KR20160086818A - Methods of determining response to therapy - Google Patents

Abstract

The present invention generally relates to methods for determining responses to meta-cognitive therapy in the treatment of Fragile X syndrome and other cognitive disorders. The present invention also relates to identifying individuals who will respond to meta-cognic therapy.

Description

[0001] METHODS OF DETERMINING RESPONSE TO THERAPY [0002]

Related application

This application is a continuation-in-part of U.S. Provisional Patent Application Serial No. 61 / 875,384, filed September 9, 2013, which is incorporated herein by reference in its entirety, U.S.Patent Application No. USSN 14/038258 filed September 26, 2013, Claims priority to and benefit from U.S. Provisional Patent Application Serial No. 61 / 991,351, filed on September 9th.

Field of invention

The present invention generally relates to methods for determining the response to metadoxine therapy for the treatment of Fragile X syndrome and other cognitive disorders. The present invention also relates to the identification of individual responsive to meta-bangin therapy.

As its name implies, Fragile X Syndrome (FXS) is associated with fragile sites expressed as isochromatid gaps in mid-range chromosomes at map location Xq 27.3. Fragile X syndrome is a genetic disorder caused by a mutation in the 5'-untranslated region of the vulnerable X mental retardation 1 (FMR1) gene located on the X chromosome. Mutations that cause FXS are associated with CGG repeats within the vulnerable X mental retardation gene FMR1. In most healthy individuals, the total number of CGG repeats is less than 10 to 40, with an average of about 29. In Fragile X syndrome, the CGG sequence is repeated from 200 to more than 1,000 times. If the subject has more than about 200 CGG repeats, the fragile X gene is hypermethylated, which silences the gene. As a result, the fragile X mental retardation protein (FMRP) is produced at an undifferentiated or reduced level, and the subject exhibits FXS findings.

The premutation expansion of the FMR1 gene (55-200 CGG repeats) is frequent in the general population, with an estimated prevalence of 1 in 259 females and 1 in 812 males. All mutation holders usually have normal IQ, but emotional problems such as anxiety are common. Elderly older male mutants (aged 50 years or older) develop intestinal tremor and ataxia. These motor impairments are often accompanied by progressive cognitive and behavioral problems, including memory loss, anxiety, and impaired executive function, seclusion or irritability, and dementia. This disorder was designated as vulnerable X-chromosome associated tremor / ataxia syndrome (FXTAS). In subjects with FXTAS, magnetic resonance imaging shows an increase in T2-weighted signal intensity in the medial cerebellar bridge and adjacent cerebellar white matter.

FXS distinguishes as an X-linked dominant disorder with reduced penetrance. If you have a fragile X mutation, both genders can show cognitive impairment and the severity is variable. Children and adults with FXS have varying degrees of intellectual or learning disabilities and behavioral and emotional problems, including autism-like characteristics and trends. Young children with FXS often delay developmental milestones such as learning how to sit, walk and speak. The affected child can cause frequent irritability, difficulty in attention, frequent seizures (eg, temporal lobe seizures), often very anxious, easily panic, sense hyperreach disorder, gastrointestinal disorder, language problems and strange There may be behavior, such as hand flapping and hand biting.

FXS can be diagnosed by an established genetic test performed on a sample of a subject (e.g., a blood sample, a mouth sample). The test determines whether there is a mutation or an entire mutation in the FMR1 gene of the subject based on the number of CGG repeats.

Subjects with FXS may also have autism. About 5% of all children diagnosed with autism have mutations in the FMR1 gene and also have Fragile X Syndrome (FXS). Autism spectrum disorder (ASD) is seen in about 30% of men with FXS and in about 20% of women, and an additional 30% of FXS subjects show autism symptoms without ASD diagnosis. Although mental retardation is a typical feature of FXS, subjects with FXS often exhibit autism characteristics ranging from mild to shyness, difficulty with eye contact, and social anxiety to severe to hand fluttering, hand biting, and obsessive language. Subjects with FXS exhibit other symptoms associated with autism such as attention deficit and hyperactivity, seizures, hypersensitivity to sensory stimuli, compulsive behaviors, and altered gastrointestinal function. The FMR1 mutation prevents or greatly reduces the expression of a single protein (FMRP). Brain development in the absence of FMRP is thought to cause major symptoms of FXS.

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

Currently available therapies for humans with FXS include, for example, behavior modification, and treatment with a range of medicines including antidepressants and antipsychotics (not approved by the FDA for FXS treatment). Cognitive behavior therapy has been used to improve language and socialization in individuals with FXS and autism. Recently, pharmacologic therapies using risperidone, an atypical antipsychotic drug, have been commonly used to increase non-pharmacologic options in the treatment of individuals with autism. Random placebo-controlled trials of risperidone in autistic children have demonstrated a significant improvement in the Aberrant Behavior Checklist and the Clinical Global Impressions-Improvement subscale (McCracken, JT , et al., N. Engl., J. Med., 347: 314-321 (2002)). However, adverse events include weight gain, increased appetite, fatigue, drowsiness, dizziness, and salivation. Social isolation and communication were not improved by the administration of risperidone, and adverse side effects such as extrapyramidal symptoms and movement disorders were associated with risperidone use in autistic children.

Summary of the Invention

The present invention comprises measuring the amount of phosphorylated ERK and Akt protein in a sample derived from a subject; Measuring the total amount of ERK and Akt protein in the sample; Calculating the ratio of the amount of phosphorylated ERK and Akt protein to the total amount of ERK and Akt protein; And comparing the calculated ratio to the calculated ratio from the subject not suffering from the disease, provides a method for evaluating the effectiveness of meta-cognitive therapy in subjects with fragile X syndrome or other cognitive disorders that have been treated with meta-cognition. Treatment is effective if the calculated ratio of the subject is similar to the calculated ratio for subjects not afflicted with the known disease.

In the present invention, the amount of phosphorylated ERK and Akt protein in the sample derived from the subject is also measured; Measuring the total amount of ERK and Akt protein in the sample; Calculating the ratio of the amount of phosphorylated ERK and Akt protein to the total amount of ERK and Akt protein; There is provided a method of determining whether a subject with Fragile X syndrome or other cognitive impairment will benefit from meta-cognitive therapy by comparing the calculated ratio of the subject to a calculated ratio measured from a subject not suffering from the disease. If the calculated ratio of the subject is higher than the calculated ratio of the subject not suffering from a known disease, the subject will benefit from meta-cognitive therapy.

Further, in the present invention, the amount of phosphorylated ERK and Akt protein in the first sample from the subject in the first period is measured; Measuring the total amount of ERK and Akt protein in the first sample in the first period; Calculating a first ratio of the amount of phosphorylated ERK and Akt protein to the total amount of ERK and Akt protein; Measuring the amount of phosphorylated ERK and Akt protein in the second sample from the subject in the second period; Measuring the total amount of ERK and Akt protein in the second sample in the second period; Meta-cognitive therapy therapy is monitored in subjects with fragile X syndrome or other cognitive impairment by calculating a second ratio of the amount of phosphorylated ERK and Akt protein to the total amount of ERK and Akt protein, and comparing the first versus second ratio Is provided. When the second ratio is lower than the first ratio, the treatment is effective.

In some aspects, the measuring step comprises an immunoassay. In some embodiments, the sample is whole blood or a fraction thereof. In some embodiments, the sample is peripheral blood mononuclear cells (PBMC). In some embodiments, the PMBC is a lymphocyte or monocyte.

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

Other features and advantages of the invention will be apparent from and elucidated with reference to the following detailed description and claims.

Figure 1 shows the effect of vehicle (V) or metadoxine (M) (100, 150, or 200 mg) on 2 month old Fmr1 knockout (KO) or wild type (WT) mice for contextual fear conditioning / kg) once a day for seven days. Specifically, panel A shows the effect of vehicle or metadocin at 150 mg / kg. Panel B shows the effect of vehicle or 100 mg / kg metadoxine. Panel C shows the effect of vehicle or 200 mg / kg metadoxine. The data presented are mean ± standard error (sem) and N = 10 mice / group. ^* p <0.05, ^**** p <0.0001, NS = not significant.
Figure 2 shows the effect of intraperitoneal administration once daily for 7 days of vehicle (V) or 150 mg / kg metadoxine (M) in 2 month old Fmr1 null out (KO) or wild type (WT) Lt; / RTI &gt; The data presented are mean ± sem, and N = 10 mice / group. ^* p < 0.05 and ^**** p < 0.0001.
FIG. 3 is a graph showing the results of a Y-maze spontaneous change (panel A), a Y-type maze-compensated change (panel B), or a Y-shaped maze aquarium space in a 2 month old Fmr1 null out (KO) (V) or 150 mg / kg metadoxin (M) for the differentiation (Panel C). The data presented are mean ± sem, and N = 10 mice / group. ^*** p <0.001, ^**** p <0.0001, NS = not significant.
Figure 4 shows vehicle (V) for a T-maze compensated change in a 2-month old Fmr1 null out (KO) or wild type (WT) Lt; RTI ID = 0.0 &gt; intraperitoneal < / RTI &gt; The data presented are mean ± sem, and N = 10 mice / group. ^**** p <0.0001.
Figure 5 shows the effect of vehicle (V) or 150 mg / kg metadoxine (M) on behavior in the successive alley task in a group of N = 10 wild-type (WT) or Fmr1 null out (KO) It shows the effect of treatment once a day on 7 days. The continuum of the device has progressively provided a more anxious environment for exploring the mouse. Thus, mainly downward movements evaluated anxiety. In addition, the overall activity level can also be quantified within this device.
FIG. 6 is a graph showing the total brain level of phosphorylation of ERK (indicating ERK activity) (panel A) and Akt (indicating Akt activity) (panel B) in 2 month old Fmr1 null out (KO) or wild type (V) or 150 mg / kg metadoxin (M) once daily for 7 days. The data presented are mean ± sem, and N = 5 mice / group. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001, NS = not significant.
Figure 7 shows the effect of ip administration of vehicle (V) or 150 mg / kg metadoxine (M) once daily for 7 days in 6 month old Fmr1 null out (KO) or wild type (WT) mice for environmental fear conditioning Lt; / RTI &gt; The data presented are mean ± sem, and N = 10 mice / group. ^**** p <0.0001 and ns = not significant.
Figure 8 shows that the social approach (panels A and C) and social memory (FT) in 6-month old Fmr1 dropout (KO) or wild-type (WT) mice, as measured by the number of sniffing bouts or duration of odor- (V) or 150 mg / kg metadoxine (M) ip for 7 days on the behavior (Panel B and D). The data presented are mean ± sem, and N = 10 mice / group. ^* p <0.05, ^**** p <0.0001, and ns = not significant.
Figure 9 shows vehicle (V) or 150 mg / kg < RTI ID = 0.0 &gt; meta < / RTI &gt; for 7 days for the entire brain level of ERK (Panel A) and Akt (Panel B) phosphorylation in 6 month old Fmr1 null out (KO) or wildtype It shows the effect of single dose ip administration of single (M). The data presented are mean ± sem, and N = 10 mice / group. ^* p <0.05, ^** p <0.01, ^**** p <0.0001, and ns = not significant.
Figure 10 is a graph showing the effect of 150 mg / kg of metadoxine (M) ip, or vehicle (V) once daily for 7 days for environmental fear conditioning in 2 month old Fmr1 null out (KO) or wild type And 300 mg / kg of metadoxine (po). The data presented are mean ± sem, and N = 10 mice / group. Specifically, Panel A shows ip and oral treatment with vehicles in Fmr1 fallout and wild type mice. Panel B shows ip and oral treatment with metadoxine in wild-type mice. Panel C shows ip and oral treatment with metadoxine in Fmr1 null mice. ^** p <0.01, ^**** p <0.0001, and ns = not significant.
Figure 11 shows vehicle (V) for 7 days for social access (Panel A) and social memory (Panel B) in 2 month old Fmr1 null out (KO) or wild type (WT) (M) once daily ip or oral administration (po). The data presented are mean ± sem, and N = 10 mice / group. ^** p <0.01, ^**** p <0.0001, and ns = not significant.
Figure 12 shows vehicle (V) for lymphocyte biomarker for 7 days or 150 or 300 mg / kg for 7 days when measured using flow cytometry in 2 month old Fmr1 null out (KO) and wild type (WT) The effect of ip or oral administration (po) once a day on metadoxin (M) is shown. The biomarkers presented are Fmr1 dropout and pAkt (panel A) and pERK (panel B) in wild type mice. The data presented are mean ± sem, and N = 10 mice / group. ^**** p <0.0001, and ns = not significant.
Figure 13 shows the effect of ip (once daily for vehicle (V) or 150 mg / kg metadoxine (M) ip for 7 days on the pERK level in the brain area of 2-month old wild-type (WT) and Fmr1 null out Lt; / RTI &gt; The analyzed regions were hippocampus (Panel A), pre-frontal cortex (Panel B), and striatum (Panel C) in Fmr1 fallout or wild type mice. The data presented are mean ± sem, and N = 10 mice / group. ^**** p <0.0001, and ns = not significant.
Figure 14 shows the effect of ip administration of vehicle (V) or 150 mg / kg metadoxine (M) once daily for 7 days on pAkt levels in the brain regions of 2-month old wild type (WT) and Fmr1 null out Show effects. The analyzed regions were hippocampus (Panel A), prefrontal cortex (Panel B), and striatum (Panel C) in Fmr1 fallout or wild type mice. The data presented are mean ± sem, and N = 10 mice / group. ^**** p <0.0001, and ns = not significant.
Figure 15 is a plot of the filopodia density (panel A), length (panel B), and width (panel C) in neuron hippocampal cultures from Fmr1 null out (KO) or wildtype (WT) 5 hours treatment with vehicle (V) or 300 [mu] M metadoxin (M). The data presented are mean ± sem (wild type, N = 20 neurons, and Fmr1 null out mice, N = 20 neurons). ^** p <0.01, ^*** p <0.001, and ns = not significant.
Figure 16 shows the results of a test using a vehicle (V) or 300 μM metadoxine (M) for de novo protein synthesis based on 400 μM hippocampal slice from Fmr1 null out (KO) or wild type (WT) It shows my treatment effect. The data presented are mean ± sem, and N = 6 intercepts / group. ^* p &lt; 0.001 and ^**** p &lt; 0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification of biomarkers associated with responses to Meta-cognitive therapy of individuals with Fragile X Syndrome (FXS) and other cognitive disorders. Specifically, meta-cognition therapy has been found to bring the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in the subject sample closer to normal ratios. Normal ratio means the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein found in normal (i.e., not diseased) subjects. It has also unexpectedly been found that alterations in the phosphorylated ERK and Akt protein to total ERK and Akt protein ratios can be detected in the blood.

Thus, the present invention provides a method for monitoring a subject undergoing metacortar treatment for FXS or other cognitive impairment by determining the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in a subject sample. The ratio is compared to the ratio obtained from the control ratios, e. The ratio of subjects similar to the normal control ratios indicates that the treatment is efficacious.

In addition, the present invention provides a method for selecting a subject with cognitive impairment that will benefit from meta-cognitive therapy by determining the ratio of phosphorylated ERK or Akt protein to total ERK and Akt protein in a subject sample. The ratio is compared to the ratio obtained from the control ratios, e. The ratio of subjects greater than the normal control ratio indicates that the subject can benefit from meta-cognitive therapy. On the other hand, subjects who do not have a greater ratio than the normal control ratio may not benefit from meta-cognitive therapy.

The calculation of the ratio is described herein as one way, but it should be understood that it includes calculating the inverse as would be apparent to one of ordinary skill in the art. Also, the calculation of the ratios described herein is useful when providing a useful relative number, while the calculation of the absolute difference between the phosphorylated ERK and Akt protein and the total ERK and Akt protein levels, as well as between the test and control subjects, And may be used effectively to practice the present invention.

Justice

"Accuracy" means the degree of agreement with the actual (or true) value of the measured or calculated amount (the reported value of the test). Clinical accuracy is related to the ratio of true performance (true positive (TP) or true negative (TN)) to misclassified performance (false positive (FP) or false negative (FN)), positive predictive value (PPV) Prediction value (NPV), or probability, odds ratio, among other measures.

"Biomarkers" in the context of the present invention include, but are not limited to, proteins, nucleic acids, and metabolites, polymorphisms, mutations, variants, modifications, subunits, fragments, protein- ligand complexes and degradation products, protein- , Urea, related metabolites, and other analytes or sample-derived scales. The biomarker may also comprise a mutated protein or a mutated nucleic acid. Biomarkers also include non-blood or non-analyte physiological markers of health, such as the "clinical parameters" as defined herein, and also the "traditional laboratory risk factors" The biomarker also includes any one or more of the above measurement combinations, including any computed index or temporal trend and difference mathematically generated. Where available, biomarkers that are gene products, unless otherwise specified herein, are assigned by the International Genome Organization Naming Committee (HGNC) and are provided by the US National Center for Biotechnology Information (NCBI) website on the basis of official letter abbreviations or genetic symbols posted at the filing date of the present application.

"Clinical indicator" is any physiological data used alone or in conjunction with other data in assessing the physiological state of a population of cells or organisms. This term includes pre-clinical indicators.

"Clinical parameters" include all non-sample or non-analyte biomarkers or other characteristics of a subject's health status, such as, but not limited to, Age, Ethnicity (RACE), Sex, ).

"FN" is a false negative, meaning that the disease entity is incorrectly classified as non-disease or normal in the disease state test.

"FP" is a false positive, meaning that the disease state test incorrectly classifies a normal subject as having a disease.

&Quot; Formulas, "" Algorithms, "or " models" refer to any arbitrary number of terms that are sometimes referred to as "exponent" or "exponent values", through one or more contiguous or categorical inputs An equation, an algorithm, an analysis or a programmed process, or a statistical technique. Non-limiting examples of "formulas " include sum, ratio, and regression operators such as coefficients or exponents, biomarker value conversion and standardization (including but not limited to clinical parameters such as sex, age, Based standardized methods), rules and guidelines, statistical classification models, and neural networks trained for the historical population. Particularly interesting in the construction of panels and combinations is the use of structured and synergistic statistical classification algorithms and risk index construction methods using pattern recognition features such as established techniques such as cross- correlation, PCA, factor rotation, LogReg, linear discriminant analysis (LDA), Eigenengine linear discriminant analysis (ELDA), Support Vector Machine SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), and other related decision tree classification techniques, in particular Shrunken Centroid (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Tree, Neural Network, Bayesian Networks, Support Vector Machine, and Hidden Markov Model. Other techniques, including the well-known Cox, Weibull, Kaplan-Meier and Greenwood models known to the ordinary artisan, can be used for survival and time to event risk analysis. Many of these techniques may be usefully combined with selection techniques, such as forward selection, backwards selection, or stepwise selection, a complete listing of all potential panels of the presented size, genetic algorithms, A biomarker selection method within the technology. They may use information criteria, such as Akaike's Information Criterion (AIC) or Bayesian &lt; (R) &gt;, to quantify the tradeoff between additional biomarkers and model improvement and to help minimize overfitting, May be associated with the Bayes Information Criterion (BIC). The resulting predictive model may be validated in other studies or may be applied to techniques such as Bootstrap, Leave-One-Out (LOO) and 10-fold cross-validation (10-fold CV) They can be cross-validated in their original trained studies. At various stages, the false discovery rate can be estimated by value permutation according to techniques known in the relevant art. A "health economic utility function" is an expression derived from a combination of expected probabilities of various clinical outcomes in the applicable patient population, which is ideal both before and after the introduction of the diagnostic or therapeutic intervention into the treatment standard. This is an estimate of the accuracy, validity and performance characteristics of the intervention, and the estimated acceptable value per quality-of-life (QALY) leading to actual health system costs (such as services, goods, equipment and drugs) and / (Usefulness) associated with each performance that can be derived from the cost and / or value measurements. Over all predicted outcomes, the sum of the product of the predicted population sizes for performance multiplied by the expected utility of each performance is the total health and economic utility of the proposed treatment standard. The difference between (i) the total health and economic utility calculated for a treatment standard in the presence of an intervention versus (ii) the total health and economic availability for a treatment standard without intervention is the total The scale is presented. This can be divided between the entire patient population (or only between intervention groups) that is analyzed to reach a cost per unit of intervention and derive the decision as an estimate of market position, pricing, and health system tolerance. The health economic usability function is typically used to compare the cost-effectiveness of an intervention, but it is also an acceptable value per QALY that the health care system is willing to pay, or an acceptable cost that requires new intervention. Lt; / RTI &gt;

For the diagnostic (or prognostic) intervention of the present invention, each economic outcome (which may be TP, FP, TN, or FN in a disease classification diagnostic test) is at a different cost, Another measure of health economic performance and value that may be more sensitive than specificity, or preferentially PPV over NPV, based on individual performance costs and values, and therefore may differ from a more direct clinical or analytical performance measure to provide. These different measures and relative equilibrium maintenance will generally converge only in the case of a complete test of error rate 0 (predicted objectivity and error classification of zero or FP and FN), and all performance measures are different but more favorable than imperfect conditions will be.

&Quot; Measuring "or" measuring ", or alternatively, "detecting" or "detecting" refers to the presence, absence, quantity, or amount of a substance, including induction of a qualitative or quantitative concentration level of a given substance in a clinical or object- Means an evaluation of the amount (which may be an effective amount) or an evaluation of the value or classification of a non-analyte clinical parameter of the subject.

The "negative predictive value" or "NPV" is calculated by the true speech fraction of TN / (TN + FN) or all the speech test results. It is also essentially influenced by the disease prevalence and pre-test probability of the population for which the trial is intended. For example, a test, for example, O'Marcaigh AS, Jacobson RM, "Estimating The Predictive Value Of A Diagnostic Test, How To &quot;, discussing the specificity, sensitivity, Prevent Misleading Or Confusing Results, "Clin. Ped. 1993, 32 (8): 485-491). Often, for binary disease status classification schemes that use continuous diagnostic test measurements, sensitivity and specificity can be determined by the method described in Pepe et al., &Quot; Limits of the Odds Ratio in Performance of a Diagnostic, Prognostic, or Screening Marker, "Am. (Or black) cut point with an area under the curve (AUC) or only a single value, summarized by the receiver operating characteristic (ROC) curves according to J. Epidemiol 2004, 159 (9): 882-890. Statistics, which is an index that allows the presentation of tests, assays, or the sensitivity and specificity of the method over its entire range. Also, for example, literature [Shultz, "Clinical Interpretation Of Laboratory Procedures," chapter 14 in Teitz, Fundamentals of Clinical Chemistry, Burtis and Ashwood (eds.), 4 th edition 1996, WB Saunders Company, pages 192-199] ; And Zweig et al., "ROC Curve Analysis: An Example Showing The Relationships Among Serum Lipid And Apolipoprotein Concentrations In Identifying Subjects With Coronary Artery Disease," Clin. Chem., 1992, 38 (8): 1425-1428. Other ways of using probability functions, multiplication ratios, information theory, predictive values, calibration (including goodness-of-fit), and reclassification measurements are described in Cook, "Use and Misuse of Receiver Operating Characteristic Curve in Risk Prediction, "Circulation 2007, 115: 928-935. Finally, the hazard ratio and absolute and relative risk ratios within the subject cohort defined by the test are additional measures of clinical accuracy and usefulness. A number of methods are frequently used to define abnormal or diseased values, including reference limits, discrimination limits, and risk thresholds.

"Analytical accuracy" means the reproducibility and predictability of the measurement process itself, and can be determined by measuring the same as the coefficient of variation, and testing the matching and calibration of the same sample or control using different times, users, devices and / Can be summarized. These and other considerations in the evaluation of new biomarkers are also summarized in the literature [Vasan, 2006].

"Performance" refers to the overall performance of a diagnostic or prognostic test, including, in particular, clinical and analytical accuracy, other analytical and process characteristics such as the use characteristics (e.g., stability, ease of use), health economic value, It is a term related to usability and quality. Any of these factors may be a source of good performance, and thus usefulness of the test, and may be measured by appropriate "performance measures ", such as the associated AUC, result acquisition time,

The "positive predictive value" or "PPV" is calculated by the true positive fraction of TP / (TP + FP) or all positive test results. This is essentially influenced by the disease prevalence and pre-test probability of the population for which the trial is intended.

In the context of the present invention, "risk" refers to the probability that an event will occur over a certain period of time, such as a response to treatment, and may mean an "absolute" or "relative" risk of the subject. The absolute risk can be measured with reference to the actual observation after the measurement for the relevant time cohort, or by reference to the exponent value resulting from a statistically valid historical cohort whose progress has been observed for the relevant time period. Relative risk refers to the absolute risk of a low-risk cohort or the ratio of the absolute risk of an object compared to the mean group risk, which may vary depending on the method of assessing the clinical risk factors. The multiplication ratio, which is the ratio of positive to negative events for the proposed test results, is also commonly used without conversion (the multiplication ratio follows the formula p / (1-p), where p is the probability of the event and -p) is the probability that an event will not occur.

In the context of the present invention, "risk assessment" or "assessment of risk" includes predicting the probability, multiplication, or likelihood that an event or disease condition may occur, The risk assessment may also include the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of FXS, either in absolute or relative terms relative to previously measured populations. The methods of the present invention can be used for performing continuous or categorical measurements of response to treatment and thus for diagnosing and defining a risk spectrum of a subject category defined as a respondent or non-respondent. In a categorical scenario, the present invention can be used to identify between normal and other target cohorts at a higher risk for response.

In the context of the present invention, a "sample" is a biological sample isolated from a subject, including but not limited to whole blood, serum, plasma, CSF, brain cells, or any other secretion, . &Lt; / RTI &gt; A "sample" may include a single cell or a plurality of cells or a fragment of a cell. The sample is also a tissue sample. The sample is or contains a brain cell or a lymphocyte. Preferably, the sample is a peripheral blood mononuclear cell, such as a lymphocyte or monocyte.

"Sensitivity" is calculated by TP / (TP + FN) or by the true positive fraction of the disease subject.

"Specificity" is calculated by the true negative fraction of TN / (TN + FP) or non-disease or normal subject.

A "statistically significant" means greater than a change can only be expected to happen by chance (which may be "false positives"). Statistical significance can be determined by any method known in the art. A commonly used significance measure includes a p-value indicating the probability of obtaining an extreme result by at least a given data point, assuming that the data point is only a coincidence result. The results are considered to be highly significant at p-values less than 0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.

"Subject" in the context of the present invention is preferably a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a pet, and is not limited to these examples. Other mammals other than humans may be usefully used as subjects to represent animal models of FXS. The subject may be male or female. The subject is suspected of, or is present, FXS or other cognitive disorders.

"TN" is true speech, meaning to correctly classify non-diseases or normal subjects in disease state testing.

"TP" is true positive, meaning accurate classification of a disease subject in a disease state test.

"Traditional experimental risk factors" correspond to biomarkers isolated or derived from a sample of the subject and currently being evaluated in clinical laboratories and used in conventional overall risk assessment algorithms. Other traditional experimental risk factors for fragile X are known to those of ordinary skill in the relevant art.

The method of the present invention

The methods disclosed herein are used for subjects undergoing meta-cognitive therapy and / or therapy for FXS and other cognitive disorders, and subjects diagnosed with FXS and other cognitive disorders.

The methods of the present invention are useful for monitoring treatment of FXS and other cognitive disorders in a subject and for selecting subjects to benefit from metacortin therapy.

In general, the signs and symptoms of FXS fall into the following five categories: intelligence and learning; Physical, social and emotional, speech and language, and perceptual disorders commonly associated with fragile X or sharing features with fragile X. For example, individuals in which FXS exists have susceptibility to impaired intellectual function, social anxiety, speech disturbances, and personality traits.

Cognitive disorders include a group of disorders in which dysfunction / impairment of mental function constitutes a central symptom. Cognitive disorders include neurogenetic cognitive disorders or behavioral cognitive disorders.

Cognitive disorders include developmental disorders, attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, Alzheimer's disease, schizophrenia and cerebrovascular disease.

Autism spectrum disorders and autism symptoms are generally associated with individuals with fragile X syndrome. Signs or symptoms of autism include significant language delay, social and communication problems, and strange behaviors and concerns. Many people with autism disorders also have intellectual disabilities.

Determination of the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in a subject sample allows monitoring of the course of treatment of FXS or other cognitive disorders. In the method, the biological sample is provided from a subject undergoing treatment. If desired, biological samples are obtained from the subject at various times before, during, or after treatment. The ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein is then calculated and compared to the control value. Control values are control individuals or populations whose ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein is known or indexed. A reference sample or index value may be derived or derived from one or more individuals that are not onset (e. G., Without FXS or other cognitive impairment). Alternatively, the reference sample or index value may be obtained or derived from the subject prior to treatment. For example, a sample may be collected after a subsequent treatment from an untreated subject to monitor the progress of the treatment. The reference sample or index value may be obtained or derived from the subject after the initial treatment. For example, a sample may be collected from an initial therapy and a subject receiving subsequent therapy for FXS to monitor the progress of the treatment.

In another embodiment, the reference value is an exponent value or a baseline value. The exponent value or baseline value is a composite sample of the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein from individuals not suffering from FXS or other cognitive impairment.

The effectiveness of treatment can be monitored by determining the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in the sample over time and comparing the ratios. For example, the first sample may be obtained before the subject is treated, and one or more subsequent samples may be taken after or during treatment of the subject.

"Efficacy" means that the treatment makes the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein similar to the corresponding ratio from subjects without FXS or other cognitive impairment. Efficacy may be determined with any known method for diagnosis, identification, or treatment of FXS.

The phosphorylated ERK and Akt and total ERK and Akt protein may be determined by any method known in the art, e.g., immunoassays.

The performance and accuracy measures of the present invention

The performance and thus the absolute and relative clinical utility of the present invention can be evaluated in a number of ways as described above. The accuracy of a diagnostic, prognostic, or prognostic test, assay, or method is based on the ratio of phosphorylated ERK and Akt protein versus total ERK and Akt protein, and tests to distinguish between subjects that are responsive to meta- , Or the ability of the method. The difference in the ratio between normal and abnormal is preferably statistically significant.

Therefore, when assessing the accuracy and usefulness of the proposed medical tests, tests, or methods for assessing the condition of a subject, always consider sensitivity and specificity, and sensitivity and specificity may be significantly different across the range of the interception point It should be noted that sensitivity and specificity are reported at any interception point. The use of statistical, including AUC, including all potential cut-off point values is desirable for most categorical risk measures using the present invention, while for continuous risk measures, the observed results or other gold standard Statistics of fit and calibration are desirable.

Using the above statistics, the "acceptable degree of diagnostic accuracy" is defined herein as an AUC (area under the ROC curve for test or assay) of at least 0.60, preferably at least 0.65, more preferably at least 0.70, 0.75, more preferably at least 0.80, and most preferably at least 0.85.

A "very high degree of diagnostic accuracy" means that the AUC (area under the ROC curve for test or assay) is at least 0.80, preferably at least 0.85, more preferably at least 0.875, preferably at least 0.90, more preferably at least 0.925 , And most preferably at least 0.95.

The predictive value of any test is determined by the sensitivity and specificity of the test, and the prevalence of the condition within the population being tested. Based on Bayes' theorem, the concept suggests that the greater the likelihood that the screened condition exists in an individual or group (the pre-test probability), the greater the likelihood that the test will be more effective and the outcome will be genuine . Thus, the problem with using a test in any population with a low likelihood of a condition is that the positive outcome has a limited value (ie, it is likely to be false positives). Similarly, in very high risk groups, the results of the negative test are more likely to be false negative.

As a result, the ROC and AUC are related to the clinical usefulness of the test in a test population of low disease prevalence (incidence less than 1% per year (incidence), or cumulative prevalence of less than 10% over a certain period of time) It can be misleading. Alternatively, the absolute risk and relative risk ratio specified elsewhere in this disclosure may be used to determine the degree of clinical utility. The population of subjects being tested is also classified as quartiles by test measures, where the upper quartiles (25% of the population) comprise the group of subjects with the highest relative risk for treatment non-responsiveness, The lower quartiles include the group of subjects with the lowest relative risk for treatment non-responsiveness. In general, values derived from tests or tests that have a relative risk greater than 2.5 times from the upper quartile to the lower quartile in the low prevalence group are considered "high degree of diagnostic accuracy" Having a seven-fold relative risk is considered to be "a very high degree of diagnostic accuracy." Nevertheless, values derived from tests or tests that have a relative risk of only 1.2 to 2.5 times for each quartile are clinically useful and widely used as risk factors for disease; This is also the case for many cholesterol and many inflammatory biomarkers for predicting future events. Often, the lower diagnostic accuracy test should be combined with additional parameters to derive a meaningful clinical threshold for therapeutic intervention, as is done using the above-mentioned overall risk rating index.

The health economic utility function is another means of measuring the performance and clinical value of the proposed test, which is based on weighting the potential categorical test performance based on the actual scale of each clinical and economic value. The health economic performance is closely related to the accuracy because the health economic usability function specifically assigns economic value to the benefit of the correct classification and the cost of the error classification of the tested object. As a performance measure, it is not uncommon to require testing to achieve a performance level that leads to an increase in the health economic value per test (before test cost reduction) exceeding the target value of the test.

Construction of clinical algorithm

Any formula may be used to combine the results into an index useful for the practice of the present invention. As indicated above, and without limitation, the index may represent probability, likelihood, absolute or relative opportunity to respond to metadoxine among various other indicators. This may be for a particular time period or time zone, or for residual lifetime risk, or simply as an exponent relative to another set of reference objects.

While various preferred formulas are described herein, various other models and formulation types are well known to those of ordinary skill in the art and other than those mentioned herein. The actual model type or formula used may itself be selected from the field of potential models based on the performance and accuracy characteristics of the results in the training population. The preferred formulas include the use of a broad class of statistical classification algorithms, and in particular identification analysis. The goal of the identification analysis is to predict the number of class members from the previously identified set of attributes. In the case of linear discriminant analysis (LDA), a linear combination of attributes that maximize separation in the group is identified by some criterion. Attributes can be identified for LDA through a unique gene based approach using a stepping algorithm based on different threshold values (ELDA) or multivariate ANOVA (MANOVA). Based on the Hotelling-Lawley statistics, it is possible to perform forward, backward, and step-by-step algorithms that minimize the probability of non-separation.

Dedicated gene-based linear discriminant analysis (ELDA) is described in Shen et al. (2006)]. The formula selects attributes (eg, biomarkers) in the multivariate framework using modified eigenvalue analysis to identify attributes associated with the most important eigenvectors. "Important" is defined as an eigenvector describing most of the variation of the differences between the samples to be classified for some thresholds.

A support vector machine (SVM) is a classification scheme that attempts to find a hyperplane that separates two classes. The hyperplane includes a support vector, a data point that is exactly a margin distance from the hyperplane. In possible events where the separating hyperplane is not present in the current dimension of the data, the dimensionality is greatly expanded by taking the nonlinear function of the original variable and projecting the data to a larger dimension (Venables and Ripley, 2002). Although not required, filtering of attributes to SVMs often improves prediction. Attributes (e.g., biomarkers) can be verified against the support vector machine using a non-parameter Kruskal-Wallis (KW) test to select the optimal univariate attribute. Random forests (RF, Breiman, 2001) or iterative partitions (RPART, Breiman et al. 1984) can also be used in combination to identify distinct or most important biomarker combinations. Both KW and RF require that many attributes be selected from the whole. RPART generates a single classification tree using a subset of available biomarkers.

 Other formulas can be used to pre-process the results of individual phosphorylation of ERK and / or Akt measurements into more valuable information forms before applying them to the predictive formula. In particular, standard mathematical conversions, such as normal distribution or other distribution locations with respect to the mean value of a population using logarithmic or logistic functions, are well known to those of ordinary skill in the art of standardizing biomarkers. A set of standardizations based on clinical parameters, such as age, gender, race, or gender, is of particular interest, wherein a particular formula is used only for a subject within a class or continuously combines clinical parameters as input. In other cases, the analyte-based biomarker may later be combined with the calculated variables presented in the formula.

In addition to the individual parameter values of one potentially normalized object, the overall predictive formula for all objects, or for any known class of objects, can be found in D'Agostino et al. (2001) JAMA 286: 180-187 , Or other similar standardization and re-calibration techniques, may be recalibrated or otherwise adjusted itself based on adjustments to the expected prevalence and average biomarker parameter values of the population. The epidemiological adjustment statistic may be stored either in a machine-readable or otherwise readable manner, through the registration of historical data presented in the model, or in a retrospective query Captured, verified, improved, and updated over the network. A further example, which may be the subject of formal recalibration or other adjustments, is Pepe, M. S. et al., 2004; Includes the statistics used in the study by Cook, N. R., 2007 on ROC curves. Finally, the numerical results of classifier formulas can be used to correct absolute risks and to establish confidence intervals for the various numerical results of classifiers or risk formulations, Lt; / RTI &gt; Examples of this are derived using actual clinical studies and are described in Genomics Health, Inc. And the confidence interval for this risk, chosen for the output of the recall score formula in the Oncotype Dx product of Genomic Health, Inc. (Redwood City, CA, USA). A further variant is to adjust the smaller sub-populations of studies defined and selected by their clinical parameters, such as age or gender, based on the output of the classifier or risk formula.

Example

Example  1: general method

The examples described herein were carried out using the reagents and methods generally described below.

Experimental animal

Fmr1 null mouse (KO2) mice (The Dutch-Belgium Fragile X Consortium, 1994) were initially obtained from Jackson Laboratory and wild-type (WT) clones were generated in the C57BL / 6J background, Repeated crosses over C57BL / 6J background for generation excess. Fmr1 null mice were housed in a temperature and humidity control room with the same genotype group and a 12 h contrast cycle (illumination from 7 am to 7 pm; the test was performed during the fixture). Room temperature and humidity were continuously recorded in the storage room, allowing feed and water to be freely available (ad libitum). During the behavioral tests, tests were performed on healthy Fmr1 calf mice at 2 or 6 months of age and their wild-type offspring (N = 10 mice / treatment group). The mice were housed in commercial plastic cages and experiments were conducted according to the requirements of the UK Animals (Scientific Procedures Act, 1986). All the experiments were performed by the experimenter who did not know about genotype and drug treatment. Animals were allowed a minimum acclimatization period of one week prior to performing any experiment. No prophylactic or therapeutic treatment was given during the adaptation period.

drug

For Study 1 (Example 2), metadoxine was dissolved in saline and administered intraperitoneally at a dose of 100, 150, or 200 mg / kg once daily for 7 days. For study 2 (Example 3, in vivo testing), metadoxin was dissolved in saline and administered intraperitoneally at a dose of 150 mg / kg / day once daily for 7 days or at a dose of 150 or 300 mg / kg / day (0.1 ml &lt; / RTI &gt; volume). For Study 2 (in vitro study), metadoxine was administered at a concentration of 300 μM for 5 hours. In all cases, saline was used as vehicle (control).

Behavior test

Social Interaction and Social Cognitive Memory : The mouse has a variety of functions, including approaching, following, smelling, grooming, aggressive encounters, sexual interactions, parenting behaviors, nesting, and sleeping in a group huddle , A social species that participates in social behaviors that are easily scored. The social approach in mice was assessed by the duration of the smell to the new mouse.

The mouse was placed in a test stage / cage (40x23x12 cm cage, with a Perspex cover for easy viewing of the mouse) that was the same size as the adult household cage, with a fresh piece of wood on the floor. The background mouse smell was generated by placing some non-experimental mice in the apparatus prior to testing. The mice were transferred to the laboratory 10-15 min before the test. The test subject and juvenile were simultaneously placed in the test cage. The total duration of the social survey defined as sniffing and closely following (2 cm from the tail) and the number of one-time surveys for the stimulus subspecies by the tested mice were evaluated for 3 min. After 30 min, the test was repeated using the same stimulus generators. The collected data parameters were the total duration and the total number of odors taken for learning and recognition. The social memory ratio defined as Test 2 / Test 1 + 2 was derived. Therefore, no memory (for example, 20 / (20 + 20)) = 0.5, and memory (for example, 10 / (20 + 10)) = 0.5.

Y-type maze change : Two tasks were carried out. The first challenge was the non-learning assessment of voluntary changes between arm positions. The second task is a spatial reference memory task where the animal learned which of the two arms had bait of rewarding feed. On the day before training began, the mice were allowed to freely navigate the maze for 5 min. They then experimented with two tests, one for the feed on the left arm and the other for the feed on the right arm. This procedure prevented the occurrence of preference for one of the arms.

Y-type maze water tank Maze : A transparent Perspex Y-type maze was filled with 2 cm of water at 20 ° C. This synchronized the mouse to swing from the far end of one arm to the exit tube and then leave the maze. I put the maze in the middle of the room surrounded by visible visual clues.

Compensated T-type maze changes : T-shaped raised or closed devices (lying horizontally) were used. The mouse was placed on the T-shaped base and allowed to select one of the target arms adjacent to the other end of the stem. Two trials were performed consecutively: in the second test the mice were asked to select a previously unarmed arm, reflecting the memory of the first choice (spontaneous change). This tendency was enhanced by compensating for the desired feed when the animal was starved and changed. Specifically, after an adaptation period of 4 days in the T-type maze, mice were trained to change arm selection to receive sweetened concentrated milk as compensation.

Continuous Main : The device consisted of four consecutive, straight-line, incremental anxiety-provoking principals made of painted wood (each succeeding main being painted in a lighter color than the preceding one and / Low / low or narrow). Each zone or main body was 25 cm in length. Mainly 1 had a 25 cm height wall, 8.5 cm wide, and painted black. The 0.5 cm steps lead mainly to 2, which was 8.5 cm in width, but gray with a 1.3 cm height wall. The 1.0 cm step consists mainly of 3, which was 3.5 cm wide and had a 0.8 cm height wall and was white. The 0.4 cm step leads mainly to 4, which is also white, but with a width of 1.2 cm and a height of 0.2 cm. The device was elevated mainly by fixing the rear side of the 1 so that it stands at a height of 50 cm. Padding was provided beneath arms 3 and 4 for the case of a mouse crash. Each mouse was placed on the closed end of the wall facing one. 1) Total test length (5 min) + latency into each arm, and 2) a timer was started during the time spent in the main one. When the mice placed all four feet on the next week, they were considered to be in the main. The total time spent in each state (all four feet) was recorded.

Environmental fear conditioning : In a fear conditioning experiment, mice were placed in a new environment (dark room), hints and electric footshock (0.2 mA, 1 second (Study 1) or 0.7 mA, 0.5 sec (Study 2) Were paired with each other. Subsequently, when tested in the original training environment, the mice showed a natural defense reaction, called freezing (Blanchard, 1969) or environmental fear conditioning. The freezing time was defined as the time spent by the mouse for inactivity, excluding respiration. The data was expressed as a percentage of the test duration. Twenty-four hours after the training period, the mice were tested in the training room for 5 min without impact presentation and observed for freezing behavior.

Statistics: Multivariate ANOVA was used to evaluate group differences across the data. Repeat measurements ANOVA were performed on behavioral data. A statistically significant effect on each ANOVA was followed by a post hoc comparison using the Newman-Keuls test (Study 1) or the Turkish (Tukey) Test (Study 2). A p value of less than 0.05 was considered significant.

Biochemical test

The phosphorylated ERK and Akt : Ras-Mek-ERK and PI3K-Akt-mToR signaling pathways are involved in mediating activity-dependent changes in gene transcription that are the basis for changes in synaptic plasticity (Klann and Dever, 2004 ). The phosphorylated ERK and Akt protein expression was measured by western blot analysis as previously described in Lopez Verrilli et al., 2009. The antibodies used were anti-phospho-specific antibodies (Cell Signaling Technology, Danber, Mass., USA) for Akt (1/1000) and kinase (ERK) 1/2 (1/2000) Antibodies to ERK1 / ERK detect phosphorylation in phospho-ERK1 / 2 (Thr202 / Tyr204) whereas antibodies against phosphoAkt detect phosphorylation in phospho-Akt (Thr308) 2 protein content and phosphorylated ERK and Akt were measured by blotting the membrane with anti-phospho-Akt (1/1000) and anti-phospho-ERK antibody (1/2000) (Cell Signaling Technology, Danbury, Mass. Akt or ERK phosphorylation was normalized to the protein content in the same sample and expressed as% change relative to basal conditions, assuming basal level as 100%. Protein loading was done by peeling the membrane and incubating with &lt; RTI ID = (Sigma-Aldrich, St. Louis, Mo. The phosphorylated ERK and Akt protein expression in blood lymphocytes was measured by flow cytometry. For lymphocyte biomarker determination, a FACStar Plus with excitation laser adjusted to 488 nm (&lt; RTI ID = 0.0 &gt; (Becton Dickinson), and green fluorescence from FITC (GST) was collected via a 515-545 nm bandpass filter. Average FITC fluorescence intensities were calculated for the fluorescence of reference cells, The intensity (MFI) is directly proportional to the average number of Ab molecules bound per cell.

Neuronal morphology : Hippocampal cell cultures were prepared from wild-type and Fmr1 KO fetal mice of embryonic day (E17.5) of gestation 17.5. The mice were sacrificed by cervical disassembly and the isolated hippocampal cells were plated in 15 mm multiwell containers (Falcon Primaria). (Ethel and Yamaguchi, 1999); [Ethell et al., 2001], [Henkemeyer et al., 1999], and then transfected with green fluorescent protein (GFP) ., 2003]). The dendrites were formed in the test tube for about 16 days (DIV). The culture was treated with metadoxine at 300 μM for 5 hr on day 17 in vitro.

The supernatant densities of GFP transfected neurons were quantified by performing a Sholl analysis of stacked Zeiss confocal generated images (40x objective, 20x0.2 mu m stack). Using the Metamorph software, concentric circles of equal spacing (every 20 탆) were counted around the cell body of each neuron and subsequently in a circle for each circle. The mean of the coefficients was compared using unpaired two-tailed Student's T-test.

The spine maturation of GFP transfected neurons was analyzed using Metamorph software (Molecular Devices, Sunnyvale, CA). Two tip truncated sections of 70-100 ㎛ were selected per neuron for visual form analysis. For each visual, length and width were measured. The length is defined as the distance from the base to the end of the protrusion; The width was defined as the maximum distance perpendicular to the long axis of the spine. Measurements were compared using unpaired two-tailed Student T-test and ANOVA corrected for multiple comparisons.

De novo hippocampus protein synthesis : Transverse hippocampal slices (400 쨉 m) were obtained from 6 week old Fmr1 dropout and WT mice. Protein synthesis assays were performed as previously described using non-radioactive fluorescence-activated cell sort-based screening, surface sensing of translation (SUNSET) methods, which were performed in individual mammalian cells and Allowing monitoring and quantification of overall protein synthesis in heterogeneous cell populations (Hoeffer, 2011). The concentration of metadoxine used in this study was 300 μM.

Example  2: Fragile X syndrome Fmr1 Out  Mouse Model (Research 1) in  For learning and memory deficits and biochemical abnormalities Metathe  (100 units 200 mg / kg) Effect of treatment

Behavior analysis

Environmental Fear Conditioning : Initial experiments tested the effect of intraperitoneal administration of vehicle or 150 mg / kg metadoxine once daily for 7 days on environmental panic conditioning in the group of N = 10 WT and Fmr1 null out mice Respectively. Vehicle-treated Fmr1 null mice showed a lack of learning in the environmental fear conditioning paradigm as reflected by reduced freeze response during the test (Panel A (p < 0.0001) in Figure 1). Metadoxine administration reversed the learning deficit effect in Fmr1 null out mice, where the inversion was partial, and therefore the metadocin-treated animals differed from metadoxin-treated WT animals (p < 0.05). Repetition of the experiment demonstrated dose-dependent effects of intraperitoneal administration of vehicle, 100 or 200 mg / kg metadoxine once daily for 7 days for environmental panic conditioning in the group of N = 10 WT and Fmr1 null out mice (Panels B and C in Fig. 1). In this experiment, vehicle-treated Fmr1 null mice showed learning deficit (p < 0.0001) as compared to vehicle-treated WT mice, which repeats the first experiment. 100 mg / kg metadoxine caused a reversal of deficiency in Fmr1 knockout mice (P <0.05), but because metadoxin-treated Fmr1 fallout mice were different from metadoxin-treated wild type mice (p <0.0001) It was a reversal. The learning deficit seen in Fmr1 null mice was completely reversed after treatment with 200 mg / kg ip metadoxin (treated Fmr1 mice were different from vehicle-treated Fmr1 null mice (P < 0.0001), metadoxin-treated WT It did not differ from the mouse). Metadokine treatment was ineffective for WT mice in both experiments (panel AC of FIG. 1).

Social Approach : Vehicle-Treated Fmr1 dropout mice showed less social access as shown by once smelling (Figure 2 (p <0.0001)). One-day intraperitoneal treatment with 150 mg / kg metadoxine for 7 days increased social access in Fmr1 null mice (p <0.0001 compared to vehicle treated Fmr1 null mice). Fmr1 null mice treated with metadoxine were different from metadoxin treated WT mice (p <0.05) but tended to approach the effect of WT mice. Metatocarcinoma treatment had no effect on WT mice.

Y-type maze spontaneous change : N = 10 mice WT or Fmr1 The effects of the vehicle for spontaneous changes in the group of null mice or the once-a-day treatment of 7 days of 150 mg / kg meta- do. Vehicle-treated Fmr1 null mice showed less spontaneous changes than vehicle treated WT mice (p < 0.0001). Metadoxin treatment increased spontaneous changes in Fmr1 null mice compared to vehicle treatment (p <0.0001), but metadoxin-treated Fmr1 null mice were deficient compared to metadoxin-treated WT mice (p <0.01). Thus, metadoxin caused a partial reversal of the deficiency seen in Fmr1 knockout mice.

Y-type maze memory task : The effect of 7 days of once-daily treatment with vehicle or 150 mg / kg meta-cognition on compensated reference memory learning in the group of N = 10 WT or Fmr1 null out mice is shown in Figure 3 In Panel B. Vehicle-treated Fmr1 null mice showed less than adequate arm entry (p < 0.0001) than vehicle-treated WT mice. Metathexone treatment reduced the deficit (p < 0.0001) compared to vehicle-treated Fmr1 null mice, and therefore, metadoxin-treated Fmr1 null mice did not differ from metadoxin-treated WT mice. Metatocarcinoma treatment had no effect on WT mice.

Y-shaped labyrinth maze Left-hand side discrimination : N = 10 WT or Fmr1 mice in a group of naïve mice with aversion to synchronized spatial discrimination learning or once daily treatment with 7 days of 150 mg / kg meta- The effect of which is shown in Panel C of FIG. Vehicle-Treated Fmr1 knockout mice showed more inaccurate arm entries than vehicle-treated WT mice. The deficiency was reduced by treatment with metadoxine.

The modified task assigned to the T-type maze : N = 10 WT or Fmr1 The effect of once-daily treatment with 7 days of vehicle or 150 mg / kg meta-cognition on compensated change working memory in the group of null mice 4. Vehicle-treated Fmr1 knockout mice showed longer waiting times to reach the correct arm than vehicle-treated WT mice (p < 0.0001). Metadocin treatment reduced this deficit in Fmr1 knockout mice compared to vehicle treatment (p < 0.0001), since the metadoxine-treated Fmr1 knockout mice responded more slowly (p < 0.0001) than WT mice .

Continuous mainly: N = shown in Figure 5 to 10 WT or Fmr1 camel vehicle or 150 mg / kg meth 1 il effects of one treatment of 7 days with a single person for action in a continuous mainly assignment from the group of-out mice, This is explained further below.

Continuous primary testing effectively measured anxiety (latency primarily to 1) and hyperactivity (mainly 2 to 4). Progression from predominantly 1 to continuous 2, 3, and 4 was associated with exposure to an incrementally brightly colored environment with increasingly lower, narrower and more exposed open arms. Indicating time spent on the open arm and anxiety in the open arm; Conversely, the increase in time consumed in the more open arms reflected hyperactivity. These factors allowed sensitive testing to encompass a range of anxiety-like behaviors with hyperactivity.

Mainly 1 : Fmr1 knockout mice showed more anxiety than WT mice (p <0.001). Fmr1 null mice treated with metadoxine showed anxiety relief (p <0.001) as compared to vehicle treated Fmr1 null mice, thus complete normalization occurred. There was no difference between the meta-cognate-treated Fmr1 fallout mouse and the meta-cx-treated WT mice. In addition, metathexone treatment was ineffective for WT mice.

Mainly 2 : WT mice showed mainly less activity in Fmr1 null mice (p <0.0001). Treatment with metadoxine reduced hyperactivity in Fmr1 null mice (p <0.001), but the hyperactivity reversal was partial because metadoxin-treated Fmr1 null mice and WT mice were different (p <0.001). Metatocarcinoma treatment had no effect on WT mice.

Mainly 3 : Fmr1 null mice showed hyperactivity (p <0.0001) compared to WT mice. Since the metadoxin-treated Fmr1 null out mice did not differ from the vehicle-treated Fmr1 null mice, the hyperactivity was not reversed by metadoxin. Metatocarcinoma treatment had no effect on WT mice.

Mainly 4 : Fmr1 null out mice showed hyperactivity compared to WT mice (p <0.01). Metadoxin - treated Fmr1 knockout mice exhibited less activity than vehicle - treated Fmr1 knockout mice (p <0.01), so metathexone therapy reversed this hyperactivity. Since the metadoxin-treated Fmr1 knockout mice did not differ from the metadoxin-treated WT mice, the effects reflected normalization. Metatocarcinoma treatment had no effect on WT mice.

Taken together, we do not want to be bound by theory, but the sequential test showed that Fmr1 null mice outgrew anxiety and hyperactivity compared to WT mice. Metathexone treatment reduced the anxiety and hyperactivity in Fmr1 null mice without affecting WT mice.

Biochemical analysis

Phosphorylation of ERK and Akt : N = 5 Fmr1 null out for ERK or Akt whole brain phosphorylation in the brain or intraperitoneal treatment once daily for 7 days using vehicle or 150 mg / kg metadoxine in WT mice Is shown in Fig. Phosphorylation levels were assessed as the ratio of phosphorylated ERK to total ERK. The increase in the ratio indicated the activation of ERK. ERK phosphorylation was increased in vehicle-treated Fmr1 null mice (p < 0.001) than in vehicle controls - this effect replicated abnormal activation of ERK seen in human subjects with Fragile X syndrome (Wang et al., 2012) . The effect was reduced by meta-coxin treatment (p < 0.01) and was therefore not different from metadoxin-treated WT mice. Metadoxin was not effective on ERK phosphorylation in WT mice or on total ERK levels in any mouse. The ratio of phosphorylated Akt to total AKT also increased (p < 0.0001) in vehicle-treated Fmr1 null mice out of vehicle-treated WT mice. Treatment with metadoxine reduced the relative levels of phosphorylated Akt in Fmr1 null mice (p < 0.01), and thus Fmr1 null mice did not differ from the control group. Metatocortical therapies were ineffective for WT mice, or for the total Akt level of any mouse.

Example  3: Fmr1 Out  In the vulnerable X mouse model Metamorphic  Evaluation (Study 2)

6 months Fmr1 Out  From the mouse Metamorphic  Behavioral effect

Environmental Fear Conditioning : Initial experiments were performed in groups of N = 10 6-month-old WT and Fmr1 null mice with intraperitoneal administration of vehicle or 150 mg / kg metadoxine once daily for 7 days for environmental panic conditioning The effect was tested. Vehicle-treated Fmr1 dropout mice (KO-V) were significantly more effective than vehicle-treated WT mice (WT-V) as reflected by a decrease in freezing response during the test period (Figure 7 The conditioning paradigm showed a lack of learning. Metadoxin administration reversed the learning deficit effect in Fmr1 null mice (p <0.0001 KO-M-150 vs. KO-V). This was a complete reversal and, therefore, the metadoxin-treated KO mice did not differ from the metadoxin-treated WT mice.

Social Approach and Social Memory : Social access data (initial test 1) are presented in Panel A (number of odor entrapment) and Panel C (duration of stench) in Figure 8. Social memory data (test 2, 24 hours after test 1) are presented in panel B (number of odor-taking) and panel D (duration of odor-taking) These results are discussed further below.

During test 1, the Fmr1 dropout mouse showed a decrease in the number of odorants (p < 0.0001) (see panel A in Figure 8) and a decrease in the duration of deodorization (p < 0.0001) See panel C). These social interaction deficits are consistent with those reported by other researchers in Fmr1 null mice (Thomas et al., 2011). For both the number of odorants and the duration of odor entrapment, treatment with metadoxine resulted in overturning in Fmr1 null mice (p <0.0001 KO-M-150 vs. KO-V for each) Thus, the metadoxin-treated Fmr1 null mice did not differ from the metadoxin-treated WT mice in terms of the number of odorant counts. Remedies were observed for the duration of the smell, but the effect was partial because Fmr1 null mice remained different than WT mice after meta-cognition treatment (p < 0.05). Metadokinesin had no effect on WT mice. These data demonstrate that abnormal social access behaviors in Fmr1 null mice were rescued by meta-cognition.

During trial 2, Fmr1 null out mice exhibited an increase in the number of odorants and an increase in the duration of deodorization (compared to wild-type mice (p <0.0001 for each measurement, panels B and D, respectively, of Figure 8) . This reflected the failure of adaptation, and therefore, social memory deficits. Meta-cognitive therapy reduced these differences (p <0.0001 for KO-M-150 vs. KO-V). Because there was a difference between the metadoxin-treated Fmr1 null mice and the metadoxin-treated WT mice (p < 0.05), the reversal to the number of odorants was partial. Since no differences were observed between the metadoxin-treated Fmr1 null mice and the metadoxin-treated WT mice, the inversion by metadokines for the odor duration was complete. Metatocarcinoma treatment had no effect on WT mice. These data show that meta-cognition reduced social memory impairment in Fmr1 null mice. The reduction in social memory deficits is illustrated by the calculation of the social memory ratio (described in Example 1) below:

The social memory ratio was defined as the duration of odor-taking once: Test 2 / Test 1 + 2. Thus, an example of no memory (for example, 20 / (20 + 20)) = 0.5 while an example of memory (for example, 10 / (20 + 10)) = 0.5.

The calculated social memory ratios were as follows:

WT-V Test 2 / Test 1 + Test 2: 12.4 / 12.4 + 26.8 = 0.3, <0.5

KO-V Test 2 / Test 1 + Test 2: 325/325 + 24.1 = 0.9, No memory

WT-M test 2 / test 1 + test 2: 12.5 / 38.5 + 12.5 = 0.2, <0.5

KO-M Test 2 / Test 1 + Test 2: 12.7 / 28.4 + 12.7 = 0.3, <0.5

6 months Fmr1 Out  From the mouse Metamorphic  Biochemical effect

N = 10 mice Fmr1 dropout or mice in the WT mice or 150 mg / kg metadoxine for the entire brain pERK (panel A of FIG. 9) and pAkt (panel B of FIG. 9) The effect of ip treatment once a day on the used 7 days is shown in Fig. Specifically, Panel A of FIG. 9 shows the brain levels of pAkt, which increased in Fmr1 null mice out of WT mice (P < 0.0001) as shown in the preceding experiments. Treatment with metadoxine reversed this increase in brain pAkt (p <0.0001 for KO-M-150 vs. KO-V), and therefore the metadoxine-treated Fmr1 null mice responded with meta- Did not do it. Panel B of FIG. 9 shows the brain levels of pERK, which increased in Fmr1 null mice as compared to WT mice (p <0.0001 for KO-M-150 vs. KO-V), as seen in previous experiments. The increase was reversed by meta-cognate treatment (p < 0.0001), and therefore the metadocyte-treated Fmr1 null out mice did not differ from the metadoxin-treated WT mice.

2 months  About the behavior of the mouse Abdominal cavity  Or after oral administration Metamorphic  effect

Figure 10 shows the effect of metadoxine administration once daily at a dose of 150 mg / kg ip, or 150 and 300 mg / kg orally for 7 days for environmental fear conditioning in 2 month old Fmr1 fallout and WT mice . Specifically, panel A of FIG. 10 shows environmental fear conditioning data from Fmr1 dropouts and WT mice after ip with vehicle and oral treatment. There was no difference in the route of administration of the vehicle. Fmr1 null out mice showed a decrease in freezing behavior (p <0.0001 in each case) compared to WT mice after vehicle treatment with ip and oral route. Panel B of FIG. 10 shows the effect of metazohalcine treatment over two routes of administration in WT mice. The effect was not seen. Panel C of Figure 10 shows that metadoxin treatment with ip 150 mg / kg and oral 150 and 300 mg / kg in Fmr1 null mice reversed the reduction in freezing behavior seen in Fmr1 null mice (KO-M- p < 0.01, p < 0.0001, and p < 0.0001 for KO-V-ip, KO-M-po 150 and KO-M-po 300 vs. KO-V-ip and KO-V po, respectively). The effect of dosing with 150 mg po methadoxine did not differ from the effect of 300 mg / kg po metadoxine. The effects of 150 and 300 mg / kg oral metadoxine in Fmr1 null mice did not differ from the effects of 150 mg / kg ip metadoxine. In each case, the metadoxin-treated Fmr1 knockout mouse was not different from the metadoxin-treated WT mouse and the inversion was complete.

FIG. 11 shows the effect of metadoxine administration once daily at doses of 150 mg / kg ip or 150 and 300 mg / kg for 7 days for social access and social memory in Fmr1 fallout and WT mice. Specifically, panel A of FIG. 11 shows the effect of 150 mg / kg ip or 150 and 300 mg / kg oral metadoxine on social access behavior in Fmr1 null out or WT mice. After ip or oral treatment with vehicle, the duration of stinking behavior in Fmr1 knockout mice was reduced compared to WT mice (p <0.0001, respectively). An arbitrary dose of metacortice treatment was ineffective for WT mice. However, methadone therapy at 150 mg / kg ip, 150 mg / kg, and 300 mg / kg orally reversed the social access deficit seen in Fmr1 null mice (KO-M-po 150 and KO-M-po 300 P < 0.0001 for the major KO-V po), respectively. The effect of oral metadoxine was not dose-dependent between 150 and 300 mg / kg. The metadoxin-treated Fmr1 null mice did not differ from the metadoxin-treated WT mice, so the inversion was complete. The effect of 150 mg / kg ip metadoxine in Fmr1 null mice did not differ from the effects of 150 mg / kg oral or 300 mg / kg oral metadoxin. Panel B of FIG. 11 shows the effect of 150 mg / kg ip or 150 and 300 mg / kg oral metadokinesin on social memory in Fmr1 null out or WT mice. After ip or oral treatment with vehicle, the duration of smelling behavior in Fmr1 knockout mice was increased compared to WT mice (p <0.0001 for each). An arbitrary dose of metacortice treatment was ineffective for WT mice. However, methadoxine treatment at 150 mg / kg ip, 150 mg / kg oral and 300 mg / kg oral administration reversed the social access deficit seen in Fmr1 null mice (KO-M-ip 150, KO-M-po P < 0.01, p < 0.05, and p < 0.01 for KO-V-ip and KO-V po, respectively. The metadoxin-treated Fmr1 null mice did not differ from the metadoxin-treated WT mice, so the inversion was complete. The effect of 150 mg / kg ip metadoxine in Fmr1 null mice did not differ from the effects of 150 mg / kg oral or 300 mg / kg oral metadoxin. There was also no dose dependence of the effect of oral metadocyns treatment between 150 mg / kg and 300 mg / kg.

2 months  From the mouse Abdominal cavity  Or biochemical after oral administration On the marker  Effects of Meta-Single Cow

Peripheral lymphocyte : Figure 12 shows the effect of 150 mg / kg for 7 days on lymphocyte pAkt (panel A in Figure 12) and pERK (panel B in Figure 12), when determined by flow cytometry in 2 month old Fmr1 null out and WT mice ip or doses of 150 mg / kg and 300 mg / kg oral doses of metadoxine once daily. Specifically, panel A of FIG. 12 shows that vehicle-treated Fmr1 null out mice showed increased phosphorylation of lymphocyte Akt (p < 0.0001 for both ip and oral administration) compared to WT mice that received equivalent vehicle treatment. Treatment with metadoxine once daily at an oral dose of 150 mg / kg ip or 150 mg / kg or 300 mg / kg for 7 days normalized the hyperactively activated Akt and thus the pAkt levels were metadoxin-treated Fmr1 Lt; / RTI &gt; mice did not differ between the null mice and the same treated WT mice. Panel B of Figure 12 shows that vehicle-treated Fmr1 null out mice showed increased phosphorylation of lymphocyte ERK (p &lt; 0.0001 for both ip and oral administration) compared to WT mice that received equivalent vehicle treatment. Treatment with metadoxine once daily at a dose of 150 mg / kg ip, or 150 mg / kg or 300 mg / kg for 7 days, normalized the hyperactive ERK and therefore the pERK levels were meta- Lt; RTI ID = 0.0 &gt; Fmr1 &lt; / RTI &gt; null mice and the same treated WT mice.

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

Figure 14 shows the effect of administration of 150 mg / kg metadoxine for 7 days on pAkt levels in hippocampus, prefrontal cortex and striatum. pAkt levels were increased in Fmr1 null mice out of WT mice in all three brain regions (p <0.0001 in all cases). pAkt levels were reduced in meta-cognate-treated Fmr1 null mice (in all cases p <0.0001) compared to vehicle-treated Fmr1 null mice in all three brain regions. In all cases, there was no difference between KO-M and WT-M groups, indicating complete reversal of activation of Akt. Metadokinesin had no effect on WT mice. Reduced brain and blood level elevations of phosphorylated ERK and Akt are correlated with improved behavioral performance of Fmr1 null mice, suggesting that phosphorylation levels are biomarkers of meta-cognitive therapy response.

In vitro Fmr1 Out  From the mouse into the first hippocampal neuron Bifurcation  Threshold Density and On maturity  About Metamorphic  effect

15 (Panel A-C) shows the effect of treatment for 5 hours using 300 μM metadoxine. The dendrites were divided into 10 sections of 10 ㎛ each based on distance from the body (from proximal to distal, from left to right). Visible density increased in neurons from Fmr1 null mice out of WT mice compared to neurons in section 3. Specifically, panel A of FIG. 15 shows the density of the neuron threshold. Primary hippocampal neurons from the Fmr1 knockout mice showed an increase in supratentorial densities (p <0.001). Treatment with 300 μM metadoxine reduced the abnormal increase in neuronal somatosensory density in Fmr1 null mice (p <0.001). Neurons from the Fmr1 knockout mice showed a mysterious stomach with a longer immature characteristic (Panel C (p < 0.01) in Figure 15) than in the longer (Panel B (p < 0.01) Treatment with metadoxine reversed the increase in the sagittal length (Panel B in Figure 15 (p < 0.01)) and reversed the decrease in width (Panel C (p < 0.001) in Figure 15).

In vitro Fmr1 Out  Mouse de Novo  For hippocampal protein synthesis Metamorphic  effect

Figure 16 shows the effect of treatment with vehicle or 300 [mu] M metadoxine for baseline de novo protein synthesis in 400 [mu] m hippocampal slices from Fmr1 null out or WT mice. Protein synthesis was higher in vehicle-treated hippocampus from Fmr1 null mice than in vehicle-treated WT control hippocampus (p <0.0001). Metadokine therapy reduced the rate of protein synthesis in Fmr1 null mouse hippocampus. Since hippocampus from Fmr1 null mice had a higher rate of protein synthesis from WT mice than the metadoxin-treated hippocampus (p <0.001), the effect was partial.

<Table 1>

Claims (8)

a) measuring the amount of phosphorylated ERK and Akt protein in a sample derived from a subject suffering from metazohalcine-treated Fragile X syndrome or other cognitive disorders;
b) measuring the total amount of ERK and Akt protein in the sample;
c) calculating the ratio of the amount of phosphorylated ERK and Akt protein determined in step a) to the amount of ERK and Akt protein determined in step b);
d) comparing the calculated ratio of step c) to the calculated ratio from an unaffected subject
Wherein the calculated ratio of step c) is similar to the calculated ratio of the subject not suffering from a disease, wherein the meta-cognitive therapy is administered to a subject having a fragile X syndrome or other cognitive disorder, A method of assessing the efficacy of a bipolar therapy. a) measuring the amount of phosphorylated ERK and Akt protein in a sample derived from a subject having Fragile X syndrome or other cognitive disorders;
b) measuring the total amount of ERK and Akt protein in the sample;
c) calculating the ratio of the amount of phosphorylated ERK and Akt protein determined in step a) to the amount of ERK and Akt protein determined in step b);
d) comparing the calculated ratio of step c) to the calculated ratio from an unaffected subject
And wherein the calculated ratio of step c) is higher than the calculated ratio of the subject not suffering from the disease indicates that the subject will benefit from the meta-cognitive therapy therapy, wherein the subject has a fragile X syndrome or other cognitive disorder A method of determining whether or not a benefit will be gained from meta-cognitive therapy. a) measuring the amount of phosphorylated ERK and Akt protein in the first sample from the subject in the first period;
b) measuring the total amount of ERK and Akt protein in the first sample in the first period;
c) calculating a first ratio of the amount of phosphorylated ERK and Akt protein determined in step a) to the total amount of ERK and Akt protein determined in step b);
d) measuring the amount of phosphorylated ERK and Akt protein in the second sample from the subject in the second period;
e) measuring the total amount of ERK and Akt protein in the second sample in the second period;
f) calculating a second ratio of the amount of phosphorylated ERK and Akt protein determined in step d) to the total amount of ERK and Akt protein determined in step e) to produce a second ratio,
g) comparing the first ratio to the second ratio
Wherein the meta-cognitive therapy is monitored in a subject having a Fragile X syndrome or other cognitive disorder. 4. The method of claim 3, wherein the second ratio is lower than the first ratio to indicate that the treatment is effective. 5. The method according to any one of claims 1 to 4, wherein the measuring step comprises an immunoassay. 6. The method according to any one of claims 1 to 5, wherein the sample is whole blood or a fraction thereof. 7. The method according to any one of claims 1 to 6, wherein the sample is peripheral blood mononuclear cells (PBMC). 8. The method of claim 7, wherein the PMBC is a lymphocyte or mononuclear cell.

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