubber polymer (such as arabic gum, Tragacanth, sodium alginate, cellulose and Magnesiumaluminumsilicate (Veegum)), and synthetic polymer (such as polymethacrylates and polyvinylpyrrolidone).

Lubricant is produced for promoting; The example of proper lubrication agent comprises such as magnesium stearate, calcium stearate, stearic acid, Tridocosanoin and Polyethylene Glycol.

Surfactant can be anion, cation, both sexes or nonionic surfactant, wherein preferred anionic surfactant.Suitable anion surfactant includes but not limited to the surfactant containing carboxylate radical, sulfonate radical and sulfate ion associated with cation (such as sodium, potassium and ammonium ion).Particularly preferred surfactant includes but not limited to: long alkyl chain sulfonate and alkylaryl sulfonates, such as dodecylbenzene sodium sulfonate; Dialkyl sulfosuccinates, such as two-(2-ethylhexyl) sodium sulfosuccinates; And alkyl sulfate such as sodium lauryl sulfate.

Stabilizing agent (such as antioxidant) includes but not limited to: propyl gallate, sodium ascorbate, citric acid, pyrosulfurous acid calcium, hydroquinone and umbelliferone.

If desired, the present composition or compositions used in the present invention also can comprise a small amount of non-toxic auxiliary substances, such as wetting agent or emulsifying agent, antiseptic etc.

Arbitrary composition of the present invention or arbitrary composition used in the present invention can be used alone or use for improving cognitive behavior with one or more of other therapeutic combination.The example of therapeutic agent is in addition: amphetamines, methylphenidate hydrochloride, hydrochloric acid dexmethylphenidate, atomoxetine, reboxetine, fluoxetine (fluoxatine), Sertraline, paroxetine, fluoroxamine, citalopram, venlafaxine, amfebutamone (bupropion), nefazodone and mirtazapine.

Can combine with carrier mass and change according to treated main body and concrete mode of administration with the amount of the compound producing single dosage form and other therapeutic agent.Preferably, compositions of the present invention should be mixed with and make to use 0.1 to 1g/kg body weight/day, the preferably dosage of 0.1 to 300mg/kg body weight.The dosage of compound depends on the disease of patient and the daily dose of the state of an illness and expectation.In human therapy, per os daily dose is 10mg to 3000mg, or preferred 100mg to 3000mg.Such as, described daily dose is 10mg, 25mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg or 3000mg.These dosage are used in a unit, and it can be used with odd-numbered day dosage or can be divided into the dosage of 2 to 3 parts less in some cases every day.

In certain embodiments, compositions of the present invention can combination with one another synergism and can under other therapeutic agent exists synergism.Therefore, in such composition, the amount of compound and other therapeutic agent will be less than amount required in the single therapy only utilizing described therapeutic agent.In such compositions, the other therapeutic agent of the dosage of 0.1 to 1g/kg body weight/day can be used.

Definition

For convenience's sake, included at this some term used in this description, embodiment and appended embodiment.Unless otherwise defined, otherwise all technology used herein and scientific terminology have understood identical implication usual with those skilled in the art.

The noun not having numeral-classifier compound to modify used herein refers to this noun one or more (that is, at least one).Such as, " key element " refers to one or more key element.

Term used herein " comprises " and means phrase and " include but not limited to ", and can use with its exchange.

Unless the context clearly indicates otherwise, otherwise term used herein " or/or " means term "and/or", and can use with its exchange.

Term used herein " such as/as " means phrase " such as/as but be not limited to ", and can use with its exchange.

Term " preventative " or " therapeutic " treatment point to object to use one or more of compositions of the present invention.If applying said compositions before less desirable disease (such as host animal clinical or other undesirably state) manifests clinically, then it is preventative, and namely it contributes to preventing (namely protecting) this object to avoid less desirable disease occurs; And if use after less desirable disease manifests, then this treatment is curative (namely it is intended to weaken, improve or stop progress or the side effect thus of undesirably disease).

Term " therapeutic effect " refer to animal (in (particularly mammal, and more especially people) by pharmacological active substance cause local or systemic effect.Therefore, this term means object is for diagnosing, curing, alleviate, treat or preventing disease in animal or human or strengthen the health of expectation or any material of intelligent development and situation.Term " treatment effective dose " refers to that described material produces certain local of expectation or the amount of systemic effect with the rational interests/Hazard ratio being applicable to any treatment.In certain embodiments, the treatment effective dose of compound or compositions will depend on its therapeutic index, dissolubility etc.Such as, some metadoxine of the present invention or metadoxine derivative formulations can be used, as those skilled in the art are confirmable with the amount being enough to produce the rational interests/Hazard ratio being applicable to selected treatment.

Term " effective dose " refers to the amount of the therapeutic agent producing at least one expected result when being applied to object with suitable dosage and scheme.

Treat that " object " or " patient " of being treated by the inventive method can mean people or non-human animal, preferred mammal.Term used herein " object " can refer to healthy individuals or suffer from the object of fragile X mental retardation or autism spectrum disorder.In some alternative embodiments, term used herein " object " and " healthy individuals " and " object in need " and " patient in need " not included in the object by alcohol function after the drinking of arbitrary form, i.e. the alcoholic of alcoholic (alcoholic) and alleviating alcohol addiction.

Term used herein " salt adduct " means the salt product containing direct two or more different ions of adduction, and wherein the total charge of salt adduct is 0.In certain embodiments, salt adduct comprises one and has the positively charged part of single positive functional groups (namely, positively charged moiety carries+1 net charge) and an electronegative part (that is, electronegative part carries-1 net charge) with single negative charge functional group.In certain embodiments, salt adduct comprises one and has the positively charged part of two positively charged functional groups (it can be identical or different) (namely, positively charged moiety carries+2 net charges) and two electronegative parts, described negative charge part can be identical or different and have single electronegative functional group (that is, each electronegative part carries-1 net charge) separately.In certain embodiments, salt adduct comprises two and has the positively charged part of a positively charged functional group (it can be identical or different) separately (namely, each positively charged part carries+1 net charge) and an electronegative part (that is, electronegative part carries-2 net charges) with two identical or different electronegative functional groups.In certain embodiments, salt adduct comprises and has+n net charge and (derive from one or more positively charged functional group, it can be identical or different) positively charged part and the electronegative part with-n net charge (derive from one or more electronegative functional group, it can be identical or different), wherein n is integer, and it can equal 1,2,3,4,5 or 6.

As used herein, the respective acids that " the positively charged part of salt adduct " of the present invention is pyridoxol, or derivatives thereof.In certain embodiments, the positive charge of described positively charged part derives from the protonated basic nitrogen-atoms of pyridoxol (as in such as compound (2)) or its any derivant (such as the compound of formula (I)).In certain embodiments, positively charged derivatives of pyridoxine is that the positively charged functional group below such as replaces: such as-NH ₃ ⁺,-CH ₂nH ₃ ⁺, NH ₂r ⁺,-NHR ₂ ⁺(wherein R is C independently of one another _1-c ₆alkyl), in some embodiments, described functional group can be present in pyridine ring together with positively charged protonated basic aromatics nitrogen-atoms.

Should understand, the part of salt adduct of the present invention can each at least one chiral centre self-contained, and therefore can exist with its any stereoisomer and can be used as its any Enantiomer separation, described stereoisomer comprises enantiomer, diastereomer or its any mixture, includes but not limited to racemic mixture.The present invention includes any possibility stereoisomer (such as enantiomer, diastereomer) of any unitary part of salt adduct of the present invention, its any mixture, include but not limited to racemic mixture.When the method for the various piece for the preparation of salt adduct of the present invention described herein produces the mixture of stereoisomer, by routine techniques (such as preparative scale chromatography) by these isomer separation.The part of salt adduct of the present invention can be prepared with any mixture (including but not limited to its racemic mixture) of its possibility stereoisomer separately, or prepares independent stereoisomer (such as enantiomer, diastereomer) by the synthesis of enantiomer specificity or by carrying out chiral chromatogram separation to racemate.Whenever mentioning aminoacid, the present invention is interpreted as containing natural and alpha-non-natural amino acid or its any derivant.

In this description in the whole text, word " comprises/comprises " or version is interpreted as inferring the group comprising described entirety or entirety, but does not get rid of any other overall or overall group.

Term " bioavailable " means at least there is a certain amount of specific compound in body circulation.The regular calculating of oral bioavailability describes (" FundamentalsofClinicalPharmacokinetics according to F value, " JohnG.Wegner, DrugIntelligencePublications; Hamilton, Ill.1975).The ratio of the parent drug concentration during the parent drug concentration that F value is circulated in (such as, blood plasma) by body after using at intravenous and body after being used by approach (such as per os) in non-vein are circulated is derived.Therefore, oral bioavailability within the scope of the present invention to relate to after oral administration detectable parent drug amount in blood plasma and is compared to the ratio or F value that intravenous uses.

Term " treatment " refers at least one symptom relaxing, improve, alleviate or alleviate disease, disease or obstacle in the mammal (such as people), or improvement relevant to disease, disease or obstacle determine measure.The treatment to healthy individuals is also contained in treatment used herein.

The term " acceptable derivates " relevant with metadoxine or metadoxine derivant refers to any salt of metadoxine, conjugate, ester, complex or other chemical derivatives or any part comprising it, it (directly or indirectly) can provide metadoxine or its metabolite or functional residue after being applied to object, or measurable metadoxine is active.Term " the upper compatible metadoxine derivant of physiology " can exchange with term " acceptable derivates " in this article and use, and refers to functional, active, the pharmaceutical usable derivatives of metadoxine.

Term " excipient " refers to the non-activity material of the carrier being used as active component in the formulation.

Term " controlled release " refers to sends medicament with controllable rate in long-time and any preparation being designed to the drug level feature obtaining expectation.

Term " sustained release " uses with its conventional sense, refers to be provided in for a long time the preparation progressively discharged providing active substance, and in certain embodiments, it also can produce substantially invariable blood level, i.e. controlled release in long-time.

Term " discharges immediately " and uses with its conventional sense, refers to provide the non-delayed of active substance or the preparation of controlled release after administration.

" half-life " of term material refers to that material loses its half pharmacologically active, physiologically active or the time needed for other activity.Biological half life is important pharmacokinetic parameter, and usually represents with abbreviation tin..

Term " Noninvasive " refers to the Therapeutic mode of not skin puncture.

Term " non-chronic administration " can exchange with term " acute administration " in this article and use, and perhaps phase ground gives the amount of measurement or non-measured or the medicine of part to object.Non-chronic administration can be single-dose treatment or multiple dose treatment, and optionally gives in time.Usually (but not always), give non-chronic administration to treat or prevent non-chronic disease.Some chronic disease also can benefit from described metadoxine or the non-chronic administration of metadoxine derivative composition herein.

Term " chronic administration " refers to the medicine of the amount giving measurement termly to object.In some embodiments, chronic administration is to treat or preventing one or more of chronic disease, problem or disease.Chronic disease has one or more feature following: they be persistent, residual lower disabled, caused by irreversible pathological change, need to carry out special training to patient so as rehabilitation or can expect needs monitoring for a long time, observe or nurse.

Term " single-dose treatment " refers to the disposable medicine giving the amount of required measurement.According to demands of individuals, give single-dose treatment aperiodically to treat non-chronic disease.

Term " t _max" refer to the time reaching peak concentration.Time when there is maximum concentration after single dose is used is calculated according to following formula:

$t_{\max }=\frac{2.303}{{\mathrm{\&lambda;}}_a-{\mathrm{\&lambda;}}_z}\log \frac{{\mathrm{\&lambda;}}_a}{{\mathrm{\&lambda;}}_z}$

Wherein λ a and λ z is respectively apparent absorption constant and elimination rate constant.

Embodiment

Embodiment 1: general approach

Embodiment described herein uses the reagent of hereafter general description and method to carry out.

Laboratory animal

Fmr1 knock-out mice (KO2) (TheDutch-BelgiumFragileXConsortium, 1994) obtain from JacksonLaboratory at first, wild type (WT) littermate produces based on C57BL/6J background and repeatedly backcrosses more than eight generations in C57BL/6J background.Fmr1 knock-out mice to be had for group is housed in the temperature and humidity controlled chamber of 12 hr light/dark cycle with homologous genes type (illumination is for 7am to 7pm; Test was carried out in the illumination phase).Room temperature in continuous record accommodating chamber and humidity, food and water can freely obtain simultaneously.At behavior experimental session, the healthy Fmr1 knock-out mice at 2 or 6 monthly ages and wild type littermates (N=10 mice/processed group) thereof are tested.Mice to be housed in commercially available plastics cage and according to UKAnimals (ScientificProcedures) Act, the requirement of 1986 is tested.All experiments are undertaken by the laboratory technician of genotype and the blind state of drug treating.Before carrying out any experiment, animal is all allowed to have the laundering period of the shortest one week.Preventative or therapeutic treatment is not used within the laundering period.

Medicine

For research 1 (embodiment 2), metadoxine to be dissolved in saline and with 100,150 or 200mg/kg dosage once a day intraperitoneal use 7 days.For research 2 (embodiment 3), in vivo in test, metadoxine to be dissolved in saline and with the intraperitoneal doses of 150mg/kg/ days, or to use 7 days once a day with the oral dosages of 150 or 300mg/kg/ days (volume with 0.1ml).For research 2, in vitro in test, metadoxine is used 5 hours with the concentration of 300 μMs.In all cases, all use saline as supporting agent (contrast).

Performance testing

Social interaction and social recognition memory: mice is sociability's species, it participates in the Social behaviors of easily scoring, comprise close, follow, smell spys, combing, aggressiveness contact (aggressiveencounter), property interaction (sexualinteraction), close sexual behaviour (parentalbehavior), nest and roll up sleep (sleepinginagroupofhuddle) in groups.Social activity in mice is evaluated close to by the spy persistent period of smelling for new mice.

Mice is placed in size and adult mice inhabitation cage and there is same order and the test site/cage (40 × 23 × 12cm cage has plexiglass cover to be conducive to observing mice) ground with fresh wood flour.Background mice abnormal smells from the patient produces by putting into some non-experiment mices before test in a device.10 to 15 minutes before testing, mice is transferred to laboratory.Study subject and young baby are placed in test-cage simultaneously.The total duration of the social research of assessment and several 3 minutes of bout (bout), it is defined as visits and intimately follow at test mice stimulating smelling of young baby (distance afterbody < 2cm).After 30 minutes, identical stimulation young baby is used to carry out retest.The data parameters of collecting is for obtain and smelling of identifying visits the total duration of bout and total degree.Derive social memorability (socialmemoryratio), it is defined as test 2/ and tests 1+2.Therefore, memoryless (such as, 20/ (20+20)=0.5, memory (such as, 10/ (20+10)=< 0.5.

Y shape labyrinth replaces: carry out two tasks.First task be arm is entered between spontaneous hocket non-study assessment.Second task is spatial reference memory task, and wherein animal must learn to remember that in two arms, which is equipped with food reward bait.Starting the same day before training, animal is allowed freely to explore labyrinth 5 minutes.Then, make it accept two tests, one test in, food is placed on left arm, one test in by foodstuff at right arm.This operation prevents deflection arm.

Y shape labyrinth water maze: transparent lucite Y shape labyrinth is filled with 20 DEG C of water of 2cm.This impels mice to the outlet of an arm far-end, to leave labyrinth wading acroos a river.Labyrinth is placed on outstanding visual cues around the centre of room.

Incentive T-shaped labyrinth replaces: the rising or the provision for sealing that use T-shaped formula (during horizontal positioned).Mice be placed on the bottom of T and allow it to select a target-arm of adjacent trunk arm (stem) other end.In extremely rapid succession carry out two tests, the arm that second test requirements document mice was not accessed before selecting, reflect the memory (spontaneous alternately) that first time is selected.If its alternate selection, then by making animal hungry and carry out award with the food of preference to it to strengthen this tendency.Especially, after 4 day laundering period on T-shaped labyrinth, training mice alternate arm is selected to accept condensed milk as award.

Continuous passage: this equipment by 4 continuous print, linearly aligned, cause the passage that anxiety effect increases progressively and form (passage after each all paints more shallow color, have lower wall and/or narrower than passage above), described passage is made up of the timber of painting.The length of each part or passage is 25cm.Passage 1 has the high wall of 25cm, and width is 8.5cm and painted black.Step-down (stepdown) guide channel 2 of 0.5cm, its width also has the high wall of 1.3cm and for Lycoperdon polymorphum Vitt for 8.5cm.The step-down guide channel 3 of 1.0cm, its width is 3.5cm, have the high wall of 0.8cm and be white.The step-down guide channel 4 of 0.4cm, it is also white but width is 1.2cm, and has the high wall of 0.2cm.By the back side of passage 1 being anchored into the high estrade lift apparatus of 50cm.Bedding and padding are provided in case mice falls below arm 3 and 4.Every mice is placed on the blind end of passage 1, towards wall.For following startup timer: the incubation period of each wall of the total duration (5 minutes) 1) tested+enter, with 2) time of spending in the channel 1.When extremity are all put into next passage by mice, think that it has entered this passage.Record the total time that (extremity are whole) in each channel spend.

Scene fear conditioning: in fear conditioning test, to be placed on mice in new environment (darkroom) and to make it accept paired hint and foot electric shock (0.2mA continues 1 second (research 1) or 0.7mA continues 0.5 second (research 2)).Subsequently, when testing in initial training situation, mice shows the natural defense reaction or scene fear conditioning that are called sluggish (freezing) (Blanchard, 1969).The sluggish time is defined as the time that mice spends in the inertia behavior except breathing.Data are expressed as the percentage ratio of testing time.After training period 24 hours, in training chest, 5 minutes are tested to mice not providing under electric shock, and observe the behavior of stagnating.

Learn around meter: employing multivariate analysis of variance carrys out the group difference in assessment data.Repeated measure ANOVA is carried out to behavioral data.Use Newman-Keuls to check (research 1) or Tukey inspection (research 2) to compare after significance,statistical effect in each ANOVA afterwards.Think that the p value being less than 0.05 is significant.

Biochemical test:

The activity dependent enzymes that phosphorylated CREB and Akt:Ras-Mek-ERK and PI3K-Akt-mToR signal transduction path participate in the genetic transcription of the potential change of mediation synaptic plasticity changes (Klann and Dever, 2004).As LopezVerrilli (LopezVerrilli etc., 2009) previously described expression of being measured phosphorylated CREB and Akt albumen by western engram analysis.The antibody adopted is the anti-phospho-specif iotac antibodies (CellSignalingTechnology, Danvers, MA, USA) for Akt (1/1000) and kinases (ERK) 1/2 (1/2000).The phosphorylation (Thr308) of the antibody test phosphoric acid-Akt for phosphoric acid-Akt for the phosphorylation (Thr202/Tyr204) of the antibody test phosphoric acid-ERK1/2 of phosphoric acid-ERK.ERK and Akt of Akt and ERK1/2 albumen total content and phosphorylation is by having anti-phosphoric acid-Akt (1/1000) and anti-phospho-ERK antibody (1/2000) (CellSignalingTechnology, Danvers, MA, USA) blotting membrane evaluate.Akt or ERK phosphorylation is normalized according to the protein content in same sample and is expressed as the change % relative to basic condition, thinks that foundation level is 100%.Following assess proteins load: stripping film also uses beta-actin antibody (1/1000) (Sigma-Aldrich, St.Louis, MO, USA) blotting membrane again.Phosphorylated CREB in blood lymphocytes and Akt protein expression are measured by flow cytometry.Lymphocyte biomarker is measured, uses excitation laser adjustment at the FACStarplus (BectonDickinson) of 488nm, and collect the green fluorescence (GST) from FITC by the band filter of 515nm to 545nm.Mean F ITC fluorescence intensity calculates relative to the fluorescence with reference to cell.The par of the Ab molecule that average cell fluorescence intensity (meancellularfluorescenceintensity, MFI) and each cell combine is directly proportional.

Neuron morphology: hippocampal cell culture is prepared by the wild type of embryo's day (E17.5) of gestation 17.5 and Fmr1KO fetus mice.Put to death mice by neck dislocation and the hippocampal cell through dissociating be layered in the porous container (FalconPrimaria) of 15mm.In vitro after 5 days, there are (Ethell and Yamaguchi, 1999 to be conducive to monitoring dendritic spine form after drug treating in transfection green fluorescent protein (GFP); Ethell etc., 2001; Henkemeyer etc., 2003).Dendritic spine are formed in vitro time about 16 days (DIV).At the 17th day, with the metadoxine extracorporeal treatment culture 5 hours of 300 μMs of concentration.

The confocal synthetic image (40 × object lens, the superposition of 20 × 0.2 μm) that neuronic filopodium density through GFP transfection adds Zeiss lens (Zeiss) through the stack carries out Sholl and analyzes and quantize.Use Metamorph software, around each neuronic cyton, draw equidistant concentric circular (every 20 μm), subsequently the filopodium quantity of each circle is counted.Use unpaired couple of tail StudentT to check to compare the meansigma methods of counting.

Neuronic sour jujube Maturity through GFP transfection uses Metamorph software (MolecularDevices, Sunnyvale, CA) to analyze.Each neuron selects the Distal dendrites sections of two 70 μm to 100 μm for carrying out sour jujube morphometric analysis.For each sour jujube, length and width are measured.Length is defined as the distance from protruding bottom to protruding terminus; And ultimate range width is defined as perpendicular to sour jujube major axis.To check for the unpaired couple of tail StudentT of multiple comparisons and ANOVA compares measurement result with being corrected to.

Hippocampal protein de novo synthesis: horizontal hippocampal slices (400 μm) knocks out and WT mice available from 6 weeks large Fmr1.Protein synthesis measures the mensuration (surface sensing (surfacesensingoftranslation of translation of use as discussed previously based on on-radiation fluorescence-activated cell sorting, SUnSET) method) carry out, its permission is monitored and quantizes overall protein synthesis (Hoeffer, 2011) in independent mammalian cell and foreign cell group.The metadoxine concentration used in this research is 300 μMs.

Embodiment 2: in the Fmr1 knock-out mice model of fragile X mental retardation, metadoxine (100 to 200mg/kg) process is to learning and memory defect and biochemical abnormal effect (research 1)

Behavior analysis

Scene fear conditioning: initial experiment is tested intraperitoneal once a day and used supporting agent or the effect to scene fear conditioning in 7 days of 150mg/kg metadoxine in the group of N=10WT and Fmr1 knock-out mice.Reduce as stagnated during the testing period reflect, the Fmr1 knock-out mice through supporting agent process shows defect in study (Fig. 1, figure A (p < 0.0001)) in scene fear conditioning pattern.Metadoxine is applied in reverse learning deficits effect in Fmr1 knock-out mice, and this reverse is part, makes the animal through metadoxine process have difference (p < 0.05) with the WT animal through metadoxine process.In the group of N=10WT and Fmr1 knock-out mice, repeat this experiment study intraperitoneal once a day and use supporting agent, 100 or 200mg/kg metadoxine 7 days dose-dependent effects to scene fear conditioning (Fig. 1, figure B and figure C).In this experiment, compared with the WT mice through supporting agent process, the Fmr1 knock-out mice through supporting agent process shows study defect (p < 0.0001), reproduction first experiment.100mg/kg metadoxine produces defect and reverses (P < 0.05) in Fmr1 knock-out mice, but this is part reverse, because the Fmr1 knock-out mice through metadoxine process has difference (p < 0.0001) with the wild-type mice through metadoxine process.After with the process of 200mg/kgi.p. metadoxine, the study defect observed in Fmr1 knock-out mice reverses completely (treated Fmr1 mice has difference (P < 0.0001) with through the Fmr1 knock-out mice of supporting agent process, but with through the WT mice of metadoxine process and zero difference).In arbitrary experiment, metadoxine process to WT mice all without effect (Fig. 1, figure A-C).

Social close: visit indicated by bout as smelt, the Fmr1 knock-out mice through supporting agent process shows poor social activity close to (Fig. 2 (p < 0.0001)).Within 7 days, improve social close to (p < 0.0001 is compared to the Fmr1 knock-out mice through supporting agent process) in Fmr1 knock-out mice with the intraperitoneal process once a day of 150mg/kg metadoxine.Fmr1 knock-out mice through metadoxine process has difference (p < 0.05) with the WT mice through metadoxine process, but has the trend of the effect close to WT mice.Metadoxine process to WT mice without effect.

Y shape labyrinth is spontaneous alternately: Fig. 3, has illustrated to carry out 7 days processing once a day the spontaneous effect replaced with supporting agent or 150mg/kg metadoxine in figure A in the group of N=10WT or Fmr1 knock-out mice.Compared with the WT mice through supporting agent process, the Fmr1 knock-out mice through supporting agent process show poor spontaneous alternately (p < 0.0001).In Fmr1 knock-out mice, compared with supporting agent process, metadoxine process raising is spontaneous alternately (p < 0.0001), but the WT mice be compared to through metadoxine process, the Fmr1 knock-out mice through metadoxine process still shows defect (p < 0.01).Therefore, metadoxine produces the defect part reverse observed in Fmr1 knock-out mice.

Y shape labyrinth reference memory task: Fig. 3, figure B in illustrated in the group of N=10WT or Fmr1 knock-out mice with supporting agent or 150mg/kg metadoxine carry out 7 days process the effect that incentive reference memory is learnt once a day.Compared with the WT mice through supporting agent process, the Fmr1 knock-out mice through supporting agent process shows more unsuitable arm and enters (p < 0.0001).Compared with the Fmr1 knock-out mice through supporting agent process, metadoxine process reduces this defect (p < 0.0001), makes Fmr1 knock-out mice through metadoxine process with the WT mice through metadoxine process and zero difference.Metadoxine process to WT mice without effect.

Distinguish about the water maze of Y shape labyrinth: Fig. 3, figure C in illustrated in the group of N=10WT or Fmr1 knock-out mice with supporting agent or 150mg/kg metadoxine carry out 7 days process once a day to detest property excite spatial discrimination to learn effect.Compared with the WT mice through supporting agent process, the Fmr1 knock-out mice through supporting agent process demonstrates higher incorrect arm and enters number of times.By using metadoxine process, this defect is reduced.

The incentive alternately task in T-shaped labyrinth: illustrated in Fig. 4 in the group of N=10WT or Fmr1 knock-out mice with supporting agent or 150mg/kg metadoxine carry out 7 days process the effect that incentive alternation is remembered once a day.Compared with the WT mice through supporting agent process, the Fmr1 knock-out mice through supporting agent process shows the incubation period (p < 0.0001) of the correct arm of longer arrival.In Fmr1 knock-out mice, compared with supporting agent process, metadoxine process reduces this defect (p < 0.0001), this reverse is part, because be compared to WT mice, through the Fmr1 knock-out mice response comparatively slow (p < 0.0001) of metadoxine process.

Continuous passage: carry out 7 days process once a day the effect of the performance in continuous passage task with supporting agent or 150mg/kg metadoxine in the group of N=10WT or Fmr1 knock-out mice shown in Figure 5, and hereafter further describing.

Anxiety (incubation period of admission passage 1) and hyperkinesia (passage 2 to 4) are measured in continuous passage test effectively.Relevant with the environmental exposure become clear gradually to color with the progress of 4 from passage 1 to passage 2,3, described environment has more and more lower wall and the more and more narrow open arms more exposed.Time to spend in open arms and enters open arms instruction anxiety; Otherwise, in more open arms, spend time reflection hyperkinesia more of a specified duration.These factors allow to carry out containing the performance of a series of anxiety sample and hyperactive sensitivity tests.

Passage 1: compared with WT mice, Fmr1 knock-out mice shows to obtain comparatively anxiety (p < 0.001).Compared with the Fmr1 knock-out mice through supporting agent process, the Fmr1 knock-out mice through metadoxine process shows the improvement (p < 0.001) of anxiety aspect, makes complete normalization occurs.Not there are differences between Fmr1 knock-out mice through metadoxine process and the WT mice through metadoxine process.In addition, metadoxine process there is no effect to WT mice.

Passage 2: when compared with Fmr1 knock-out mice, WT mice shows more inactive (p < 0.0001) in passage 2.In Fmr1 knock-out mice, metadoxine process reduces hyperkinesia (p < 0.001), but hyperactive this reverse is part, because there is difference (p < 0.001) through the Fmr1 knock-out mice of metadoxine process and WT mice.Metadoxine process there is no effect to WT mice.

Passage 3: compared with WT mice, Fmr1 knock-out mice shows hyperkinesia (p < 0.0001).This hyperkinesia does not reverse by metadoxine, because the Fmr1 knock-out mice through metadoxine process is with Fmr1 knock-out mice through supporting agent process and zero difference.Metadoxine process there is no effect to WT mice.

Passage 4: compared with WT mice, Fmr1 knock-out mice shows hyperkinesia (p < 0.01).Metadoxine process reverses this hyperkinesia, shows more inactive because be compared to through the Fmr1 knock-out mice of metadoxine process with the Fmr1 knock-out mice through supporting agent process.This action-reaction normalization, because Fmr1 knock-out mice through metadoxine process is with the WT mice through metadoxine process and zero difference.Metadoxine process there is no effect to WT mice.

In a word, be undesirably limited by theory, continuous passage test display, compared with WT mice, the anxiety of Fmr1 knock-out mice and hyperkinesia increase.Metadoxine process reduces this anxiety and hyperkinesia in Fmr1 knock-out mice, and there is no effect to WT mice.

Biochemical analysis

The phosphorylation of ERK and Akt: illustrated in Fig. 6 N=5Fmr1 knock out or WT mice group in carry out 7 days with supporting agent or 150mg/kg metadoxine the process of intraperitoneal once a day to brain in the effect of overall brain phosphorylation of ERK and Akt.Phosphorylation level is assessed with the ratio of phosphorylated CREB and total ERK.This ratio increases expression ERK and is activated.Compared with contrasting with supporting agent, in the Fmr1 through supporting agent process knocks out, the phosphorylation of ERK increases (p < 0.001), abnormal ERK activation (Wang etc., 2012) that the reproduction of this effect is observed in the people's object suffering from fragile X mental retardation.Metadoxine process reduces this effect (p < 0.01), makes to be compared to the WT mice through metadoxine process and zero difference.Metadoxine to the total ERK level in the ERK phosphorylation in WT mice or any mice without effect.Compared with the WT mice through supporting agent process, in the Fmr1 knock-out mice through supporting agent process, the ratio of phosphorylation Akt and total AKT also increases (p < 0.0001).In Fmr1 knock-out mice, reduce the relative level (p < 0.01) of phosphorylation Akt with metadoxine process, make Fmr1 knock-out mice and contrast and zero difference.The total Akt level of metadoxine process to WT mice or any mice there is no effect.

Embodiment 3. knocks out evaluation (research 2) the behavior effect of metadoxine in 6 monthly age Fmr1 knock-out mices to metadoxine in fragile X mouse model at Fmr1

Scene fear conditioning: initial experiment N=10WT and Fmr1 knock out 6 the monthly age mice group in test intraperitoneal once a day and use supporting agent or the effect to scene fear conditioning in 7 days of 150mg/kg metadoxine.As during the testing period stagnate reduce reflect, compared with the WT mice (WT-V) through supporting agent process, the Fmr1 knock-out mice (KO-V) through supporting agent process shows the defect (Fig. 7 (p < 0.0001)) in study in scene fear conditioning pattern.In Fmr1 knock-out mice, metadoxine uses reverse learning deficits effect (p < 0.0001, KO-M-150 is relative to KO-V).This reverses completely, makes the KO mice through metadoxine process not have difference with the WT mice through metadoxine process.

Social activity is remembered close to social: social activity is shown in Fig. 8 close to data (initial trial 1), in figure A (smelling spy bout number of times) and figure C (smelling the spy persistent period).Social data memory (test 2, test 1 latter 24 hours) is shown in Fig. 8, in figure B (smell and visit bout number of times) and figure D (smelling the spy persistent period).These results are hereafter being discussed further.

During test 1, compared with WT mice, the display of Fmr1 knock-out mice is smelt and is visited bout number of times increase (p < 0.0001) (see Fig. 8, figure A) and smell spy persistent period reduction (p < 0.0001) (see Fig. 8, figure C).These social interaction defects and other researcheres are to the report of Fmr1 knock-out mice those (Thomas etc., 2011) unanimously.Bout number of times and persistent period is visited for smelling, the reverse using metadoxine to carry out processing the exception produced in Fmr1 knock-out mice (is p < 0.0001, KO-M-150 is relative to KO-V), make the Fmr1 knock-out mice through metadoxine process with through metadoxine process WT mice smell visit bout number of times measure in there is no difference.Meanwhile, display is smelt and is visited duration measure and remedy to some extent, but this effect is part, because Fmr1 knock-out mice still having difference (p < 0.05) compared with WT mice after metadoxine process.Metadoxine there is no effect to WT mice.These data show, the exception that metadoxine is remedied in Fmr1 knock-out mice is social close to behavior.

During test 2, compared with wild-type mice, the display of Fmr1 knock-out mice smell visit bout number of times increase and smell visit the persistent period increase (for each measuring, p < 0.0001; Be respectively Fig. 8, figure B and figure D).This reflects adjustment failure, and therefore reflects social memory impairment.Metadoxine process reduces these differences (p < 0.0001, KO-M-150 is relative to KO-V).Part to smelling the reverse visiting bout number of times, because still there are differences (p < 0.05) between the Fmr1 knock-out mice through metadoxine process and the WT mice through metadoxine process.By the reverse of metadoxine, for smelling, to visit the persistent period be completely, because do not observe difference between the Fmr1 knock-out mice through metadoxine process and the WT mice through metadoxine process.Metadoxine does not act on WT mice.These data show, it is impaired that metadoxine reduces social memory in Fmr1 knock-out mice.This reduction of calculating to social memory impairment below by social memorability (being described in embodiment 1) is illustrated:

Social memorability is defined as the persistent period of smelling and visiting bout: test 2/ test 1+2.Therefore, a memoryless example be (such as 20/ (20+20)=0.5, and memory an example be (such as 10/ (20+10)=< 0.5.

The social memorability calculated is as follows:

WT-V test 2/ test 1+ tests 2:12.4/12.4+26.8=0.3, < 0.5 and remembers

KO-V test 2/ test 1+ tests 2:325/325+24.1=0.9, memoryless

It is memoryless that WT-M test 2/ test 1+ tests 2:12.5/38.5+12.5=0.2, < 0.5

KO-M test 2/ test 1+ tests 2:12.7/28.4+12.7=0.3, < 0.5 and remembers.

The biochemical action of metadoxine in 6 monthly age Fmr1 knock-out mices

After above-mentioned performance testing, illustrated in Fig. 9 N=10Fmr1 knock out or carry out 7 days in WT mice with supporting agent or 150mg/kg metadoxine the process of ip once a day to the overall brain pERK (Fig. 9 in brain, figure A) and the effect of pAkt (Fig. 9, scheme B).Particularly, Fig. 9, figure A shows the brain level of pAkt, as viewed in experiment before, is compared to WT mice, and in Fmr1 knock-out mice, the brain level of pAkt increases (P < 0.0001).Metadoxine is used to carry out processing this increase (the p < 0.0001 having reversed brain pAkt, KO-M-150 is relative to KO-V), make the Fmr1 knock-out mice through metadoxine process there is no difference with the WT mice through metadoxine process.Fig. 9, figure B shows the brain level of pERK, as viewed in experiment before, is compared to WT mice, and in Fmr1 knock-out mice, the brain level of pERK increases (p < 0.0001, KO-M-150 is relative to KO-V).Metadoxine process has reversed this increase (p < 0.0001), makes the Fmr1 knock-out mice through metadoxine process not have difference with the WT mice through metadoxine process.

Metadoxine after intraperitoneal or oral administration to 2 the monthly age mice the effect of behavior

Figure 10 show 2 monthly age Fmr1 knock out with WT mice in use the metadoxine effect to scene fear conditioning in 7 days once a day with the dosage of 150mg/kgip or 150 and 300mg/kg per os.Particularly, the Fmr1 after Figure 10, figure A shows personal supporting agent ip and per os process knocks out the scene fear conditioning data with WT mice.For the route of administration of supporting agent, not there are differences.After carrying out supporting agent process by ip and peroral route, be compared to WT mice, Fmr1 knock-out mice shows the reduction (in either case, p < 0.0001) of sluggish behavior.Figure 10, figure B shows and carries out the effect of metadoxine process in WT mice by two kinds of route of administration.Do not observe effect.Figure 10, figure C display, ip150mg/kg in Fmr1 knock-out mice and per os 150 and the 300mg/kg metadoxine process viewed sluggish behavior in Fmr1 knock-out mice that reversed alleviate (being respectively p < 0.01, p < 0.0001 and p < 0.0001, for KO-M-ip, KO-M-po150 and KO-M-po300 relative to KO-V-ip and KO-Vpo).The effect of using 150mgpo metadoxine and the effect of using 300mg/kgpo metadoxine do not have difference.In Fmr1 knock-out mice, 150 and the effect of 300mg/kg per os metadoxine and the effect of 150mg/kgip metadoxine there is no difference.In either case, reversing is all completely, because the Fmr1 knock-out mice through metadoxine process does not have difference with the WT mice through metadoxine process.

Figure 11 show Fmr1 knock out with WT mice in the dosage of 150mg/kgip or 150 and 300mg/kg per os use once a day metadoxine 7 days to social close to and the effect of social memory.Particularly, Figure 11, figure A show Fmr1 knock out or in WT mice the metadoxine of supporting agent or 150mg/kgip or 150 and 300mg/kg per os to the effect of social activity close to behavior.After carrying out ip or per os process with supporting agent, compared with WT mice, in Fmr1 knock-out mice, smell the decreased duration (being p < 0.0001) of spy behavior.The metadoxine process of any dosage to WT mice all without effect.But, the metadoxine process of 150mg/kgip, 150mg/kg and 300mg/kg per os produces viewed social activity in Fmr1 knock-out mice and reverses (being respectively p < 0.0001, for KO-M-po150 and KO-M-po300 relative to KO-Vpo) close to defect.Acting between 150mg/kg and 300mg/kg of per os metadoxine is not dose dependent.This reverse is completely, because the Fmr1 knock-out mice through metadoxine process does not have difference with the WT mice through metadoxine process.In Fmr1 knock-out mice, the effect of the effect of 150mg/kgip metadoxine and 150mg/kg per os or 300mg/kg per os metadoxine does not have difference.Figure 11, figure B shows the effect that Fmr1 knocks out or in WT mice, the metadoxine of supporting agent or 150mg/kgip or 150 and 300mg/kg per os is remembered social activity.After carrying out ip or per os process with supporting agent, compared with WT mice, in Fmr1 knock-out mice, smell the duration extension (being p < 0.0001) of spy behavior.The metadoxine process of any dosage to WT mice all without effect.But, the metadoxine process of 150mg/kgip, 150mg/kg per os and 300mg/kg per os produces viewed social activity in Fmr1 knock-out mice and reverses (being respectively p < 0.0001, < 0.05 and p < 0.01, for KO-M-ip150, O-M-po50 and KO-M-po300 relative to KO-V-ip and KO-Vpo) close to defect.This reverse is completely, because the Fmr1 knock-out mice through metadoxine process does not have difference with the WT mice through metadoxine process.In Fmr1 knock-out mice, the effect of the effect of 150mg/kgip metadoxine and 150mg/kg per os or 300mg/kg per os metadoxine does not have difference.In addition, there is not dose dependent in acting between 150mg/kg and 300mg/kg of per os metadoxine process.

In 2 monthly age mices metadoxine after intraperitoneal or oral administration to the effect of biochemical marker

Periphery lymphocyte: Figure 12 shows as by measured by flow cytometry, 2 monthly age Fmr1 knock out with WT mice in use metadoxine 7 days once a day to lymphocyte pAkt (Figure 12 with the dosage of 150mg/kgip or 150mg/kg and 300mg/kg per os, figure A) and the effect of pERK (Figure 12, scheme B).Particularly, Figure 12, figure A display: compared with accepting the WT mice of equivalent supporting agent process, the Fmr1 knock-out mice through supporting agent process shows the lymphocyte Akt phosphorylation (p < 0.0001, for ip and oral administration) of increase.Process the overactive Akt normalization of 7 angel with metadoxine once a day with the oral dosages of 150mg/kgip or 150mg/kg or 300mg/kg, make pAkt level there is no difference between the Fmr1 knock-out mice and the WT mice accepting same treatment of metadoxine process.Figure 12, figure B display: compared with accepting the WT mice of equivalent supporting agent process, the Fmr1 knock-out mice through supporting agent process shows the lymphocyte ERK phosphorylation (p < 0.0001, for ip and oral administration) of increase.Process the overactive ERK normalization of 7 angel with metadoxine once a day with the oral dosages of 150mg/kgip or 150mg/kg or 300mg/kg, make pERK level there is no difference between the Fmr1 knock-out mice and the WT mice accepting same treatment of metadoxine process.

Brain region: Figure 13 shows and uses the effect to the pERK level in Hippocampus, prefrontal cortex and striatum in 7 days of 150mg/kg metadoxine.In all three brain regions, be compared to WT mice, in Fmr1 knock-out mice, pERK level all improves (in all cases, p < 0.0001).Compared with the Fmr1 knock-out mice through supporting agent process, in the Fmr1 knock-out mice through metadoxine process, pERK level reduces (in all cases, p < 0.0001).In Hippocampus and striatum, there is no difference between KO-M and WT-M group, show the reverse completely that ERK activates.Effect in prefrontal cortex is part, KO-V with KO-M group keeps different (p < 0.05).Metadoxine there is no effect to WT mice.

Figure 14 shows and uses the effect to the pAkt level in Hippocampus, prefrontal cortex and striatum in 7 days of 150mg/kg metadoxine.In all three brain regions, be compared to WT mice, in Fmr1 knock-out mice, pAkt level all improves (in all cases, p < 0.0001).In all three brain regions, compared with the Fmr1 knock-out mice through supporting agent process, in the Fmr1 knock-out mice through metadoxine process, pAkt level all reduces (in all cases, p < 0.0001).In all cases, between KO-M and WT-M group, all there is no difference, show the reverse completely that Akt activates.Metadoxine there is no effect to WT mice.It is relevant that brain and the phosphorylation ERT of high blood levels and the reduction of Akt and the behavior of Fmr1 knock-out mice improve result, shows that phosphorylation level is the biological marker of metadoxine processing response.

Metadoxine is to the dendron filopodium density in the primary hippocampal neurons from Fmr1 knock-out mice and ripe interaction in vitro.

Figure 15 (figure A to C) shows with 300 μMs of metadoxine process effect of 5 hours.Dendron is divided into the sections of 10 10 μm, each all based on the distance from cell space (near-end to far-end, from left to right).In sections 3, compared with the neuron from WT mice, in the neuron from Fmr1 knock-out mice, sour jujube density increases.Particularly, Figure 15, figure A shows the density of neuron filopodium.Primary hippocampal neurons display filopodium density from Fmr1 knock-out mice increases (p < 0.001).The exception reducing neuron filopodium density with 300 μMs of metadoxine process in Fmr1 knock-out mice increases (p < 0.001).Neuron from Fmr1 knock-out mice illustrates the longer (Figure 15 with immature feature, figure B (p < 0.01)) and the filopodium of narrower (Figure 15, figure C (p < 0.01)).Reverse this increase (Figure 15, figure B (p < 0.01)) of filopodium length with metadoxine process and reverse the reduction (Figure 15, figure C (p < 0.01)) of width.

Metadoxine is to the interaction in vitro of the hippocampal protein matter de novo synthesis in Fmr1 knock-out mice

Figure 16 show to knock out from Fmr1 or WT mice 400 μMs of hippocampal slices in supporting agent or 300 μMs of metadoxine process to the effect of protein basis de novo synthesis.Compared with contrasting Hippocampus with the WT through supporting agent process, through supporting agent process from the Hippocampus of Fmr1 knock-out mice in protein synthesis higher (p < 0.0001).In the Hippocampus of Fmr1 knock-out mice, metadoxine process reduces protein synthesis rate.This effect is part, because be compared to through the higher protein synthesis rate (p < 0.001) of the maintenance of the Hippocampus from WT mice of metadoxine process from the Hippocampus of Fmr1 knock-out mice.

Claims (10)

1. treatment or alleviate the method for symptom of fragile X mental retardation or relevant disease, it comprises the compositions to having the object of these needs to use to comprise metadoxine.

2. method according to claim 1, it comprises the metadoxine of the every TDD using 100mg to 3000mg.

3. method according to claim 1, wherein said metadoxine every day, every other day or weekly uses.

4. method according to claim 1, wherein said metadoxine is used with 1,2 or 3 dosage forms every day.

5. method according to claim 1, wherein said metadoxine is used with sustained release oral, and wherein said metadoxine is formulated as the combination of slow releasing form and releasing pattern immediately.

6. method according to claim 5, wherein:

A () described slow releasing form provides the sustained release of at least 8 hours of described metadoxine, and

B () wherein slow releasing metadoxine is about 60: 40 to 80: 20 with the relative scale discharging metadoxine immediately.

7. method according to claim 6, wherein slow releasing metadoxine is about 65: 35 with the relative scale discharging metadoxine immediately.

8. method according to claim 1, wherein said symptom is for study is impaired or social competence is impaired.

9. method according to claim 1, wherein said object suffers from fragile X mental retardation or autism spectrum disorder.

10. method according to claim 1, wherein said relevant disease is autism spectrum disorder.