EP3044589A1 - Procédés de détermination d'une réponse à un traitement - Google Patents

Procédés de détermination d'une réponse à un traitement

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Publication number
EP3044589A1
EP3044589A1 EP14776939.2A EP14776939A EP3044589A1 EP 3044589 A1 EP3044589 A1 EP 3044589A1 EP 14776939 A EP14776939 A EP 14776939A EP 3044589 A1 EP3044589 A1 EP 3044589A1
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EP
European Patent Office
Prior art keywords
mice
metadoxine
erk
subject
ratio
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14776939.2A
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German (de)
English (en)
Inventor
Jonathan Rubin
Yaron DANIELY
Johanna SCHUMANN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arcturus Therapeutics Ltd
Original Assignee
Alcobra Ltd
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Filing date
Publication date
Priority claimed from US14/038,258 external-priority patent/US20150073023A1/en
Application filed by Alcobra Ltd filed Critical Alcobra Ltd
Publication of EP3044589A1 publication Critical patent/EP3044589A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention rel ates generally to methods of determining the response to metadoxine therapy for the treatment of Fragile X Syndrome and other cognitive disorders.
  • the invention also relates to identifying individuals that will be responsive to metadoxine therapy.
  • Fragile X Syndrome is associated with a fragile site expressed as an isochromatid gap in the metaphase chromosome at map position Xq 27.3
  • Fragile X syndrome is a genetic disorder caused by a mutation in the S'-untranslated region of the fragile mental retardation 1 (FMRl) gene, located on the X chromosome.
  • the mutation that causes FXS is associated with a CGG repeat in the fragile X mental retardation gene FMRl. In most healthy individuals, the total number of CGG repeats ranges from less than 10 to 40, with an average of about 29. In fragile X syndrome, the CGG sequence is repeated from 200 to more than 1 ,000 times.
  • Premutation expansions (55-200 CGG repeats) of the FMRl gene are frequent in the general population, with estimated prevalences of 1 per 259 females and 1 per 812 males.
  • Carriers of the premutation typically have normal IQ, although emotional problems such as anxiety are common.
  • Older male carriers of the premutation (50 years and older) develop progressive intention tremor and ataxia.
  • These movement disorders are frequently accompanied by progressive cognitive and behavioral difficulties, including memory loss, anxiety, and deficits of executive function, reclusive or irritable behavior, and dementia.
  • This disorder has been designated fragile X-associated tremor/ataxia syndrome (FXTAS). Magnetic resonance imaging in subjects with FXTAS reveals increases in T2- weighted signal intensity in the middle cerebellar peduncles and adjacent cerebel lar white matter.
  • FXS segregates as an X-linked dominant disorder with reduced penetrance. Either sex when carrying the fragile X mutation may exhibit intellectual disability, which is variable in severity. Children and adults with FXS have varying degrees of intellectual disability or learning disabilities and behavioral and emotional problems, including autistic-like features and tendencies. Young children with FXS often have delays in developmental milestones, such as learning how to sit, walk and talk. Affected children may have frequent tantrums, difficulties in paying attention, frequent seizures (e.g., temporal lobe seizures), are often highly anxious, easi ly overwhelmed, can have sensory hyperarousal disorder, gastrointestinal disorders, and may have speech problems and unusual behaviors, such as hand flapping and hand biting.
  • FXS can be diagnosed by an established genetic test performed on a sample (e.g., blood sample, buccal sample) f om the subject. The test determines whether a mutation or premutation is present in the FMR1 gene of the subject based upon the number of CGG repeats.
  • a sample e.g., blood sample, buccal sample
  • Subjects with FXS can also have autism. About 5% of all children diagnosed with autism have a mutation in the FMR1 gene and also have fragi le X syndrome (FXS). Autism spectrum disorder (ASD) is seen in approximately 30% of males and 20% of females with FXS, and an additional 30% of FXS individuals display autistic symptoms without having the ASD diagnosis. Although intellectual disability is a hallmark feature of FXS, subjects with FXS often display autistic features ranging from shyness, poor eye contact, and social anxiety in mild cases to hand flapping, hand biting and perseverafive speech in the severely affected. Subjects with FXS display other symptoms associated with autism such as attention deficit and hyperactivity, seizures, hypersensitivity to sensory stimuli obsessive-compulsive behavior and altered
  • the FMR1 mutation prevents or greatly decreases expression of a single
  • FMRP protein
  • the invention provides methods of assessing the effectiveness of a metadoxine treatment regimen in a subject having Fragile X Syndrome or other cognitive disorder who has received the metadoxine treatment, by measuring the amount of phosphorylated ERK and Akt protein in a sample derived from the subject; measuring the total amount of ERK and Akt protein in the sample; calculating a 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 a calculated ratio measured from a non-diseased subject.
  • the treatment is effective.
  • Also provided by the invention are methods of determining whether a subject with Fragile X Syndrome or other cognitive disorder would derive a benefit from a metadoxine treatment regimen by measuring the amount of phosphorylated ERK and Akt protein in a sample derived from the subject; measuring the total amount of ERK and Akt protein in the sample; calculating a ratio of the amount of phosphorylated ERK and Akt protein to the total amount of ERK and Akt protein and comparing the subject calculated ratio to a calculated ratio measured from a non-diseased subject. When the subject calculated ratio is higher than the calculated ratio of a known non-diseased subject, the subject would derive a benefit from the metadoxine treatment regimen.
  • the measuring steps comprise an immunoassay.
  • the sample is whole blood or a fraction thereof.
  • the sample is a peripheral blood mononucleated cell (PBMC).
  • PBMC peripheral blood mononucleated cell
  • the PMBC is a lymphocyte or a monocyte.
  • Fig. 1 shows the effect of seven days of once daily intraperitoneal (ip) administration of vehicle (V) or metadoxine (M) (100, 150, or 200 mg/kg) in 2-months oldFmrl knockout ( O) or Wild Type (WT) mice on contextual fear conditioning.
  • Panel A shows the effect of vehicle or 150 mg/kg of metadoxine.
  • Panel B shows the effect of vehicle or 100 mg/kg of metadoxine.
  • Panel C shows the effect of vehicl e or 200 mg kg of metadoxme.
  • Fig. 3 shows the effect of seven days of once daily intraperitoneal administration of vehicle (V) or 150 mg/kg metadoxine (M) on Y-maze spontaneous alternation (Panel A), Y- maze rewarded alternation (Panel B) or Y-maze water maze spatial discrimination (Panel C) in 2-months old Fmrl knockout (KO) or Wild Type (WT) mice.
  • V vehicle
  • M metadoxine
  • the successi ve aileys of the apparatus presented progressively more anxiogenic environments to explore mice. Movement down the alleys therefore assessed anxiety, in addition, overall activity levels could also be quantitated in the apparatus.
  • Panel A shows ip and oral treatment with vehicle in Fmrl knockout and Wild Type mice.
  • Panel B shows ip and oral treatment with metadoxine in Wild Type mice.
  • Fig. 12 shows the effect of once daily ip or oral administration (po) of vehicle (V) or metadoxine (M) at 150 or 300 mg/kg for 7 days on lymphocyte biomarkers as assessed using flow cytometry in 2 month old Fmrl knockout (KO) and Wild Type (WT) mice.
  • Biomarkers shown are pAkt (Panel A) and pER (Panel B) in Fmrl knockout or Wild Type mice.
  • Fig. 13 shows the effect of once daily ip of vehicle (V) or 150 mg/kg metadoxine (M) for 7 days on pERK levels in brain regions of two month old Wild Type (WT) and Fmrl knockout (KO) mice.
  • Fig. 14 shows the effect of once daily ip of vehicle (V ) or 150 mg/kg metadoxine (M) for 7 days on pAkt levels in brain regions of two month old Wild Type (W f T) and Fmrl knockout (KO) mice.
  • Fig. 15 shows the effect of 5 hour treatment with vehicle (V) or 300 ⁇ metadoxine (M) in vitro on filopodia density (Panel A), length (Panel B), and width (Panel C) in neuronal tiippocampal cultures from Fmrl knockout (KO) or Wild Type (WT) mice.
  • the present invention relates to the identification of biomarkers associated with the response to metadoxine therapy for individuals with Fragile X Syndrome (FXS) and other cognitive disorders.
  • FXS Fragile X Syndrome
  • metadoxine treatment returns the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in a subject sample closer to normal ratios.
  • normal ratios it is meant the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein found in normal ( i.e, non-diseased ) subjects.
  • normal ratios it is meant the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein found in normal ( i.e, non-diseased ) subjects.
  • these alterations in phosphorylated ERK and Akt protein to total ER and Akt protein ratio could be detected in the blood.
  • the invention provides methods for monitoring subjects undergoing metadoxine treatment for FXS or other cognitive disorders by determining the ratio of phosphorylated ERK and Akt proteins to total ERK and Akt protein in a subject sample.
  • the ratio is compared to a control ratio, such as the ratio obtained from a subject not afflicted with the cognitive disorder.
  • a subject ratio similar to a normal control ratio indicates that the treatment is efficacious.
  • the invention provides methods of selecting subjects who have cognitive disorders that would derive a benefit from metadoxine treatment, 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 a control ratio, such as the ratio obtained from a subject not afflicted with the cognitive disorder. A subject ratio greater than a normal control ratio indicates that the subject may derive a benefit from metadoxme treatment. Whereas subjects that do not have a ratio greater than a normal control ratio may not derive a benefit from metadoxine treatment
  • Biomarker in the context of the present invention encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein - ligand complexes, elements, related metabolites, and other anaiytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids. Biomarkers also encompass non-blood borne factors or non-analyte physiological markers of health status, such as "clinical parameters" defined herein, as well as “traditional laboratory risk factors”, also defined herein.
  • Biomarkers also include any calculated indices created mathematically or combinations of any one or more of the foregoing measurements, including temporal trends and differences. Where available, and unless otherwise described herein, biomarkers which are gene products are identified based on the official letter abbreviation or gene symbol assigned by the international Human Genome Organization Naming Committee (HGNC) and listed at the date of this filing at the US National Center for Biotechnology Information (NCBI) web site.
  • HGNC Human Genome Organization Naming Committee
  • NCBI National Center for Biotechnology Information
  • a "Clinical indicator” is any physiological datum used alone or in conjunction with other data in evaluating the physiological condition of a collection of cells or of an organism. This term includes pre-clinical indicators.
  • “Clinical parameters” encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), or fami ly history (FamHX).
  • “FN" is false negative, which for a disease state test means classifying a disease subject incorrectly as non-disease or normal.
  • FP is false positive, which for a disease state test means classifying a normal subject incorrectly as having disease
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an "index” or “index value.”
  • Parameters continuous or categorical inputs
  • Non-limiting examples of “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value
  • transformations and normalizations including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity
  • rules and guidelines including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity
  • statistical classification models including, without limitation, those classification schemes based on clinical parameters, such as gender, age, or ethnicity
  • neural networks trained on historical populations.
  • classification algorithms and methods of risk index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELD A), Support V ector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), Ste AIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, among others.
  • PCA Principal Components Analysis
  • LogReg Logistic Regression
  • LDA Linear Discriminant Analysis
  • ELD A Eigengene Linear Discriminant Analysis
  • SVM Support V ector Machines
  • RF Random Forest
  • RPART Recursive Partitioning Tree
  • SC Shrunken Centroids
  • SC Ste AIC
  • BIC Criterion
  • the resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV).
  • LEO Leave-One-Out
  • 10-Fold cross-validation 10-Fold CV.
  • false discover rates may be estimated by value permutation according to techniques known in the art.
  • a "health economic utility function" is a formula that is derived from a combination of the expected probability of a range of clinical outcomes in an idealized applicable patient population, both before and after the introduction of a diagnostic or therapeutic intervention into the standard of care.
  • a cost and/or value measurement associated with each outcome, which may be derived from actual health system costs of care (services, supplies, devices and drugs, etc.) and/or as an estimated acceptable value per quality adjusted life year (QALY) resulting in each outcome.
  • the sum, across all predicted outcomes, of the product of the predicted population size for an outcome multiplied by the respective outcome's expected utility is the total health economic utility of a given standard of care.
  • the difference between (i) the total health economic utility calculated for the standard of care with the intervention versus (ii) the total health economic utility for the standard of care without the intervention results in an overall measure of the health economic cost or value of the intervention.
  • This may itself be divided amongst the entire patient group being analyzed (or solely amongst the intervention group) to arrive at a cost per unit intervention, and to guide such decisions as market positioning, pricing, and assumptions of health system acceptance.
  • Such health economic utility functions are commonly used to compare the cost-effectiveness of the intervention, but may also be transformed to estimate the acceptable value per QALY the health care system is willing to pay, or the acceptable cost-effective clinical performance characteristics required of a new intervention.
  • a health economic utility function may preferentially favor sensitivity over specificity, or PPV over NPV based on the clinical situation and individual outcome costs and value, and thus provides another measure of health economic performance and value which may be different from more direct clinical or analytical performance measures.
  • Measuring or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's non-analyte clinical parameters.
  • NBV Neuronal predictive value
  • NPV neurotrophic factor
  • TN/(TN+FN) the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. See, e.g., O'Marcaigh A S, Jacobson R M, "Estimating The Predictive Value Of A Diagnostic lest, Flow To Prevent Misleading Or Confusing Results," Clin. Fed. 1993, 32(8): 485-491, which discusses specificity, sensitivity, and positive and negative predictive values of a test, e.g., a clinical diagnostic test. Often, for binary disease state classification approaches using a continuous diagnostic test measurement, the sensitivity and specificity is summarized by Receiver Operating
  • ROC Characteristics
  • Performance is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g., stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate "performance metrics," such as AUC, time to result, shelf life, etc. as relevant.
  • PSV Positive predictive value
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the responsiveness to treatment, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1-p) is the probability of no event) to no-conversion.
  • "Risk evaluation” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conv ersion from one disease state.
  • Ris k evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of FXS, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categori cal measurements of the responsi veness to treatment thus diagnosing and defining the risk spectrum of a category of subjects defined as being responders or non- responders.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for responding.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, cerebrospinal fluid (CSF), brain cells, or any other secretion, excretion, or other bodily fluids.
  • sample may include a single cell or multiple cells or fragments of cells.
  • the sample is also a tissue sample.
  • the sample is or contains a brain cell or a lymphocyte.
  • the sample is peripheral blood mononuclear cell such as a lymphocyte or monocyte.
  • Specificity is calculated by TN/(TN+FP) or the true negative fraction of non- disease or normal subjects.
  • significance can be determined by any method known in the art. Commonly used measures of significance include the p-vahie, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is considered highly significant at a p-value of 0.05 or less. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.
  • a "subject" in the context of the present invention is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
  • Mammals other than humans can be advantageously used as subjects that represent animal models of FXS.
  • a subject can be male or female.
  • the subject has or is suspected of having FXS or other cogniti ve disorders.
  • TN is true negative, which for a disease state test means classifying a non-disease or normal subject correctly.
  • the methods disclosed herein are used with subjects undergoing metadoxine treatment and/or therapies for FXS and other cognitive disorders and for subjects who have been diagnosed with FXS and other cognitive disorders.
  • the methods of the present invention are useful to monitor the treatment of FXS and other cognitive disorders in a subject and to select subjects who would derive a benefit from metadoxine treatment.
  • FXS FXS
  • intelligence and learning physical, social and emotional, speech and language and sensory disorders commonly associated or sharing features with Fragile X.
  • individuals with FXS have impaired intellectual functioning, social anxiety, language difficulties and sensitivity to certain sensations.
  • Cognitive disorders include the group of disorders in which a dysfunction/ impairment of mental processing constitutes the core symptomatology. Cognitive disorders include neurogenetic cognitive disorders or behavioral cognitive disorders
  • Cognitive disorders include developmental disorders, attention deficit hyperactivity disorder (ADHD ), autism spectrum disorders, Alzheimers disease, schizophrenia and cerebrovascular disease.
  • ADHD attention deficit hyperactivity disorder
  • autism spectrum disorders Autism spectrum disorders and autistic symptoms are commonly associated with individuals with Fragile X syndrome. Signs and symptoms of autism include significant language delays, social and communication chal lenges, and unusual behaviors and interests. Many people with autistic disorder also have intellectual disability.
  • ⁇ 60 Determination of the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in a subject sample allows for the course of treatment of FXS or other cognitive disorder to be monitored.
  • a biological sample is provided from a subject undergoing treatment. If desired, biological samples are obtained from the subject at various time points before, during, or after treatment. The ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein are then calculated and compared to a control value.
  • a control value is a control individual or population whose ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein state is known or an index value.
  • the reference sample or index value may be taken or derived from one or more individuals who are not diseased (e.g., not affected with the FXS or other cognitive disorder).
  • the reference sample or index value may be taken or derived from the subject before treatment.
  • samples may be collected from a subject who has not received an initial treatment and after subsequent treatment to monitor the progress of the treatment.
  • the reference sample or index value may be taken or derived from the subject after the initial treatment.
  • samples may be collected a subject who have received initial treatment and subsequent treatment for FXS to monitor the progress of the treatment.
  • the reference value is an index value or a baseline value.
  • An index 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 that do not suffer from FXS or other cognitive disorder.
  • the effectiveness of treatment can be monitored by determining the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein in a sample obtained from a subject over time and comparing the ratios. For example, a first sample can be obtained prior to the subject receiving treatment and one or more subsequent samples are taken after or during treatment of the subject. [8(5063] By “efficacious”, it is meant that the treatment leads to a ratio of phosphorylated ER and Akt protein to total ERK and Akt protein similar to that from a subject that does not have FXS or other cognitive disorder. Efficacy can be determined in association with any known method for diagnosing, identifying, or treating FXS.
  • Phosphorylated ERK and Akt and total ERK and Akt protein can be determined by any method known in the art, such as immunoassay.
  • the performance and thus absolute and relative clinical usefulness of the invention may be assessed in multiple ways as noted above.
  • the accuracy of a diagnostic, predictive, or prognostic test, assay, or method concerns the ability of the test, assay, or method to distinguish between subjects responsive to metadoxine treatment and those that are not, is based upon the ratio of phosphorylated ERK and Akt protein to total ERK and Akt protein.
  • the difference in the ratio betwen normal and abnormal is preferably statistically significant.
  • an "acceptable degree of diagnostic accuracy” is herein defined as a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.
  • a "very high degree of diagnostic accuracy” it is meant a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.80, desirably at least 0.85, more desirably 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 depends on the sensitivity and specificity of the test, and on the prevalence of the condition in the population being tested.
  • ROC and AUC can be misleading as to the clinical utility of a test in low disease prevalence tested populations (defined as those with less than 1% rate of occurrences (incidence) per annum, or less than 1.0% cumulative prevalence over a specified time horizon).
  • absolute risk and relative risk, ratios as defined elsewhere in this disclosure can be employed to determine the degree of clinical utility.
  • Populations of subjects to be tested can also be categorized into quartiles by the test's measurement values, where the top quartile (25% of the population) comprises the group of subjects with the highest relative risk for therapeutic unresponsiveness, and the bottom quartile comprising the group of subjects having the lowest relative risk for therapeutic unresponsiveness
  • the top quartile (25% of the population) comprises the group of subjects with the highest relative risk for therapeutic unresponsiveness
  • the bottom quartile comprising the group of subjects having the lowest relative risk for therapeutic unresponsiveness
  • values derived from tests or assays having over 2.5 times the relative risk from top to bottom quartile in a low prevalence population are considered to have a "high degree of diagnostic accuracy," and those with five to seven times the relative risk for each quartile are considered to have a "very high degree of diagnostic accuracy.”
  • values derived from tests or assays having only 1.2 to 2.5 times the relative risk for each quartile remain clinically useful are widely used as risk factors for a disease; such is the case with total cholesterol and for many inflammatory biomark
  • a health economic utility function is an yet another means of measuring the performance and clinical value of a given test, consisting of weighting the potential categorical test outcomes based on actual measures of clinical and economic value for each.
  • Health economic performance is closely related to accuracy, as a health economic utility function specifically assigns an economic value for the benefits of correct classification and the costs of miscutzificatioti of tested subjects.
  • As a performance measure it is not unusual to require a test to achieve a level of performance which results in an increase in health economic value per test (prior to testing costs) in excess of the target price of the test.
  • any formula may be used to combine results into indices useful in the practice of t he invention.
  • indices may indicate, among the various other indications, the probability, likelihood, absolute or relative chance of responding to metadoxine . This may be for a specific time period or horizon, or for remaining lifetime risk, or simply be provided as an index relative to another reference subject population.
  • model and formula types beyond those mentioned herein and in the definitions above are well known to one skilled in the art.
  • 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 its results in a training population.
  • Preferred formulas include the broad class of statistical classification algorithms, and in particular the use of discriminant analysis.
  • the goal of discriminant analysis is to predict class membership from a previously identified set of features.
  • LDA linear discriminant analysis
  • features can be identified for LDA using an eigengene based approach with different thresholds (ELDA) or a stepping algorithm based on a multivariate analysis of variance (MANOVA). Forward, backward, and stepwise algorithms can be performed that minimize the probability of no separation based on the Hotelling-Lawley statistic.
  • ELDA Linear Discriminant Analysis
  • ELDA Eigengene-based Linear Discriminant Analysis
  • the formula selects features (e.g. biomarkers) in a multivariate framework using a modified eigen analysis to identify features associated with the most important eigenvectors. "Important" is defined as those eigenvectors that explain the most variance in the differences among samples that are trying to be classified relative to some threshold.
  • SVM support vector machine
  • a support vector machine (SVM) is a classification formula that attempts to find a hyperplane that separates two classes. This hyperplane contains support vectors, data points that are exactly the margin distance away from the hyperplane.
  • an overall predictive formula for all subjects, or any known class of subjects may itself be recalibrated or otherwise adjusted based on adjustment for a population's expected prevalence and mean biomarker parameter values, according to the technique outlined in
  • numeric result of a classifier formula itself may be transformed postprocessing by its reference to an actual clinical population and study results and observed endpoints, in order to calibrate to absolute risk and provide confidence intervals for varying numeric results of the classifier or risk formula.
  • An example of this is the presentation of absolute risk, and confidence intervals for that risk, derived using an actual clinical study, chosen with reference to the output of the recurrence score formula in the Oncotype Dx product of Genomic Health, Inc. (Redwood City, Calif.).
  • a further modification is to adjust for smaller sub- populations of the study based on the output of the classifi er or ri sk formula and defined and selected by their Clinical Parameters, such as age or sex.
  • Fmrl knockout mice ( 02) mice (The Dutch-Belgium Fragile X Consortium, 1994), initially obtained from the Jackson Laboratory, and wild type (WT) littermates were generated on a C57BL/6J background and repeatedly backcrossed onto a C57BL/6J background for more than eight generations.
  • the Fmrl knockout mice were housed in groups of the same genotype in a temperature and humidity controlled room with a 12-h light-dark cycle (lights on from 7 am to 7 pm; testing was conducted during light phase).
  • metadoxine in vivo testing, metadoxine was dissolved in saline and administered at an intraperitoneal dose of 150 mg/kg per day or at an oral dose of 150 or 300 mg/kg/day (in a volume of 0.1 ml) once daily for seven days.
  • metadoxine in vitro testing, metadoxine was administered at concentration of 300 ⁇ for five hours. In all cases, saline was used as a vehicle (control).
  • mice are a social species that engage in easily scored social behaviors including approaching, following, sniffing, grooming, aggressive encounters, sexual interactions, parental behaviors, nesting, and sleeping in a group huddle. Social approach in mice was evaluated by sniffing duration directed to a novel mouse.
  • [0008S] Mi ce were placed in a test arena/cage of the same order of magnitude in size as the adult's home cage (40 x 23 x 12 cm cage, with a Perspex lid to facilitate viewing the mice) with fresh wood chippings on the floor.
  • a background mouse odor was created by putting in some non-experimental mice into the apparatus prior to testing. Mice were transferred to the experimental room 10-15 min prior to testing.
  • a test subject and a juvenile were placed simultaneously into the test cage. The total duration and number of bouts of social investigation, defined as sniffing and close following ( ⁇ 2 cm from the tail) of the stimulus juvenile by the tested mouse, was assessed for 3 min. 30 min later, the test was repeated using the same stimulus juvenile.
  • Y-Maze Water Maze A clear Perspex Y-maze was filled with 2 cm of water at 20°C. This motivated the mouse to leave the maze after paddling to an exit tube at the distal end of one arm. The maze was placed in the middle of a room surrounded by prominent visual cues.
  • the apparatus consisted of four successive, linearly arranged, increasingly anxiogenic alleys (each succeeding alley was painted a lighter color, had lower walls and/or was narrower than the previous alley) made of painted wood. Each section or alley was 25 cm long. Alley 1 had 25 cm high walls, was 8.5 cm wide, and was painted black. A 0.5 cm step down led to alley 2, which was again 8.5 cm wide, but had 1.3 cm high walls and was grey. A 1.0 cm step down led to alley 3, which was 3.5 cm wide, had 0.8 cm high walls, and was white. A 0.4 cm step led down to alley 4, which was also white, but had 1.2 cm wide and 0.2 cm high walls.
  • the apparatus was elevated by anchoring the back of alley 1 to a stand, 50 cm high. Padding was provided under arms 3 and 4 in case a mouse fell off. Each mouse was placed at the closed end of alley 1 facing the wall. Timers were started 1) for the overall length of the test (5 min) + the latency to enter each arm, and 2) for the time spent in alley 1. When the mouse placed all 4 feet on to the next alley, it was considered to have entered the al ley. Total time spent in each alley (all four feet) was recorded.
  • mice were placed into a novel environment (dark chamber) and received pairings of a cue and electric footshock (0.2 mA for 1 sec (Study 1) or 0.7 mA for 0.5 sec (Study 2)). Subsequently, when tested in the original training context, mice displayed a natural defensive response termed freezing
  • Freezing time was defined as the time that the mice spent in immobile behavior, except for respiration. The data was expressed as the percentage of the test period. 24 hours after a training session, mice were tested for 5 min in the training chamber with no shock presentation and observed for freezing behavior.
  • Phosphorylated ERK and Akt The Ras-Mek-ER and PI3K-Akt-mToR signaling pathways are involved in mediating activity dependent alterations in gene transcription underlying changes in synaptic plasticity ( lann and Dever, 2004). Phosphorylated ERK and Akt protein expression was measured by western blot analysis as previously described by Lopez Verri lli (Lopez Verriili et al., 2009). The antibodies employed were anti-phosphospecific antibodies against Akt (1/1000) and kinase (ERK) 1/2 (1/2000) (Cell Signaling Technology, Danvers, MA, USA).
  • the antibody against phospho-ERK detects phosphorylation at phospho- ER l/2 (Thr202/Tyr204) whereas the antibody against phospho-Akt detects phosphorylation at phospho-Akt (Thr308).
  • Total Akt and ERK 1/2 protein content and phosphorylated ERK and Akt were evaluated by blotting membranes with antiphospho-Akt (1 /1000) and antiphospho- ERK antibodies (1/2000) (Ceil Signaling Technology, Danvers, MA, USA). Akt or ERK phosphorylation was normalized to protein content in the same sample and expressed as % of change with respect to basal conditions, considering basal levels as 100%.
  • Protein loading was evaluated by stripping and re-b lotting membranes with ⁇ -actin antibody (1/1000) (Sigma- Aldrich, St. Louis, MO, USA). Phosphorylated ERK. and Akt protein expression in blood lymphocytes was measured by flow cytometry.
  • a FACStar plus Bee ton Dickinson was used with the excitation laser tuned at 488 nm and green fluorescence from FITC (GST) was collected through a 515-545 nm bandpass filter.
  • the mean FITC fluorescence Intensity was calculated in relation to the fluorescence of reference cells.
  • the mean cellular fluorescence intensity (MFI) is directly proportional to the mean number of Ab molecules bound per cell.
  • Neuronal morphology ippocampal cell cultures were prepared from wild type and Fmrl KO fetal mice at embryonic day of gestation 17.5 (El 7.5). Mice were killed by cervical dislocation and dissociated hippocampal cells were plated in 15 mm multi well vessels (Falcon Primaria). After 5d in vitro, green fluorescent protein (GFP) was transfected to facilitate monitoring dendritic spine morphogenesis after drug treatment (Ethell and Yamagucbi, 1999; Ethell et ai., 2001 , Henkemeyer et al., 2003). Dendritic spines were formed at around 16 days in vitro (DIV). Cultures were treated with metadoxine at 300 uM concentration at day 17 in vitro for 5 hrs.
  • GFP green fluorescent protein
  • Filopodia density of GFP transfected neurons was quantified by performing Sholl analyses of stacked Zeiss confocal generated images (40 x objective, stack of 20 ⁇ 0.2 ⁇ ). With Metamorph software, concentric equally spaced circles (every 20um) were drawn around the cell soma of each neuron and subsequently, the amount of filopodia was counted per circle.
  • Transverse hippocampal slices 400 ⁇ im were obtained from 6-week-old Fmrl knockout and WT mice.
  • a protein synthesis assay was performed as previously described using the nonradioactive fluorescence-activated cell sorting- based assay, surface sensing of translation (SU SET) method, which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. (Hoeffer, 2011). The concentration of metadoxme used in this study was 300 ⁇ ,
  • EXAMPLE 2 THE EFFECT OF METADOXINE (100 to 200 mg/kg) TREATMENT ON LEARNING AND MEMORY DEFICITS AND BIOCHEMICAL ABNORMALITIES IN THE Fmrl KNOCKOUT MOUSE MODEL OF FRAGILE X SYNDROME (Study 1)
  • Metadoxme administration reversed the learning deficit effect in / ⁇ ' /;/,' ⁇ / knockout mice, this reversal being partial such that metadoxme-treated animals differed from the metadoxine-treated WT animals (p ⁇ 0.05).
  • metadoxine produced a reversal of the deficit in Fmrl knockout mice (P ⁇ 0.05) but this was a partial reversal since metadoxine-treated Fmrl knockout mice differed from the metadoxine-treated Wild Type mice (p ⁇ 0.0001).
  • the learning deficit seen in Fmrl knockout mice was completely reversed following treatment with 200 mg/kg i.p, metadoxme (treated Fmrl mice differed from vehicle-treated Fmrl knockout mice (P ⁇ 0.0001) but did not differ from metadoxine-treated WT mice), Metadoxine treatment had no effect on WT mice in either experiment (Fig, 1, Panels A-C).
  • Vehicle-treated Fmrl knockout mice showed less spontaneous alternation than vehicle treated WT mice (pO.OGOl).
  • Metadoxine treatment increased spontaneous alternation compared to vehicle treatment in Fmrl knockout mice (pO.0001), although metadoxine-treated Fmrl knockout mice showed a deficit compared to metadoxine-treated WT mice (p ⁇ 0.01 ). Metadoxine therefore produced a partial reversal of the deficit seen in Fmrl knockout mice.
  • Vehicle-treated Fmrl knockout mice made less appropriate arm entries than vehicle-treated WT mice (p ⁇ 0.0001).
  • Metadoxine treatment reduced this deficit (p ⁇ 0.0001) compared to vehicle-treated / ⁇ ;;>/ ⁇ / knockout mice, such that metadoxine-treated Fmrl knockout mice did not differ from metadoxine-treated WT mice.
  • Metadoxme treatment had no effect on WT mice.
  • the successive alleys test effectively measured anxiety (latency to enter the Alley 1) and hyperactivity (Alleys 2 to 4). Progression from Alley 1 through the successive Alleys 2, 3, and 4 was associated with exposure to an increasingly brightly colored environment with increasingly lower walls and narrower, more exposed open arms. Time spent on, and entries into, the open arms indicated anxiety; conversely, increasing time spent in more open arms reflected hyperactivity. These factors allowed for a sensitive test bracketing a range of anxietylike behaviors together with hyperactivity.
  • Alley 1 The Fmrl knockout mice showed more anxiety than WT mice (pO.QOl). Fmrl knockout mice treated with metadoxine showed an amelioration in anxiety compared with the vehicle treated Fmrl knockout mice (pO.001), such that complete normalization occurred. There was no difference between the metadoxine-treated Fmrl knockout and metadoxine-treated WT mice. Also, metadoxine treatment had no effect on WT mice.
  • Alley 2 WT mice showed less activity in Alley 2 when compared with the Fmrl knockout mice (p ⁇ 0.0001). Treatment with metadoxme reduced hyperactivity in the Fmrl knockout mice (p ⁇ 0.001), although this reversal of hyperactivity was partial since metadoxine- treated Fmrl knockout and WT mice differed (p ⁇ 0.001 ). Metadoxine treatment had no effect on WT mice. [8(50187] Alley 3: Fmrl knockout mice showed hyperactivity compared to WT mice (p ⁇ Q.0001). This hyperactivity was not reversed by metadoxme, since metadoxine-treated Fmrl knockout mice did not differ from vehicle-treated Fmrl knockout mice. Metadoxine treatment had no effect on WT mice.
  • Metadoxine treatment reduced this anxiety and hyperactivity in the Fmrl knockout mice whilst leaving WT mice unaffected.
  • EXAMPLE 3 THE EVALUATION OF METADOXINE IN THE Fmrl KNOCKOUT
  • Figure 10 shows the effect of administration of once daily metadoxine at doses of 150 mg/kg ip or 150 and 300 mg/kg orally for seven days on contextual fear conditioning in two month old Fmrl knockout and WT mice.
  • Panel A shows contextual fear conditioning data from Fmrl knockout and WT mice after ip and oral treatment with vehicle. There were no differences related to the route of administration of vehicle. Fmrl knockout mice showed a reduction in freezing behavior compared to WT mice after vehicle treatment via ip and oral routes (p ⁇ 0.0001 in each case).
  • Panel B shows the effect of metadoxine treatment via both routes of administration in WT mice. No effects were seen.
  • Panel C shows that ip 1 50 mg/kg and oral 1 0 and 300 mg/kg metadoxine treatment in Fmrl knockout mice reversed the decrease in freezing behavior seen in Fmrl knockout mice (p ⁇ 0.01 , p ⁇ 0.0001, and p ⁇ 0.0001 , for KO-M-ip, KO-M-pol50, and KO-M-po 300 vs. KO-V-ip and KO-V po, respectively).
  • the effect of administration with 150 mg po metadoxme did not differ from the effect of administration of 300 mg/kg po metadoxine.
  • the effect of 150 and 300 mg/kg oral metadoxine in Fmrl knockout mice did not differ from the effect of 150 mg/kg ip metadoxme. In each case, the reversal was complete since metadoxine-treated Fmrl knockout mice did not differ from metadoxine-treated WT mice.
  • Figure 1 1 shows the effect of administration of once daily metadoxine at doses of 150 mg/kg ip or 150 and 300 mg/kg orally for seven days on social approach and social memory in Fmrl knockout and WT mice.
  • Panel A shows the effect of vehicle or metadoxine at 150 mg/kg ip or 150 and 300 mg/kg orally on social approach behavior in Fmrl knockout or WT mice.
  • the duration of sniffing behavior in Fmrl knockout mice was reduced compared to WT mice (pO.GOOl for each). Metadoxine treatment at any dose was without effect on WT mice.
  • metadoxine treatment at 150 mg/kg ip, 150 mg kg, and 300 mg/kg orally produced reversals of the social approach deficit seen in Fmrl knockout mice (p ⁇ Q.0001 for O ⁇ M-pol50 and KQ-M-po300 vs. O-V po, respectively).
  • the effect of oral metadoxme was not dose dependent between 150 and 300 mg/kg. This reversal was complete since metadoxme-treated Fmrl knockout mice did not differ from metadoxine-treated WT mice.
  • the effect of 150 mg/kg ip metadoxme in Fmrl knockout mice did not differ from the effect of 150 mg/kg oral or 300 mg kg oral metadoxme.
  • Panel B shows the effect of vehicle or metadoxme at 150 mg/kg ip or 150 and 300 mg/kg orally on social memory in Fmrl knockout or WT mice.
  • the duration of sniffing behavior in Fmrl knockout mice was increased compared to WT mice (p ⁇ 0.0001 for each).
  • Metadoxme treatment at any dose was without effect on WT mice.
  • FIG. 12 shows the effect of administration of once daily rnetadoxine at doses of 150 mg/kg ip or 150 mg kg and 300 mg/kg orally for 7 days on lymphocyte pAkt (Fig. 12, Panel A) and pERK (Fig. 12, Panel B) as determined by flow cytometry in two month old Fmrl knockout and WT mice.
  • Panel A shows that vehicle-treated Fmrl knockout mice exhibited increased phosphorylation of lymphocyte Akt (p ⁇ 0.0001 for both ip and oral administration) compared to WT mice receiving equivalent vehicle treatment.
  • Panel B shows thai vehicle-treated Fmrl knockout mice showed increased phosphorylation of lymphocyte ERK (p ⁇ 0.G001 for both ip and oral administration) compared to WT mice receiving equivalent vehicle treatment.
  • pERK levels were increased in Fmrl knockout mice compared to WT mice in all three brain regions (p ⁇ 0.0001 in all cases).
  • pERK levels were decreased in metadoxine-treated Fmrl knockout mice compared to vehicle-treated Fmrl knockout mice (p ⁇ 0.0001 in all cases).
  • the effect in pre-frontal cortex was partial, the O-V and KO-M groups remained different (p ⁇ 0.05). Metadoxme was without effect on WT mice.
  • Figure 14 shows the effect of administration of 150 mg/kg metadoxine for seven days on pAkt levels in hippocampus, pre-frontal cortex and striatum.
  • pAkt levels were increased in Fmrl knockout mice compared to WT mice in all three brain regions (p ⁇ 0.0001 in ail cases).
  • pAkt levels were decreased in metadoxine-treated Fmrl knockout mice compared to vehicle- treated Fmrl knockout mice in all three brain regions (p ⁇ 0.0001 in all cases). In all cases, there were no differences between KO-M and WT-M groups, showing complete reversal of activation of Akt, Metadoxme was without effect on WT mice.
  • Figure 15 shows the effect of treatment for five hours with 300 ⁇ metadoxine. Dendrites were divided into 10 segments of 10 ⁇ , each based on distance from the soma (proximal to distal, left to right). Spine density was increased in neurons from Fmrl knockout m ce compared to neurons from WT mice in segment 3. Specifically, Figure 15, Panel A shows the density of neuronal filopodia. Primary hippocampal neurons from Fmrl knockout mice displayed an increased density of filopodia (p ⁇ Q.001). Treatment with 300 ⁇ metadoxine reduced the aberrant increase in density of neuronal filopodia in Fmrl knockout mice (p ⁇ 0.001).
  • Figure 16 shows the effect of treatment with either vehicle or 300 ⁇ metadoxine on basal de novo protein synthesis in 400 ⁇ hippocampal slices from Fmrl knockout or WT mice. Protein synthesis was higher in vehicle-treated hippocampi from Fmrl knockout mice than vehicle-treated WT control hippocampi (p ⁇ 0.0001 ). Metadoxine treatment reduced protein synthesis rates in Fmrl knockout mouse hippocampi. This effect was partial since hippocampi from Fmrl knockout mice retained higher protein synthesis rates than metadoxine-treated hippocampi from WT mice (p ⁇ 0.001).

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Abstract

La présente invention concerne généralement des procédés de détermination de la réponse à un traitement à la métadoxine pour le traitement du syndrome du X fragile et d'autres troubles cognitifs. L'invention concerne en outre l'identification d'individus qui seront répondeurs au traitement à la métadoxine.
EP14776939.2A 2013-09-09 2014-09-09 Procédés de détermination d'une réponse à un traitement Withdrawn EP3044589A1 (fr)

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