WO2014085480A1 - Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique - Google Patents

Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique Download PDF

Info

Publication number
WO2014085480A1
WO2014085480A1 PCT/US2013/072049 US2013072049W WO2014085480A1 WO 2014085480 A1 WO2014085480 A1 WO 2014085480A1 US 2013072049 W US2013072049 W US 2013072049W WO 2014085480 A1 WO2014085480 A1 WO 2014085480A1
Authority
WO
WIPO (PCT)
Prior art keywords
mepe
mice
syndrome
treated
treatment
Prior art date
Application number
PCT/US2013/072049
Other languages
English (en)
Inventor
Lawrence Irwin GLASS
Michael John Bickerdike
Michael Frederick Snape
Patricia Perez DE COGRAM
Original Assignee
Neuren Pharmaceuticals Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Neuren Pharmaceuticals Limited filed Critical Neuren Pharmaceuticals Limited
Priority to CA2929286A priority Critical patent/CA2929286A1/fr
Priority to AU2013352294A priority patent/AU2013352294A1/en
Priority to EP13858943.7A priority patent/EP2928300A4/fr
Priority to JP2015545191A priority patent/JP2016506380A/ja
Priority to BR112015012506A priority patent/BR112015012506A2/pt
Publication of WO2014085480A1 publication Critical patent/WO2014085480A1/fr
Priority to US14/602,600 priority patent/US20150224164A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • 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
    • 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
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala

Definitions

  • This invention relates generally to therapy of Autism Spectrum Disorders (ASD), including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA).
  • ASSD Autism Spectrum Disorders
  • RTT Rett Syndrome
  • PDA Pathological Demand Avoidance
  • G-2-MePE Glycyl-2-methyl-Prolyl-Glutamate
  • Autism Spectrum Disorders and neurodevelopment disorders are becoming increasingly diagnosed.
  • DSM-4 Diagnostic and Statistical Manual of Mental Disorders
  • ASD Autism spectrum disorders
  • Current classification of ASD according to the DSM-4 recognises five distinct forms: classical autism or Autistic Disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD- NOS).
  • a sixth syndrome, pathological demand avoidance (PDA) is a further specific pervasive developmental disorder.
  • DSM-5 Diagnostic and Statistical Manual of Mental Disorders
  • NDDs Neurodevelopment Disorders
  • FXS Fragile X Syndrome
  • Angelman Syndrome Tuberous Sclerosis Complex
  • Phelan McDermid Syndrome Phelan McDermid Syndrome
  • Rett Syndrome CDKL5 mutations (which also are associated with Rett Syndrome and X-Linked Infantile Spasm Disorder) and others.
  • Many but not all NDDs are caused by genetic mutations and, as such, are sometimes referred to as monogenic disorders. Some patients with NDDs exhibit behaviors and symptoms of autism.
  • Fragile X Syndrome is an X-linked genetic disorder in which affected individuals are intellectually handicapped to varying degrees and display a variety of associated psychiatric symptoms.
  • Fragile X Syndrome is characterized by intellectual handicap, hyperactivity and attentional problems, autism spectrum symptoms, emotional lability and epilepsy (Hagerman, 1997a).
  • the epilepsy seen in Fragile X Syndrome is most commonly present in childhood, but then gradually remits towards adulthood. Hyperactivity is present in approximately 80 percent of affected males (Hagerman, 19973 ⁇ 4). Physical features such as prominent ears and jaw and hyper-extensibility of joints are frequently present but are not diagnostic.
  • Intellectual handicap is the most common feature defining the phenotype.
  • males are more severely affected than females.
  • Early impressions that females are unaffected have been replaced by an understanding of the presence of specific learning difficulties and other neuropsychiatric features in females.
  • the learning disability present in males becomes more defined with age, although this longitudinal effect is more likely a reflection of a flattening of developmental trajectories rather than an explicit neurodegenerative process.
  • Fragile X Syndrome The compromise of brain function seen in Fragile X Syndrome is paralleled by changes in brain structure in humans. MRI scanning studies reveal that Fragile X Syndrome is associated with larger brain volumes than would be expected in matched controls and that this change correlates with trinucleotide expansion in the FMRP promoter region (Jakala et al, 1997). At the microscopic level, humans with Fragile X Syndrome show abnormalities of neuronal dendritic structure, in particular, an abnormally high number of immature dendritic spines (Irwin et al, , 2000).
  • EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu.
  • GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.
  • W095/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.
  • CNS central nervous system
  • WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).
  • ChAT choline acetyltransferase
  • GAD glutamic acid decarboxylase
  • NOS nitric oxide synthase
  • WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS to increase TH-mediated dopamine production in the treatment of diseases such as Parkinson's disease.
  • GPE tyrosine hydroxylase
  • WO02/16408 discloses certain GPE analogs having amino acid substitutions and certain other modification that are capable of inducing a physiological effect equivalent to GPE within a patient.
  • the applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including injury or disease in the CNS.
  • This invention relates to synthetic analogs and peptidomimetics of glycyl-L-prolyl-L- glutamic acid (GPE).
  • GPE glycyl-L-prolyl-L- glutamic acid
  • this invention relates to GPE analogs and peptidomimetics that . are anti-apoptotic and anti-necrotic, to methods of making them, to pharmaceutical compositions containing them, and to their use to enhance cognitive function and/or treat memory disorders and to improve neuronal connectivity in animals.
  • this application relates to the methods of use of the GPE analog, L-Glycyl-2-methyl-L-Prolyl-L-Glutamate (G-
  • this invention provides compounds of Formula 1 and Formula 2:
  • n 0 or 1 ;
  • n 0 or 1 ;
  • X is H or— R 6 R 7 ;
  • Y is H, alkyl, - ⁇ C0 2 R 5 , or— CONR 6 R 7 ;
  • Z is H, alkyl, -TM-C0 2 R 5 or --CONR 6 R 7 ;
  • R 1 is H, alkyl, or aralkyl
  • R 2 , R 3 , and R 4 are independently H or alkyl
  • each R 5 is independently H, alkyl, or a fatty alcohol residue
  • each R 6 and R 7 is independently H, alkyl, or aralkyl, or -NR 6 R 7 is pyrrol idino, piped dino, or morpholino;
  • the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt thereof.
  • Another aspect the invention provides methods for treatment of an animal having a Autism Spectrum Disorder comprising administration of an effective amount of Glycyl-L-2- Methylprolyl-L-GIutamic Acid (G-2-MePE) to the animal.
  • G-2-MePE Glycyl-L-2- Methylprolyl-L-GIutamic Acid
  • FIG. 1 is a general scheme for preparation of synthetic analogs of GPE of the invention.
  • FIGs. 2 and 3 depict schemes for modifying glycine residues on GPE.
  • FIGs. 4 through 9 depict schemes for modifying glutamic acid residues of GPE.
  • FIGs. 10 and 11 depict schemes for modifying peptide linkages of GPE.
  • FIGs, 12 - IS depict graphs summarizing results of testing neurons in vitro with GPE or G-2-MePE and okadaic acid.
  • FIG. 12 depicts a graph showing effects of GPE on cortical neurons injured with okadaic acid.
  • FIG. 13 depicts a graph showing effects of G-2-MePE on cortical neurons injured with okadaic acid.
  • FIG. 14 depicts a graph showing effects of G-2-MePE, GPE on cerebellar microexplants injured with okadaic acid.
  • FIG. 15 depicts a graph showing effects of G-2-MePE or GPE on striatal cells injured with okadaic acid.
  • FIG. 16 shows the effects of subcutaneous injection of G-2-MePE (at doses of 0.012, 0.12, 1 ,2 and 12 mg kg) on the number of ChAT-positive neurons in the striatum of 18-month old rats.
  • FIG. 17 shows effects of G-2-MePE treatment on spatial memory retention in middle- aged 12-month old rats.
  • FIGs. 18A and 18B show effects of G-2-MePE on spatial working memory of aged (17- month old) rats in an 8-arm radial maze following 3 -weeks of treatment and a nine day washout.
  • FIG. 18A shows the maze acquisition profiles across days for the different groups.
  • FIG. 18B shows the proportion of correct maze choices averaged across days for the groups.
  • FIG. 19A shows effects of a single intraperitoneal administration of 4 doses of G-2- MePE on neuroblast proliferation as assessed by the number of PCNA positive cells in the subventricular zone (S VZ) of aged rats.
  • FIG. 19B shows effects of a single intraperitoneal administration of 4 doses of G-2-
  • FIG. 19C shows effects of G-2-MePE on neuroblast proliferation as assessed by PCNA immunohistochemical staining in middle-aged rats.
  • FIG. 20A shows a significant increase in the number of reactive astrocytes as assessed by
  • FIG. 20B shows a photograph of a section of cerebral cortex of an aged rat, showing astrocytes as assessed with GFAP staining, some of which are associated with formation of capillaries (arrows).
  • FIG. 20C shows dose-dependent effects of G-2-MePE treatment (at doses of 0.12, 0.12, 1.2 and 12 mg kg day) on reduction of the number of astrocytes as assayed using GFAP staining in the CA4 sub-region of the hippocampus in aged rats.
  • FIG. 20D shows dose-dependent effects of G-2-MePE treatment (at doses of 0.12, 0.12, 1.2 and 12 mg kg/day) on reduction of the number of astrocytes as assayed using GFAP staining in the cerebellar cortex.
  • FIG. 21 shows pharmacokinetic properties of GPE and G-2-MePE in the circulation of rats after intravenous injection.
  • FIG. 22 shows the effects of G-2-MePE on increased survival duration in MeCP2 deficient mice compared to saline-treated MeCP2 deficient mice.
  • FIG. 23 shows the effects of G-2-MePE on the hippocampal long-term potentiation as measured by the fEPSP slope in MeCP2 deficient mice, compared to saline-treated MeCP2 deficient mice,
  • FIG. 24 depicts a graph showing effects of G-2-MePE on dendrite length as a function of distance from the cell soma.
  • FIGs. 25 and 26 depict graphs illustrating the open field behaviour of wild-type and finrl knockout mice, treated with either saline vehicle or G-2-MePE (100 mg/kg, i.p. in saline; 28 days). Movement (FIG. 25) and rearing activity (FIG. 26) are both elevated in finrl knockout mice. These effects are attenuated by treatment with G-2-MePE on trials 2 and 3 regarding locomotion (most likely indicative of enhanced cognition), and on all three trials regarding rearing (indicative of ablation of the hyperactivity seen in the vehicle-treated finrl knockout mice).
  • FIG. 26 is a graph showing effects of G-2-MePE (100 mg/kg, i.p.; 28 days) on behavior of wild-type and finrl knockout mice in the Successive Alley Test as compared to vehicle (saline) treated finrl KO and wild-type animals.
  • Fmrl KO mice showed no alley preference, attributed to the hyperactive phenotype of the model.
  • G-2-MePE treatment for 28 days reversed this behavioural pattern. Filled circles: vehicle treated wild-type; open circles: vehicle treated fmrl KO; filled triangles: G-2-MePE treated wild- type; open triangles: G-2-MePE treated finrl KO.
  • FIG. 27A-D show behavior of wild-type and finrl knockout mice given either saline vehicle or G-2-MePE (100 mg kg, i.p.; 28 days) on the elevated plus maze.
  • FIG, 27 A is a graph showing the effects of G-2-MePE on the total number of arm entries. Open arm entries, expressed as a percentage of total arm entries are depicted in FIG. 27B.
  • FIG. 27C is a graph showing the effects of G-2-MePE on the time spent in the center of the apparatus. Open bars: vehicle-treated wild type; shaded bars: G-2-MePE treated wild- type; hatched bars: vehicle treated fmrl KO; filled bars: G-2-MePE treated fmrl KO.
  • FIG. 28A - B depict graphs of the effects of G-2-MePE on numbers of arm entries in wild type and fmrl knock-out mice.
  • Open bars vehicle-treated wild type; shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle treated fmrl KO; filled bars: G-2-MePE treated fmrl KO.
  • FIG. 28C depicts a graph of the time spent in the center of a maze in G-2-MePE treated and vehicle-treated wild type and fmrl knockout mice. Open bars: vehicle-treated wild type; shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle treated fmrl KO; filled bars: G-2-MePE treated fmrl KO.
  • FIG. 29 shows a graph depicting the effects of G-2-MePE (100 mg/kg, i.p.; 28 days) on contextual-fear conditioning in wild-type and fmrl knockout mice.
  • Filled bar G-2-MePE treated wild-type; open bar: G-2-MePE treated fmrl KO; shaded bar: vehicle treated wild-type; hatched bar: vehicle treated frml KO,
  • FIG. 30A depicts a graph showing the effects of G-2-MePE treatment on social behavior: sniffing of a conspecific odor.
  • FIG. 30B shows the count of marbles buried by wild-type and fmrl knockout mice given either saline vehicle or G-2-MePE (100 mg/kg, i.p.; 28 days).
  • FIG. 30C shows the nest building score of wild-type and fmrl knockout mice given either saline vehicle or G-2-MePE. Open bars: vehicle-treated wild type; shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle treated fmrl KO; filled bars: G-2-MePE treated fmrl KO.
  • FIG. 31A shows an illustration depicting the microfluidic chamber used to culture the hippocampal cells
  • FIG. 31B shows a confocal micrograph showing GFP-positive untreated WT axons.
  • FIG. 31C shows fmrl KO axons showing spine supernumeracy.
  • FIG. 31D shows a clear reduction in spine number when hippocampal fmrl KO neurons were cultured with G-2-MePE at a concentration of 50nM, compared to untreated fmrl KO neurons (FIG. 31E).
  • FIG. 31F shows the effects of 0.5 nM G-2-MePE on fhirl KO hippocampal neurons..
  • FIG. 31G shows a substantial reduction in spine number observed in fmrl KO neurons cultured with G-2ePE at a concentration of 5nM, indicating a mild, treatment-positive effect.
  • FIG 31H shows a vehicle treated neuron indicating no toxicity, and no effect in the number of neuronal spines.
  • FIG. 311 shows fmrl KO hippocampal culture treated with MPEP at 20uM. The mean ( ⁇ SD) spine density was measured as number of spines per micrometer.
  • FIG. 32 is a graph showing the testis weight of each animal group (** p ⁇ 0.01 versus vehicle-treated wild-type mice).
  • FIG. 33 A is a photograph of a Western blot of pERK expression in lymphocytes obtained from the wild-type and fmrl KO animals treated either with vehicle or G-2- MePE (100 mg/kg, i.p.; 28 days).
  • FIG. 33B is a graph representing levels of pERK in the animal groups.
  • FIG. 33C is a Western blot analysis of total ERK and
  • FIG. 33D is a graph showing total levels of ERK in each animal group of the study.
  • FIG. 34A is a photograph of a Western blot of pAkt and Akt levels in lymphocytes obtained from wild-type and fmrl knockout mice administered either vehicle or G-2-MePE (100 mg/kg, i.p.; 28 days).
  • FIG. 34B is a graph showing levels of pAkt in the animal groups of the study. DETAILED DESCRIPTION
  • alkyl means a linear saturated hydrocarbyl group having from one to six carbon atoms, or a branched or cyclic saturated hydrocarbyl group having from three to six carbon atoms.
  • exemplary alkyl groups include straight and branched chain, or cyclic alkyl groups, methyl, ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, and hexyl.
  • animal includes humans and non-human animals, such as domestic animals
  • aralkyl means a group of the formula— (CH 2 )]. 2 Ar, where Ar is a 5- or 6-membered carbocyclic or heterocyclic aromatic ring, optionally substituted with 1 to 3 substituents selected from CI, Br,— OH,— O— alkyl,— C0 2 R s (where R 8 is H or alkyl), or— NR 8 R 9 , where R s is as described previously and R 9 is H or alkyl.
  • exemplary aralkyl groups include benzyl, 2-chlorobenzyl, 4-(dimethylamino)benzyl, phenethyl, 1 -pyrrolylmethyl, 2- thienylmethyl, and 3-pyridylmethyl.
  • disease includes any unhealthy condition of an animal including particularly Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, diabetes, motor disorders, seizures, cognitive dysfunctions due to aging and Autism Spectrum Disorders including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA).
  • Parkinson's disease Huntington's disease
  • Alzheimer's disease Alzheimer's disease
  • multiple sclerosis diabetes
  • motor disorders seizures
  • cognitive dysfunctions due to aging Autism Spectrum Disorders including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA).
  • fatty alcohol residue is a linear hydrocarbyl group having from seven to twenty carbon atoms, optionally containing up to three carbon-carbon double bonds.
  • exemplary fatty alcohol residues include decyl, pentadecyl, hexadecyl (cetyi), octadecyl (stearyl), oleyl, linoleyi, and eicosyl.
  • growth factor means an extracellular polypeptide-signaling molecule that stimulates a cell to grow or proliferate.
  • injury includes any acute damage of an animal including non-hemorrhagic stroke, traumatic brain injury, perinatal asphyxia associated with fetal distress such as that following abruption, cord occlusion or associated with intrauterine growth retardation, perinatal asphyxia associated with failure of adequate resuscitation or respiration, severe CNS insults associated with near miss drowning, near miss cot death, carbon monoxide inhalation, ammonia or other gaseous intoxication, cardiac arrest, coma, meningitis, hypoglycemia and status epilepticus, episodes of cerebral asphyxia associated with coronary bypass surgery, hypotensive episodes and hypertensive crises, cerebral trauma and toxic injury.
  • Memory disorders or “cognitive disorders” are disorders characterized by permanent or temporary impairment or loss of ability to learn, memorize or recall information.
  • Memory disorder can result from normal aging, injury to the brain, tumors, neurodegenerative disease, vascular conditions, genetic conditions (Huntington's disease), hydrocephalus, other diseases (Pick's disease, Creutzfeld-Jakob disease, AIDS, meningitis), toxic substances, nutritional deficiency, biochemical disorders, psychological or psychiatric dysfunctions.
  • the presence of memory disorder in a human can be established thorough examination of patient history, physical examination, laboratory tests, imagining tests and neuropsychological tests.
  • Standard neuropsychological tests include but are not limited to Brief Visual Memory Test-Revised (BVMT-R), Cambridge Neuropsychological Test Automated Battery (CANTAB), Children's Memory Scale (CMS), Contextual Memory Test, Continuous Recognition Memory Test (CMRT), Controlled Oral Word Association Test and Memory Functioning Questionnaire, Denman Neuropsychology Memory Scale, Digit Span and Letter Number Sequence sub-test of the Wechsler Adult Intelligence Scale-Ill, Fuld Object Memory Evaluation (FOME), Graham- Kendall Memory for Designs Test, Guild Memory Test, Hopkins Verbal Learning Test, Learning and Memory Battery (LAMB), Memory Assessment Clinic Self-Rating Scale (MAC-S), Memory Assessment Scales (MAS), Randt Memory Test, Recognition memory Test (RMT), Rey Auditory and Verbal Learning Test (RAVLT), Rivermead Behavioral Memory Test, Russell's Version of the Wechsler Memory Scale (RWMS), Spatial Working Memory, Test of Memory and Learning (TOMAL), Vermont Memory Scale (VMS), Wechsler Memory Scal
  • pharmaceutically acceptable excipient means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • salts means a salt that is pharmaceutically acceptable and has the desired pharmacological properties.
  • Such salts include salts that can be formed where acidic protons present in the compounds react with inorganic or organic bases.
  • Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum.
  • Suitable organic salts include those formed with organic bases such as amines e.g. ethanolamine, diethanolamine, triethano!amine, tromethamine, N- methylglucamine, and the like. Salts also include acid addition salts formed by reaction of an amine group or groups present in the compound with an acid.
  • Suitable acids include inorganic acids (e.g.
  • hydrochloric and hydrobromic acids and organic acids (e.g. acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid).
  • a pharmaceutically acceptable salt may be a mono-acid mono-salt or a di-salt; and similarly where there are more than two acidic groups present, some or all of such groups can be salified. The same reasoning can be applied when two or more amine groups are present in a compound.
  • protecting group is a group that selectively blocks one or more reactive sites in a multifunctional compound such that a chemical reaction can be carried out selectively on another unprotected reactive site and such that the group can readily be removed after the selective reaction is complete.
  • terapéuticaally effective amount means the amount of an agent that, when administered to an animal for treating a disease, is sufficient to effect treatment for that disease as measured using a test system recognized in the art.
  • treating or “treatment” of a disease may include preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development), providing relief from the symptoms or side-effects of the disease (including palliative treatment), and relieving the disease (causing regression of the disease).
  • the term "functional deficit” means a behavioral deficit associated with neurological damage. Such deficits include deficits of gait, as observed in patients with Parkinson's disease, motor abnormalities as observed in patients with Huntington's disease. Functional deficit also includes abnormal foot placement and memory disorders described herein.
  • seizure means an abnormal pattern of neural activity in the brain that results in a motor deficit or lack of motor control resulting in abnormal motion, including spasmodic motion.
  • Sensizure includes electroencephalographic abnormalities, whether or not accompanied by abnormal motor activity.
  • Implicit hydrogen atoms (such as hydrogen atoms on a pyrrolidine ring, etc.) are omitted from the formulae for clarity, but should be understood to be present.
  • ASDs Autism spectrum disorders
  • DSM-IV Diagnostic and Statistical Manual of Mental Disorders
  • Classical autism is a highly variable neurodevelopmental disorder. It is typically diagnosed during infancy or early childhood, with overt symptoms often apparent from the age of 6 months, and becoming established by 2-3 years. According to the criteria set out in the DSM- IV, diagnosis of autism requires a triad of symptoms to be present, including (a) impairments in social interaction, (b) impairments in communication and (c) restricted and repetitive interests and behaviours. Other dysfunctions, such as atypical eating, are also common but are not essential for diagnosis. Of these impairments, social interaction impairments are particularly important for diagnosis, and two of the following impairments must be present for a diagnosis of autism:
  • Communication impairments in autism may be manifest in one or more of the following ways: delay in (or total lack of) the development of spoken language; marked impairment in the ability to initiate or sustain a conversation; stereotyped and repetitive use of language; and/or a lack of spontaneous make-believe play. Restricted, repetitive and stereotyped patterns of behavior is also required for diagnosis, such as preoccupation with one or more interest considered abnormal in intensity, inflexible adherence to routines or rituals, repetitive motor mannerisms and/or persistent focus on parts of objects.
  • Autism is commonly associated with epilepsy or epileptiform activity in the electroencephalogram (EEG). As many as 60 percent of patients with autism have epileptiform activity in their EEGs (Spence and Schneider, 2009 Peel Res 65 : 599-606).
  • IGF-1 Central Nervous System
  • Asperger syndrome or Asperger Disorder is similar to autism, and shares certain features. Like autism, Asperger syndrome is also characterized by impairment in social interaction and this is accompanied by restricted and repetitive interests and behavior. Thus, diagnosis of Asperger syndrome is characterized by the same triad of impairments as autism. However, it differs from the other ASDs by having no general delay in language or cognitive development and no deficit in interest in the subject's environment. Moreover, Asperger syndrome is typically less severe in symptomology than classical autism and Asperger's patients may function with self-sufficiency and lead relatively normal lives.
  • Childhood disintegrative disorder also known as He!ler syndrome, is a condition in which children develop normally until age 2-4 years (i.e. later than in Autism and Rett syndrome), but then demonstrate a severe loss of social, communication and other skills. Childhood disintegrative disorder is very much like autism and both involve normal development followed by significant loss of language, social play and motor skills. However, childhood disintegrative disorder typically occurs later than autism, involves a more dramatic loss of skills and is far less common.
  • Diagnosis of CDD is dependent on dramatic loss of previously acquired skills in two or more of the following areas: language, social skills, play, motor skills (such as a dramatic decline in the ability to walk, climb, grasp, etc), bowel or bladder control (despite previously being toilet- trained).
  • the loss of developmental skills may be abrupt and take place over the course of days to weeks or may be more gradual.
  • PDD-NOS Pervasive Developmental Disorder - Not Otherwise Specified
  • PDD-NOS Pervasive Developmental Disorder - Not Otherwise Specified
  • the key criteria for diagnosis of an ASD include difficulty socializing with others, repetitive behaviors, and heightened sensitivities to certain stimuli. These are all found in the ASDs described above.
  • autism, Asperger syndrome, Rett syndrome and childhood disintegrative disorder all have other features that enable their specific diagnosis. When specific diagnosis of one of these four disorders cannot be made, but ASD is apparent, a diagnosis of PDD-NOS is made. Such a diagnosis may result from symptoms starting at a later age than is applicable for other conditions in the spectrum.
  • Rett Syndrome is a neurodevelopmental disorder that almost exclusively affects females (1 in 10:000 live births). RTT is classified as an autism spectrum disorder (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition - Revised (DSM-IV-R). Approximately 16,000 patients are currently affected by it in the U.S.A. (Rett Syndrome Research Trust data). For a diagnosis of Rett syndrome, the following symptoms are characteristic: impaired development from age 6-18 months; slowing of the rate of head growth starting from between age 3 months and 4 years; severely impaired language; repetitive and stereotypic hand movements; and gait abnormalities, e.g. toe-walking or unsteady stiff-legged walk.
  • RTT The onset of RTT usually begins between 6-18 months of age with a slowing of development and growth rates. This is followed by a regression phase (typically in children aged 1-4 years of age), pseudo-stationary phase (2-10 years of age) and a subsequent progressive late motor deterioration state.
  • RTT symptoms include sudden deceleration of growth and regression in language and motor skills including purposeful hand movements being replaced by stereotypical movements, autistic features, panic-like attacks, sleep cycle disturbances, tremors, seizures, respiratory dysfunctions (episodic apnea, hyperpnea), apraxia, dystonia, dyskinesia, hypotonia, progressive kyphosis or scoliosis and severe cognitive impairment. Most RTT patients survive into adulthood with severe disabilities and require 24-hour-a-day care.
  • Mecp2 maps to the X-chromosome (location Xq28) and for this reason, mutations to the gene in males are usually lethal.
  • RTT is a genetic disorder, less than 1 % of recorded cases are inherited; almost all mutations of Mecp2 occur de novo, with two thirds caused by mutations at 8 CpG dinucleotides (R106, R133, T158, R168, R255, R270, R294 and R306) located on the third and fourth exons.
  • MeCP2 is a protein that binds methylated CpG dinucleotides to exert transcriptional silencing of DNA in the CNS.
  • the key effect of a reduction or absence of MeCP2 appears to be an impairment of dendritic spine development and the formation of synapses.
  • MeCP2 expression appears to temporally correlate with brain maturation, explaining why symptoms typically appear around 18 months of age.
  • NLGN3 and NLGN4 are postsynaptic cell-adhesion molecules present in glutamatergic synapses. They play a role in coordinating presynaptic contact to the postsynaptic site and also interact with the postsynaptic scaffolding protein shank3. Mutations to NLGN3 and NLGN4 have been observed in the ASD population and account for perhaps 1% of all ASD cases (Lintas & Persico, 2008). Jamain and colleagues first reported a missense to NLGN3 and a frameshift to NLGN4 in two unrelated subjects, resulting in Asperger syndrome and classical autism respectively (Jamain et al, 2003).
  • the R451 C mutant therefore mouse represents a model for ASD based upon NLGN3 mutation.
  • mutation at the R451 position of NLGN3 results in a 'gain-of-function' mutation.
  • Presynaptic neurexin proteins induce postsynaptic differentiation in opposing dendrites through interactions with postsynaptic neuroligin counterparts. Mutations of the neurexin-l ct (NRXNl) gene have been reported in numerous studies (Sebat et al, 2007; Marshall et al, 2008; Kim et al, 2008; Yan et al, 2008) and these have been observed in the form of copy-number variants. As with NLGN mutations, when a mutation of the NRXNl gene is introduced to mice (in the form of gene knockout), a mutant strain with certain ASD-like features is produced (Etherton et al, 2009).
  • NRXNl knockout mice show a decrease in hippocampal miniature excitatory postsynaptic current (mEPSC) frequency and a decreased input-output relationship of evoked currents. These electrophysiological effects relate to decreased excitatory transmission in the hippocampus. In addition to decreased excitatory neurotransmission, NRX l knockout mice exhibit a decrease in pre-pulse inhibition, though social behaviour appears to be unaffected (Etherton et al, 2009).
  • mEPSC hippocampal miniature excitatory postsynaptic current
  • cell adhesion molecule 1 is an immunogolbulin family protein present both pre- and post- synaptically that is also involved in synaptic trans-cell adhesion activity (Biederer et a!, 2002). Mutations to the CADM1 gene have been detected in ASD patients and appear to represent a further possible cause of these conditions (Zhiling et al, 2008).
  • CADM1 knockout mice show increased anxiety-related behavior, impaired social interaction and impaired social memory and recognition.
  • CADM1 knockout mice demonstrate poorer motor skills (Takayanagi et al, 2010). These dysfunctions are again consistent with ASD symptomatology.
  • 22q 13 deletion syndrome (also known as Phelan-McDermid Syndrome), is a rare genetic disorder caused by a micro-deletion at the ql3.3 terminal end of chromosome 22. This micro- deletion is rarely uncovered by typical genetic screening and a fluorescence in situ hybridization test is recommended to confirm the diagnosis. Recent work indicates the syndrome is caused by errors in the gene shank3, a postsynaptic density protein critical for normal neuronal functioning. Interestingly, errors En this gene have also been associated with ASD and 22q ) 3 deletion syndrome can commonly lead to an ASD diagnosis (Durand et al, 2007; Moessner et al, 2007; Sykes et al, 2009). Given the close association of 22ql 3 deletion syndrome and the consequential di gnosis of ASD, a mutant mouse model of this mutation has been developed.
  • the shank3 knockout mouse exhibits several deficits that mirror ASD symptoms, including reduced ultrasonic vocalizations (i.e. diminished social communication) as well as impaired social interaction time between mice.
  • these mice have impaired hippocampal CA1 excitatory transmission, measured by input-output relationship of evoked currents and impaired long-term potentiation (LTP).
  • LTP is believed to be a physiological process underlying memory formation and consolidation.
  • the model exhibits a similar phenotype to the NLGN4 knockout, consistent with ASD.
  • 22ql 3 deletion syndrome itself is very rare. However, it provides important information that involvement specific genes may have in the etiology of ASDs. In addition to shank3, this disorder reveals a further possible gene defect in ASD. Of the 50 or so cases of 22q 13 deletion syndrome described, all but one have a gene deletion that extends beyond shank3 to include a further gene, known as the Islet Brain-2 gene (IB2) (Sebat et al, 2007). The IB2 protein interacts with many other proteins including MAP kinases and amyloid precursor protein, appears to influence protein trafficking in neurites, and is enriched at postsynaptic densities (Giza et al, 2010).
  • IB2 Islet Brain-2 gene
  • mice lacking the protein exhibit impaired social interaction (reduced social sniffing and interaction time), reduced exploration and cognitive and motoric deficits (Giza et al, 2010). This behavioral phenotype was associated with reduced excitatory transmission in cerebellar cells. As with shank3 knockout, the phenotype of IB2 mutation is therefore also consistent with ASD.
  • Fragile X Syndrome is caused by the expansion of a single trinucleotide gene sequence (CGG) on the X-chromosome that results in failure to express the protein coded by the fmrl gene.
  • FMR1 fragment X mental retardation 1
  • FXS can cause a child to have autism (Hagerman et al, 2010); in 2-6% of all children diagnosed with autism the cause is FXS gene mutation.
  • approximately 30% of FXS children have some degree of autism and a further 30% are diagnosed with PDD-NOS (Hagerman et al, 2010).
  • Fragile X Syndrome is the most common known single gene cause of autism.
  • FMR1 knockout mice have been developed as a model of FXS and, therefore, as a further model of ASD. Knockout mutation of the FMR1 gene has been shown to result in neuronal connectivity deficits such as abnormal dendritic spine development and pruning (Comery et al, 1997), along with an associated dysregulation of dendritic scaffold proteins (including shank 1) and glutamate receptor subunits in postsynaptic densities (Schutt et al, 2009).
  • Rett syndrome appears to have an almost monogenetic basis and may be modelled in mice with good face validity. Rett syndrome is thought be caused, in up to 96% of cases, by a defect in the Mecp2 gene (Zoghbi, 2005). As a result, MeCP2 knockout mutant mice provide an animal model with all the hallmarks of clinical Rett syndrome, with a phenotype showing some overlap with the NLGN4, shank3 and IB2 knockout models of ASD.
  • MeCP2 knockout mice display a clear impairment in LTP in the hippocampus along with a corresponding decrease in social and spatial memory (Moretti et al, 2006) and impaired object recognition (Schaevitz et al, 2010). This impairment in LTP is accompanied by a decrease in dendritic spine density. Patients with Rett Syndrome show reduced dendritic spine density (Belichenko et al., 1994 Neuroreport 5:1509- 1513).
  • ASDs in human beings share many features of cognitive or developmental disorders in animals, including rodents. Therefore, studies of therapies of ASDs in rodents such as mice and rats are reasonably predictive of results obtained in human beings.
  • a common feature seen in autism, Fragile X Syndrome and Rett Syndrome is the presence of neuronal connectivity deficits, reflected in either decreased dendritic spine density or enhanced dendritic spine density with immature synapses. The functional consequences of these morphological changes are similar in animal models of these disorders, reflected as deficits in LTP, for example,
  • ASDs that comprises impaired neurite development, impaired synaptic connectivity and a corresponding impairment in social and cognitive functioning as a result.
  • Such synaptic dysfunctions result from genetically altered functions of postsynaptic density proteins.
  • Normal neurite growth and postsynaptic development may be regulated and augmented by growth factors such as brain derived neurotrophic factor (BDNF; Chapleau et al, 2009) and insulin-like growth factor-1 (IGF-1; Riikonen et al, 2006; Tropea et al, 2009).
  • BDNF brain derived neurotrophic factor
  • IGF-1 insulin-like growth factor-1
  • IGF-1 is essential for normal dendritic spine growth and synapse formation (Cheng et al., 2003 JNeurosci Res. 73:1-9).
  • cG- 2AllylP is a small molecule analog of the terminal tripeptide of IGF-1, IGFI (I-3). As an IGF-1 mimetic analog, cG-2AllylP exerts trophic and neuroprotective effects in various animal models. cG-2AllylP is therefore effective at treating ASD and FXS symptoms such as those relating to synaptic dysfunctions resulting from the gene mutations described above.
  • ASD patients In clinical terms, ASD patients, presenting with autism, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and PDD-NOS, as well as patients with 22ql3 deletion syndrome, Fragile X Syndrome and pathological demand avoidance are treated with cG- 2AllylP. Patients exhibit social and communication impairments as well as cognitive deficit. Treatment with cG-2AllylP, for example, on a daily basis and in another example, by the oral route, is observed to induce an improvement in stereotypic repetitive movements, improved social functioning and improved cognitive performance following drug treatment
  • G-2MePE In animal models of ASDs, daily G-2MePE treatment by oral gavage or intraperitoneal injection to knockout mice will improve ASD-!ike symptoms.
  • G-2MePE is effective in the following ASD mutant mouse models: NLGN3 (R451C) mutant, NLGN4 knockout, NRXNl knockout, CADMI knockout, shank3 knockout, IB2 knockout, FMR1 knockout and MeCP2 knockout.
  • NLGN3 (R451C) mutant NLGN4 knockout
  • NRXNl knockout CADMI knockout
  • shank3 knockout shank3 knockout
  • IB2 knockout FMR1 knockout
  • MeCP2 knockout MeCP2 knockout.
  • G-2MePE When administered sub-chronically (1-10 weeks) on a daily basis, G-2MePE is effective at improving LTP in the hippocampus following burst stimulation or high frequency stimulation.
  • G-2MePE increases excitatory neurotransmission as measured by field extracellular postsynaptic potential electrophysiological recordings in cortex, hippocampus and cerebellum.
  • G-2MePE is observed to improve cognitive and motoric outcome tests of cognitive performance.
  • G-2-MePE improves performance in the Morris water maze and radial arm maze tests.
  • G-2MePE administered to ASD mutant mice, increases time spent by knockout males in social interaction with wild-type females.
  • ultrasonic vocalizations to female wild type mice is increased.
  • G-2MePE is a member of the compounds of GPE analogs disclosed herein, any of the disclosed compounds also can be effective in treating symptoms of ASDs.
  • this invention can provide more than short-term management of symptoms, Rather, compounds and methods of this invention can improve neural function, promote neuronal cell migration, promote neurogenesis, promote neuronal stem cell differentiation, promote axonal and dendritic outgrowth, and promote synaptic transmission, thereby relieving adverse symptoms of ASDs.
  • this invention provides compounds of Formula 1 and Formula 2:
  • n 0 or 1 ;
  • n 0 or 1 ;
  • X is H or— NR 6 R 7 ;
  • Y is H, alkyl,— C0 2 R 5 , or— CONR 6 R 7 ;
  • Z is H, alkyl,— C0 2 R 5 or— CONR 6 R 7 ;
  • R 1 is H, alkyl, or aralkyl
  • R 2 , R 3 , and R 4 are independently H or alkyl
  • each R 5 is independently H, alkyl, or a fatty alcohol residue
  • each R 6 and R 7 is independently H, alkyl, or aralkyl, or -NR 6 R 7 is pyrrolidino, piperidino, or morpholino;
  • the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt thereof.
  • this invention includes:
  • n is i ;
  • R , R J , R", and R s is not hydrogen;
  • X is— NR 6 R 7 ;
  • Y is— C0 2 R 5 or— C0 2 NR 6 R 7 ;
  • (g) Z is— C0 2 R 5 or— C0 2 NR 6 R 7 .
  • R 6 and R 7 are independently alkyl or aralkyl.
  • the more preferred embodiment is a compound of Formula I wherein X is -NR 6 R 7 and both R 6 and R 7 are alkyl.
  • Compounds of this invention can have anti -inflammatory, anti-apoptotic, anti-necrotic and neuroprotective effects. Their activity in vivo can be measured by cell counts, specific staining of desired markers, or by methods such as those discussed in Klempt ND et al: Hypoxia- ischemia induces transforming growth factor ⁇ mRNA in the infant rat brain. Molecular Brain Research: 13: 93- 101. Their activity can also be measured in vitro using methods known in the art or described herein.
  • Memory loss and memory impairment are distressing to patients affected and their families. Memory loss or impairment can result from normal aging, injury to the brain, neurodegenerative disease and psychological or psychiatric dysfunctions. It is therefore of great benefit to patients, their families and to society that novel compounds are identified and characterized that enhance memory and/or cognitive function, and treat or prevent memory loss or impairment.
  • One such useful system is the rat. It is known that with aging, rats and other animals (including human beings) can exhibit symptoms of memory loss, memory impairment and other cognitive dysfunctions. Further, it is known that studies in rats of therapeutic agents are predictive of therapeutic effects in humans. Thus, studies of effects of GPE and G-2-MePE and cognitive function in aging rats are reasonably predictive of therapeutic effects of those agents in aging human beings that have or are prone to acquiring memory deficits or other cognitive dysfunction. Compounds of this invention can enhance cognitive function and/or treat memory disorders. The cognitive enhancing activity and therapeutic activity in vivo can be measured by standard neuropsychological or behavioral tests known to individuals skilled in the art.
  • Such tests can be chosen from a wide range of available tests described above, and will vary depending on the cognitive function to be tested and the condition of the animal.
  • Standard behavioral tests useful for testing cognitive function in experimental animals include but are not limited to the Morris Water Maze test, passive avoidance response test, novel object recognition test, olfactory discrimination test, the 8-arm radial maze test and the T-maze test. These tests are directly applicable to studies of effects of GPE and G-2-MePE on cognitive function in aging rats.
  • the compounds of this invention are also expected to have pharmacological and therapeutic activities similar to those of GPE, and these activities may be measured by the methods known in the art, and discussed in the documents cited herein, and by methods used for measuring the activity of GPE.
  • the therapeutic ratio of a compound can be determined, for example, by comparing the dose that gives effective anti-inflammatory, anti-apoptotic and anti-necrotic activity in a suitable in vivo model such as a hypoxic-ischemic injury (Sirimanne ES, Guan J, Williams CE and Gluckman PD: Two models for determining the mechanisms of damage and repair after hypoxic- ischemic injury in the developing rat brain (Journal of Neuroscience Methods: 55: 7-14, 1994) in a suitable animal species such as the rat, with the dose that gives significant observable side-effects in the test animal species.
  • a suitable in vivo model such as a hypoxic-ischemic injury (Sirimanne ES, Guan J, Williams CE and Gluckman PD: Two models for determining the mechanisms of damage and repair after hypoxic- ischemic injury in the developing rat brain (Journal of Neuroscience Methods: 55: 7-14, 1994) in a suitable animal species such as the rat,
  • the therapeutic ratio of a compound can also be determined, for example by comparing the dose that gives effective cognitive function enhancement or treats a memory disorder in a suitable in vivo model (Examples 4, 5 and 6 below) in a suitable animal species such as the rat, with the dose that gives significant weight loss (or other observable side-effects) in the test animal species.
  • Compounds of this invention can be useful in treatment of a variety of neurodegenerative disorders, including hypoxi a/ischemia and neuronal degeneration (U.S. Pat. No. 7,041 ,314), traumatic brain injury, motor disorders and seizures, stroke, and cardiac artery bypass graft surgery (U.S. Pat. No. 7,605, 177), non-convulsive seizures (U.S. Pat. No. 7,714,020), and disorders of cognitive function (U.S. Appl. No. 12/903,844). Additionally, as described more fully herein below, compounds of this invention can be useful for treating Rett Syndrome, including prolonging life, increasing neuronal activity and treating seizures associated with Rett Syndrome.
  • GPE being a naturally occurring peptide, is rapidly degraded in vivo and in vitro, and its utility in chronic therapy of patients with Rett Syndrome is therefore unclear.
  • compounds of this invention can be administered in therapeutically effective amounts by any of the usual modes known in the art, either singly or in combination with at least one other compound of this invention and/or at least one other conventional therapeutic agent for the disease being treated.
  • a therapeutically effective amount may vary widely depending on the disease or injury, the severity of the disease, the age and rel tive health of the animal being treated, the potency of the compound(s), and other factors.
  • anti-inflammatory, anti-apoptotic, anti-necrotic, anti-neurodegenerative, therapeutically effective amounts of compounds of this invention can range from about 0.001 milligrams per kilogram (mg/kg) to about 100 (mg/kg) mass of the animal, for example, about 0.1 to about 10 mg/kg, with lower doses such as about 0.001 to about 0.1 mg/Kg, e.g. about 0.01 mg Kg, being appropriate for administration through the cerebrospinal fluid, such as by intracerebroventricular administration, and higher doses such as about 1 to about 100 mg Kg, e.g. about 10 mg/Kg, being appropriate for administration by methods such as oral, systemic (e.g. transdermal), or parenteral (e.g. intravenous) administration.
  • a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically effective amount of a compound of this invention for a given disease or injury.
  • compounds of this invention can be administered as pharmaceutical compositions by one of the following routes: oral, topical, systemic (e.g. transdermal, intranasal, or by suppository), or parenteral (e.g. intramuscular, subcutaneous, or intravenous injection), by administration to the CNS (e.g. by intraspinal or intercisternal injection); by implantation, and by infusion through such devices as osmotic pumps, implantable pumps, transdermal patches, and the like.
  • routes e.g. oral, topical, systemic (e.g. transdermal, intranasal, or by suppository), or parenteral (e.g. intramuscular, subcutaneous, or intravenous injection), by administration to the CNS (e.g. by intraspinal or intercisternal injection); by implantation, and by infusion through such devices as osmotic pumps, implantable pumps, transdermal patches, and the like.
  • compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulation, solutions, suspensions, elixirs, aerosols, soluble gels or any other appropriate compositions; and comprise at least one compound of this invention in combination with at least one pharmaceutically acceptable or physiological acceptable excipient.
  • Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions, may be found in such standard references as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 th ed., Lippincott, Williams & Wilkins, 2000.
  • Suitable liquid carriers especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, glycols, and the like, with isotonic solutions being preferred for intravenous, intraspinal, and intracisternal administration and vehicles such as artificial cerebrospinal fluid being also especially suitable for administration of the compound to the CNS.
  • isotonic solutions being preferred for intravenous, intraspinal, and intracisternal administration and vehicles
  • artificial cerebrospinal fluid being also especially suitable for administration of the compound to the CNS.
  • Compounds of this invention can be administered orally, in tablets or capsules.
  • compounds of this invention can be prepared in water-in-oil emulsions in the form or microemulsions, coarse emulsions, liquid crystals, or nanocapsules (U.S. Appl. No. 12/283,684, now U.S. Pat. No. 7,887,839 issued February 15, 201 1).
  • compounds of this invention can have substantial oral bioavailability, they can be advantageously used for convenient and chronic administration.
  • orally available compositions include soluble hydrogels containing active compounds, thus permitting oral administration of neuroprotective compounds without the need for a patient to swallow a tablet or capsule. Such slow-release materials and gels are known in the art.
  • Compounds of this invention can be administered after or before onset of a condition that is likely to result in neurodegeneration or a symptom thereof. For example, it is known that hypoxia/ischemia can occur during coronary artery bypass graft (CABG) surgery. Thus, a patient can be pre-treated with a compound of this invention before being placed on an extracorporeal oxygenation system. In some embodiments, it can be desirable to administer a compound of this invention beginning about 4 hours before surgery or before an event that is likely to lead to traumatic or other neurological injury. In other embodiments, it can be desirable to infuse a compound of this invention during the surgery or during a surgical procedure to repair a neurological injury.
  • CABG coronary artery bypass graft
  • Compounds of this invention can also be used in emergency situations, for example in a patient that has just experienced a stroke, hypoxic event, traumatic brain injury or other acute insult. In such situations, a compound of this invention can be administered immediately after a diagnosis of neural injury is made.
  • kits containing compound of this invention can be prepared in advance of use in the field.
  • a kit can contain a vial containing a compound of the invention in a pharmaceutically acceptable formulation (e.g., for injection or oral administration), along with a syringe or other delivery device, and instructions for use.
  • a compound of this invention can be administered along with an anticonvulsant.
  • Many anticonvulsants are known in the art and need not be described in detail herein.
  • secondary neurological injuries can occur after a primary insult such as a traumatic injury, stroke or surgical procedure.
  • a primary insult such as a traumatic injury, stroke or surgical procedure.
  • inflammation of neural tissue can lead to neurodegeneration.
  • Secondary injuries can be reflected by increased activation of inflammatory cells (e.g., astrocytes and/or microglia), and actions of inflammatory mediators can cause neurological damage.
  • inflammatory cells e.g., astrocytes and/or microglia
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polylactides (U.S. Pat. No.
  • gel compositions are soluble in aqueous solutions, are biocompatible, non-toxic and therefore can be used for administering compounds of this invention to any mucosal surface, including the oral cavity, nasopharynx, urogenital tract, intestine or rectum.
  • Sustained-release compositions also include a liposomally entrapped compound.
  • Liposomes containing the compound are prepared by methods known per se: DE 3,218,121; Epstein et al., 1985; Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102, 324.
  • liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted for the most efficacious therapy.
  • Compounds of this invention can also be attached to polyethylene glycol ("PEGylated”) to increase their lifetime in vivo, based on, e.g., the conjugate technology described in WO 95/32003.
  • PEGylated polyethylene glycol
  • compounds of this invention when administered as an anti- inflammatory, an anti-apoptotic agent, an anti-necrotic agent, or an anti -neurodegenerative agent, compounds of this invention can be administered orally.
  • the amount of a compound of this invention in the composition can vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of ordinary skill in the art.
  • the final composition can comprise from about 0.0001 percent by weight (% w) to about 10% w of the compound of this invention, preferably about 0.001% w to about 1% w, with the remainder being an excipient or excipients.
  • a composition may optionally contain, in addition to a compound of this invention, at least one agent selected from, for example, growth factors and associated derivatives (insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), transforming growth factor- ⁇ , activin, growth hormone, nerve growth factor, brain-derived neurotrophic factor (BDNF), growth hormone binding protein, IGF-binding proteins (especially IGFBP-3), basic fibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor.
  • growth factors and associated derivatives IGF-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-I), transforming growth factor- ⁇ , activin, growth hormone, nerve growth factor, brain-derived neurotrophic factor (BDNF), growth hormone binding protein, IGF-binding proteins (especially IGFBP-3), basic fibroblast growth
  • Additional members of the FGF family include, for example, int-2, fibroblast growth factor homologous factor- 1 (FHF-1 ), FHF-2, FHF-3 and FHF-4, karatinocyte growth factor 2, glial-activating factor, FGF- 30 and FGF- 16, ciliary neurotrophic factor, brain derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial-cell line derived neurotrophic factor, activity-dependant neurotrophic factor, cytokine leukaemia inhibiting factor, oncostatin M, interleukin), ⁇ -, ⁇ -, ⁇ -, or consensus interferon, and TNF-a.
  • FHF-1 fibroblast growth factor homologous factor- 1
  • FHF-2 fibroblast growth factor homologous factor- 1
  • FHF-2 fibroblast growth factor homologous factor-2
  • FHF-3 and FHF-4 karatinocyte growth factor 2
  • FGF- 30 and FGF- 16 ciliary neurotrophic
  • neuroprotective therapeutic agents include, for example, clomethiazole; kynurenic acid, Semax, tacrolimus, L-threo-l -phenyl-2-decanoylamino- 3-morpholino-l -propanol, andrenocorticotropin-(4-9) analog [ORG 2766] and dizolcipine (MK- 801 ), selegiline; glutamate antagonists such as mematine (Namenda) NPS1506, GV1505260, MK-801 , GV150526; AMPA antagonists such as 2,3-dihydroxy-6-nitro- 7-sulfamoylbenzo(f)quinoxaiine (NBQX), LY303070 and LY300164; anti-inflammatory agents directed against the addressin MAdCAM-3 and/or its integrin a4 receptors ( ⁇ 4 ⁇ 1 and ⁇ 4 ⁇ 7), such as anti-MAdCAM- l inAb MECA-367 (ATCC accession no.
  • a composition may include a selective serotonin reuptake inhibitor such as fluoxetine, a selective norepinephine reuptake inhibitor such as viloxazine, or an atypical anti-psychotic such as risperidone. Most of these agents, especially the peptides such as the growth factors, etc., are not orally active, and will require administration by injection or infusion. Preparation of Compositions
  • the starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St.Louis, Mo.), or are prepared by methods well known to the person of ordinary skill in the art following procedures described in such references as Fieser and Fieser's Reagents for Organic Synthesis, vols 1 -17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1 89; Organic Reactions, vols.
  • Starting materials, intermediates, and compounds of this invention may be isolated and purified using conventional techniques, including filtration, distillation, crystallization, chromatography, and the like. They may be characterized using conventional methods, including physical constants and spectra! data.
  • Compounds of Formula 1 are analogs of GPE, or modifications thereof, such as esters or amides. In general, they may be prepared by methods such as are already well-known to persons of ordinary skill in the art of peptide and modified peptide synthesis, following the reaction schemes set forth in the FIGs 1-11 accompanying this specification, or by following other methods well-known to those of ordinary skill in the art of the synthesis of peptides and analogs.
  • synthetic production of the polypeptides of the invention may be according to the solid-phase synthetic method described by Merrifield et al. Solid phase peptide synthesis.
  • I The synthesis of a tetrapeptide: J. Amer. Chem. Soc: 85, 2149-2156, 1963.
  • This technique is well understood and is a common method for preparation of peptides.
  • the general concept of this method depends on attachment of the first amino acid of the chain to a solid polymer by a covalent bond. Succeeding protected amino acids are added, one at a time (stepwise strategy), or in blocks (segment strategy), until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved off. By this procedure, reagents and by-products are removed by filtration, thus eliminating the necessity of purifying intermediaries.
  • Amino acids may be attached to any suitable polymer as a resin.
  • the resin must contain a functional group to which the first protected amino acid can be firmly linked by a covalent bond.
  • Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethylmethacrylate and polystyrene. Suitable resins are commercially available and well known to those of skill in the art.
  • protective groups usable in such synthesis include tert-butyloxycarbonyi (BOC), benzyl (Bzl), t-amyloxycarbonyl (Aoc), tosyl (Tos), o-bromo- phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BzlCl 2 ), and phenylmethoxycarbonyl (Z or CBZ). Additional protective groups are identified in Merrifield, cited above, as well as in McOmie JFW: Protective Groups in Organic Chemistry, Plenum Press, New York, 1973, both references expressly incorporated fully herein.
  • General procedures for preparing peptides of this invention involve initially attaching a carboxyl-terminal protected amino acid to the resin. After attachment the resin is filtered, washed and the protecting group (desirably BOC) on the I-amino group of the carboxyl-terminal amino acid is removed. The removal of this protecting group must take place, of course, without breaking the bond between that amino acid and the resin. The next amino, and if necessary, side chain protected amino acid, is then coupled to the free I-amino group of the amino acid on the resin. This coupling takes place by the formation of an amide bond between the free carboxyl group of the second amino acid and the amino group of the first amino acid attached to the resin.
  • the protecting group desirably BOC
  • peptide synthesis is described in Bodanszky et al, Peptide Synthesis, 2nd ed, John Wiley and Sons, New York, 1976.
  • the peptides of the invention may also be synthesized using standard solution peptide synthesis methodologies, involving either stepwise or block coupling of amino acids or peptide fragments using chemical or enzymatic methods of amide bond formation.
  • standard solution peptide synthesis methodologies involving either stepwise or block coupling of amino acids or peptide fragments using chemical or enzymatic methods of amide bond formation.
  • analogs in which the glycine residue of GPE is replaced by an alternative amino acid, or by a non-amino acid may conveniently be prepared by the preparation of a C- protected proline-glutamic acid dipeptide (such as the dibenzyl ester), and coupling that dipeptide with an N-protected glycine analog, such as BOC-N-methylglycine, BOC-L-valine, N- pyrrolidineacetic acid, and the like, followed by deprotection, as illustrated in FIGs.
  • a C- protected proline-glutamic acid dipeptide such as the dibenzyl ester
  • an N-protected glycine analog such as BOC-N-methylglycine, BOC-L-valine, N- pyrrolidineacetic acid, and the like
  • Analogs in which the glutamic acid residue of GPE is replaced by an alternative amino acid or an amino acid amide or ester may conveniently be prepared by the preparation of an N-protected glycine-L-proline dipeptide (such as BOC-glycyl-L-proline), and coupling that dipeptide with a C-protected glutamic acid or analog thereof, such as ieri-butyl ⁇ -aminobutyrate, methyl 4-amino- 4-dimethylcarbamoylbutyrate, L-glutamine methyl ester, dimethyl I-methylglutamate, etc.
  • an N-protected glycine-L-proline dipeptide such as BOC-glycyl-L-proline
  • C-protected glutamic acid or analog thereof such as ieri-butyl ⁇ -aminobutyrate, methyl 4-amino- 4-dimethylcarbamoylbutyrate, L-glutamine methyl ester,
  • Lactones may be prepared by the preparation of an appropriate mono-acid-mono-ester derivative and reduction Analogs in which R 2 is alkyl may conveniently be prepared simply by use of the appropriate 2-alkyIproline in the synthesis, and similarly analogs in which R 3 is alkyl may conveniently be prepared by the use of the appropriate N-alkylglutamic acid or analog in the synthesis. Where modifications are to be made to two or more amino acids, the coupling techniques will still be the same, with just more than one modified amino acid or analog being used in the synthesis.
  • the choice of appropriate protecting groups for the method chosen (solid- phase or solution-phase), and of appropriate substrates if solid-phase synthesis is used, will be within the skill of a person of ordinary skill in the art.
  • Compounds of Formula 2 may be prepared from suitably protected 5-oxo-L-proline or analogs or derivatives thereof, following methods such as the coupling of the proline carboxyl group with a protected glutamic acid or analog or derivative to give an analog of intermediate A of FIG. 2, comparable to the coupling reaction shown in FIG. 2, and then alkylating the pyrrolidine nitrogen with a group of the formula A— -(CH 2 ) m — CH(R')— CH 2 R, protected at A if necessary, where R is a leaving group under alkylation conditions.
  • the suitably protected 5-oxo-L-proline may first by alkylated at the pyrrolidine nitrogen to give an analog of intermediate B of FIG. 4, and then coupling this with a suitably protected glutamic acid or analog or derivative in the manner shown in FIGs. 4 though 9.
  • L-2-Methylproline and L-glutamic acid dibenzyl ester -toluenesulphonate were purchased from Bachem, jV-benzyloxycarbonyl-glycine from Acros Organics and bis(2-oxo-3- oxazo]idinyl)phosphinic chloride (BoPCl, 97%) from Aldrich Chem. Co.
  • Dipeptide 5 was shown to be exclusively the frafw-orientated conformer by NMR analysis: R f 0.50 (20% MeOH - CH 2 C1 2 ); [ ] D -62.3 (c 0.20 in CH 2 C1 2 ); v max (filmycm "1 3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251 , 1219, 1 179, 1053, 1027, 965, 912, 735 and 698; ⁇ 3 ⁇ 4 (300 MHz; CDC1 3 ; Me 4 Si) 1.59 (3H, s, Proa-CH 3 ), 1 .89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Prop-3 ⁇ 4H B ), 2.01 (2H, dtt, J 18.7, 6.2 and 6.2, Proy-H 2 ), 2.25-2.40 (IH, m, Prop-H A 3 ⁇ 4), 3.54 (2H, t, / 6.6
  • Triethylamine (0.50 cm 3 , 3.59 mmoi) was added dropwise to a solution of dipeptide 5 (0.36 g, 1.12 mmoi) and L-glutamic acid dibenzyl ester Moluenesulphonate 6 (0.73 g, 1.46 mmoi) in methylene chloride (60 cm 3 ) under nitrogen at room temperature, and the reaction mixture stirred for 10 min.
  • Bis(2-oxo-3-oxazoIidinyl)phosphinic chloride (BoPCl, 97%) (0.37 g, 1.41 mmoi) was added and the colourless solution stirred for 17 h.
  • Tripeptide 7 was shown to be exclusively the irarar-orientated conformer by NMR analysis: R f 0.55 (EtOAc); [a] D -41.9 (c 0.29 in CH 2 C1 2 ); v roax (film)/cm " ' 3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1 166, 1051, 91 1 , 737 and 697; ⁇ 3 ⁇ 4 (400 MHz; CDC1 3 ; Me 4 Si) 1.64 (3H, s, Proot-CH 3 ), 1.72 (IH, dt, J 12.8, 7.6 and 7.6, Prop-3 ⁇ 4H B ), 1.92 (2H, 5 lines, J 6.7, Proy-H 2 ), 2.04 (IH, 6 lines, J 7.3 Glup-3 ⁇ 4H B ), 2.17- 2.27 (IH, m, GluP-H A 3 ⁇ 4), 2.35-2
  • Glycyl-L-2-methylprolyl-L-glutamic acid G-2-MePE
  • G-2-MePE was shown to be a 73 :27 trans cis mixture of conformers by ⁇ NMR analysis (the ratio was estimated from the relative intensities of the double doublet and triplet at ⁇ 4.18 and 3.71 , assigned to the Glu -H protons of the major and minor conformers, respectively): mp 144 °C*; [oc] D -52.4 (c 0.19 in H 2 0); ⁇ (300 MHz; D 2 0; internal MeOH) 1.52 (3H, s, Proa-CHj), 1.81-2.21 (6H, m, Prop-3 ⁇ 4, Proy-H, and GluP-H 2 ), 2.34 (1.46H, t, J 7.2, Gluy-H 2 ), 2.42 * (0.54H, t, 77.3, Gluy-H 2 ), 3.50-3.66 (2H, m, Pro6-H 2 ), 3.71 * (0.27H, t, J 6.2, GIuoc-H), 3.85 (
  • GPE analogs were examined in a series of experiments in vitro to determine their effects on neurodegeneration of neural cells of different origin.
  • the in vitro systems described herein are well-established in the art and are known to be predictive of neuroprotective effects observed in vivo, including effects in humans suffering from neurodegenerative disorders.
  • DMEM fetal calf serum
  • a dam was sacrificed by C0 2 -treatment, and then was prepared for caesarean section.
  • a whole brain was removed from the skull with the ventral side facing upwards in DMEM/F12 medium.
  • the striatum was dissected out from both hemispheres under a stereomicroscope and the striatal tissue was placed into a Falcon tube on ice. Striatal tissue was then triturated using a PI 000 pipettor in 1 ml of volume. The tissue was triturated by gently pipetting the solution up and down into the pipette tip about 15 times, using shearing force on alternate outflows. The tissue pieces settled to the bottom of the Falcon tube within 30 seconds. The supernatant containing a suspension of dissociated single cells was then transferred to a new sterile Falcon tube on ice.
  • tissue pieces were triturated again to avoid excessively damaging already dissociated cells, by over triturating them.
  • 1 milliliter of ice-cold DMEM/F12 medium was added to the tissue pieces in the first tube and triturated as before.
  • the tissue pieces were allowed to settle and the supernatant was removed to a new sterile Falcon tube on ice.
  • the cells were centrifuged at 250g for 5 minutes at 4°C.
  • Striatal cells were plated into Poly-L-Lysine (O. l mg/ml) coated 96-weIl plates (the inner
  • Cortices were placed on ice in PBS+0.65%(+)- glucose and centrifuged at 350g for 5 minutes. The supernatant was removed and trypsin/EDTA (0.05%/0.53mM) was added for 8min at 37°C. The reaction was stopped by adding an equal amount of DMEM and 10% fetal calf serum. The supernatant was removed by centrifugation followed by two subsequent washes in
  • the cells were triturated once with a glass Pasteur pipette in 1 ml of Neurobasal B27 medium and subsequently twice by using a 1 ml insulin syringe with a 22 gauge needle.
  • the cell suspension was passed through a ⁇ ⁇ cell strainer and rinsed by 1 ml of Neurobasal B27 medium. Cells were counted and adjusted to 50,000 cells per 60 ⁇ 1.
  • 96-well plates were coated with 0.2mg/ml Poly-L-Lysine and subsequently coated with 2 g/mI laminin in PBS, after which 60 ⁇ of cortical astrocyte-conditioned medium was added to each well. Subsequently, 60 ⁇ 1 of cortical cell suspension was added. The cells were cultivated in the presence of 10% C0 2 at 37°C under 100% humidity. At day 1, there was a complete medium change (1 : 1 - Neurobasal/B27 and astrocyte-conditioned medium) with addition of 1 ⁇ cytosine-B-D-arabino-furanoside (mitosis inhibitor). On days 2 and 5, 2/3 of the medium was changed. Cerebellar Microexplants from P8 Animals: Preparation, Cultivation and Fixation
  • Laminated cerebellar cortices of the two hemispheres were explanted from a P8 rat, cut into small pieces in PBS +0,65% D(+) glucose solution and triturated with a 23gauge needle and subsequently pressed through a 125 ⁇ pore size sieve.
  • the obtained microexplants were centrifuged (60g) twice (media change) into serum-free BSA-supplemented STARTV-medium (Biochrom).
  • 40 ⁇ 1 of cell suspension was adhered for 3 hours on a O.lmg/ml Poly- L-Lysine coated cover slip placed in 35 mm sized 6 well plates in the presence of 5% C0 2 under 100% humidity at 34°C.
  • okadaic acid is an art-recognized toxin that is known to cause injury to neurons. Further, recovery of neural cells or neural cell function after injury by okadaic acid is recognized to be predictive of recoveries from injuries caused by other toxins.
  • GPE I nM- ImM
  • G-2-MePE I nM- ImM
  • the survival rate was determined by a colorimetric end-point MTT-assay at 5 5nm in a multi-well plate reader.
  • four windows field of 0.65 mm 2 ) with highest cell density were chosen and cells displaying neurite outgrowth were counted.
  • GPE analog G-2-MePE exhibited comparable neuroprotective effects within all three tested in vitro systems (FIGs 12-15).
  • G-2-MePE Cerebellar microexplants: FIG. 14 and striatal cells: FIG. 15. Striatal cells demonstrated neuroprotection within the range of InM to ImM of G-2-MePE (FIG. 15), while the postnatal cerebellar microexplants demonstrated neuroprotection with G-2-MePE in the dose range between aoout inivi ana aoouti uunivi ( i . 14;. t hus, we conclude that G-2-MePE is a neuroprotective agent and can have therapeutic effects in humans suffering from neurodegenerative disorders. Because G-2-MePE can be neuroprotective when directly administered to neurons in culture, that G-2-MePE can be effective in vivo when directly administered to the brains of affected animals.
  • Choline acetyltransferase is an enzyme that is involved in the biosynthesis of the neurotransmitter for cholinergic nerves, acetylcholine. It is well known that immunodetection of ChAT can be used to determine the numbers of cholinergic nerves present in a tissue. It is also known that the numbers of cholinergic nerves present is associated with the physiological function of cholinergic neural pathways in the brain.
  • the sections were washed using PBS/TritonTM ( 15 minutes x 3d) and then incubated with goat anti-rabbit biotinylated secondary antibodies (1 :1000) at room temperature overnight.
  • the sections were washed and incubated in ExtrAvidinTM (Sigma) (1 : 1000) for 3 hours and followed by H 2 0 2 (0.01%) in 3,3- diaminobenzine tetrahydrochloride (DAB, 0.05%) to produce a coloured reaction product.
  • DAB 3,3- diaminobenzine tetrahydrochloride
  • the striatal neurons in both hemispheres exhibiting specific immunoreactivities corresponding to ChAT were counted using a light microscope and a 1 mm 2x1000 grid. The size of the striatal region used for the count was measured using an image analyser. The total counts of neurons/mm 2 were compared between the groups.
  • FIG, 16 shows that the number of ChAT-immunopositive neurons increased in the brains of animals treated with G-2-MePE. This clearly indicates that administration of G-2-MePE is effective in increasing the level of ChAT in the brains of aged rats. Because ChAT is an enzyme involved in the synthesis of the cholinergic neurotransmitter acetylcholine, we conclude that G-2- MePE can increase the amount of cholinergic transmitter in the brains of middle-aged rats.
  • G-2-MePE can increase ChAT and therefore has the potential to improve cholinergic neural function
  • G-2-MePE can be useful in treating age-related changes in cognition and/or memory. Therefore, we carried out a series of studies in rats using well-established tests for memory.
  • the Morris water maze test is a well-recognized test to assess spatial reference memory in rats.
  • the Morris water maze test was conducted using a black plastic pool filled to a depth of 25 cm with water colored black with a non-toxic dye.
  • the pool had a circular black insert so that the walls also appeared uniform black
  • the pool was divided into four quadrants (north, south, east and west) by two imaginary perpendicular lines crossing at the pool's center
  • a metal platform was placed in the geographical centre of the SE quadrant 50cm from the edge of the pool, so that it was 2 cm below the water surface and invisible. The platform remained in that position though the training.
  • the experiment used extra-maze cues (i.e. objects in the room surrounding the pool) that the rats could use to navigate to the platform. Distinctive posters or paintings were hung on the walls. Furniture in the room was not moved during the testing period. The placement of the pool allowed the experimenter an easy access to it from all sides. The pool was emptied and refilled daily during testing, with water at 25°C +/- 2°C.
  • the furthermost point in the pool (relative to the position of the experimenter) was designated as "north”, and the other compass points "east”, “south” and “west” were the right- most, bottom and left-most points of the pool respectively. These points were marked with tape on the outside of the pool.
  • Rats in each group were trained to swim to the submerged platform.
  • the rats received six 60-second trials per day for four consecutive days.
  • a trial began by placing the rat into the water facing the wall of the pool, at one of four start locations (north, south, east, west).
  • the sequence of start locations was chosen pseudorandomly, so that the start location of any given trial was different from that of the previous trial, and no start location was used more than twice during daily training.
  • the same sequence of locations was used for all the rats on a given day but varied between days.
  • the trial ended when the rat had found the platform, or in 60 seconds, which ever occurred First.
  • the trials were timed with a stop watch. If the rat found the platform, it was allowed to remain there for 15 seconds before being removed to a holding container.
  • the rat was guided there manually and placed on the platform for 15- seconds.
  • the inter-trial interval was 60 seconds.
  • the holding container was covered in order to minimize any inter-trial interference.
  • the animal was towel-dried and placed under the heat lamp in the holding bucket until his coat was dry. The time needed to locate the platform (latency, sees) was obtained for each rat in each training trial. If the rat did not find the platform in a given trial their latency score was the maximum length of that trial (60 seconds).
  • mini-osmotic pumps (Alzet) were implanted subcutaneously under halothane anesthesia) to dispense drug or vehicle continuously for 1 or 3 weeks. At the completion of the infusion the pumps were removed and the wounds re-sutured.
  • the 5 treatment groups were:
  • the ability of the rats to remember or to relearn the original platform location was tested four weeks after original training. This means that residual drug would have been washed out for a minimum of 7 days in the case of the 3-week pumps, and 21 days in the case of the 1-week pumps.
  • the retention testing procedure was identical to that of acquisition.
  • Pharmacokinetic studies indicate that the plasma concentration of subcutaneously administered G-2-MePE rose to a peak and then declined with an approximately first order kinetic pattern, with a plasma half-life (t 1 ⁇ 2) of between about 30 and 60 minutes.
  • t 1 ⁇ 2 plasma half-life
  • the 3-week vehicle and 3-week high dose G-2-MePE were compared in acquisition and retention.
  • the high dose of G-2-MePE, given over 3 weeks improved the retention of the original water maze task after a 4-week delay.
  • FIG. 17 shows the comparison between high-dose (4.8 mg/day) G-2-MePE-treated and low-dose-treated (0.96 mg/day) aged rats and saline treated aged rats, with the young controls (4 months) used as controls.
  • Prior to treatment with G-2-MePE there were no differences between the aged (12 month old) groups. In contrast, the 4 month old animals required less time to reach the platform than older animals.
  • animals that received saline only did not show improved ability to reach the platform, as indicated by the similar times required at test day 4 of the acquisition phase and test day 1 of the retention phase.
  • the apparatus consists of a central platform communicating with 8 identical arms, each with a food cup at the end of the arm
  • Rats were partially food-deprived for at least 10 days prior to, and throughout the radial maze procedure.
  • the maze was assembled and positioned so that the experimenter could clearly observe the rats' behavior from a predetermined location.
  • the experimenter numbered the arms of the maze according to their orientation from one to eight in a clock- wise direction.
  • the treatment groups were:
  • Saline and the low dose groups are comprised of all the rats that received those treatments in phase 1 of this experiment (when the rats were 12 months old) regardless of whether they had the one or three week treatment.
  • One rat in each of the saline and high dose groups have been dropped because of skin tumors.
  • One of the low dose rats did not participate in this experiment due to the fact that it could not be pre-trained (see below).
  • Rats received 10 daily training sessions over 12 days. The procedure was the same as for pre-training but only the food cups were baited. Rats had 6 minutes to make up to 16 choices by visiting any of the eight arms. A choice was defined as occurring when all four paws were inside an arm. The experimenter recorded the sequence of arm entries with pen and paper. Sessions were terminated after all eight arms had been entered, 16 choices made, or 6 minutess had elapsed. The time taken to enter all eight arms, when this occurred, was recorded.
  • Correct Choice (CC) 8-12 is the number of correct choices made divided by the total number of choices made. For animals that failed to visit all 8 arms in a test, the denominator of this ratio is considered to be 12.
  • WCC 8-12 is the measure from which the working memory data are derived. Data were collected as described for CC 8-12 above, but for this parameter, only the rats that entered all 8 arms in a session were included. Rats that made fewer than 8 arm entries were not used to ascertain working memory because they didn't remember which arms they had previously visited and therefore had memory so impaired that they could not complete the test, as opposed to the animals that, for whatever reason, did not explore the maze.
  • the high dose G-2Me-PE group showed the greatest improvement across days, followed by the young controls. There was very little difference between the low dose G- 2Me-PE and saline.
  • Neurons are derived from neuroblasts, a less differentiated cell than a neuron, but within the neural lineage.
  • a neuroblast is exposed to conditions that cause it to mature into a mature phenotype, having a defined soma, neural processes (axons and dendrites) and ultimately, making connections with other neurons (e.g., synapses).
  • measuring neuroblast proliferation has become a well- known early marker for nerve cell proliferation.
  • detecting an increase in neuroblast proliferation induced by a pharmaceutical agent is an accepted method for predicting growth of neural cells in animals. Because rats and humans share similar mechanisms in neural cell proliferation, detection of changes in neuroblast proliferation in rats in vivo is predictive of similar effects in human beings.
  • G-2-MePE G-2-MePE
  • GFAP glial fibrillary acidic protein
  • PCNA proliferating cell nuclear antigen
  • the sections were then incubated with following primary antibodies: monoclonal mouse anti-GFAP antibody (Sigma, St. Louis, MO, U.S.A. diluted 1 :500); mouse anti-PCNA antibody (DAKA, A/S, Denmark, diluted 1 : 100). After incubation with primary antibodies at 4°C for 2 d (except for PCNA staining which was incubated overnight) the sections were incubated with biotinylated horse anti-mouse or goat anti-rabbit secondary antibody (1 :200, Sigma) at 4°C overnight.
  • ExtrAvidinTM (Sigma, 1 :200), which had been prepared 1 h before use, was applied for 3 h at room temperature, and then reacted in 0.05% 3,3-diaminobenzidine (DAB) and PBS to produce a brown reaction product. Sections were dehydrated in a series of alcohols to xylene and coverslipped with mounting medium.
  • Control sections were processed in the same way except the primary antibody was omitted from the incubation solution.
  • the number of PCNA positive cells was counted in the subventricular zone and the GFAP positive cells was scored in the cerebral cortex.
  • the subventricular zone (SVZ) and the dentate gyrus (DG) are two brain regions hosting adult neurogenesis.
  • the reduction of neurogenesis in both SVZ and the DG has been well reported to be co-related to the memory decline with aging and effects of Nerve Growth Factor and Epidermal Growth Factor on memory improvement are reported to be due to increase in progenitors proliferation of the SVZ.
  • PCNA as a marker of cell proliferation, cellular proliferation in the SVZ was examined by counting the numbers of cells that are positive for PCNA. In selected animals, at least some of the proliferating cells were identified as neuroblasts, as stained with the neural-cell specific agent, doublecortin.
  • FIG. 19B is a photograph of a portion of a rat's brain showing an increase in both PCNA (green, x20) and doublecortin (red, x20) in the rat treated with the highest dose of G-2-MePE (right panel) compared to the vehicle treated rat (left panel).
  • the two markers clearly co-localised ( Figure 1 B, photo, l OO).
  • G-2-MePE can stimulate proliferation of brain cells, including neuroblasts. Because neuroblasts are precursor cells for neurons, we further conclude that G-2-MePE can increase the population of neurons in the brains of animals treated with the compound of this invention.
  • G-2-MePE Effects of G-2-MePE (1.2mg/kg) were studied in a group of middle-aged, 9 month old rats.
  • G-2-MePE (1.2 mg/kg) or vehicle was administered intraperitoneally (i.p.).
  • the proliferation of cells in the SVZ was examined 3 days after the treatment using PCNA immunohistochemical staining.
  • Experiment 3 Astrocytosis in Aging Brains
  • GFAP positive astrocytes also play a role in angiogenesis (FIG. 20B, arrows), which also contribute to inflammatory response in brains. Therefore the elevated GFAP astrocytes seen in aged brains may indicate a chronic stage of brain degeneration.
  • G-2-MePE Treatment with G-2-MePE reduced number of reactive astrocytes in the CA4 region of the hippocampus compared to the vehicle treated group (FIG. 20C; *p ⁇ 0.05), particularly the groups treated with doses of 0.12 and 12 mg/kg. A similar effect was observed for G-2-MePE in the cerebral cortex (FIG. 20D).
  • GFAP-positive astrocytes located in the deep layer of cortex of rat brains and those that are present are usually in close association with white matter tracks.
  • G-2-MePE G-2-MePE
  • G-2-MePE increases the amount of ChAT present in the brain cells of animals exposed to the neurotoxins okadaic acid or 3-NP.
  • This effect of G-2-MePE mimicked that of a well-known neuroprotective agent, GPE.
  • G-2-MePE increased ChAT in the striatum, indicating that cholinergic neurons are sensitive to G-2-MePE. These observed chemical and histological changes were paralleled by behavioral changes. Aged animals treated with G-2-MePE exhibited improved memory in two well-known test systems compared to vehicle-treated controls. Next, G-2-MePE induced neuroblast proliferation in aging brains. Finally, treatment with G-2-MePE reversed the increase in astrocytosis observed in the hippocampus and cortex of aging brains. The effects of G-2-MePE were not due to acute effects of the agent; because in many of the studies cited herein, sufficient time had elapsed from cessation of drug delivery to the test, that there was likely little or no drug present.
  • Blood samples (about 220 ⁇ each) were collected into heparinized tubes containing Sigma protease inhibitor cocktail for mammalian tissues at 10 and 0 min before injection of either GPE or G2MePE, and 1, 2, 4, 8, 16, 32, 64 and 128 min after injection of either GPE or G2MePE.
  • the samples were centrifuged at 3000g for 15 min at 4°C and the plasma removed and stored at -80°C until extraction and assay by either radioimmunoassay ("RIA") or reverse phase HPLC.
  • RIA radioimmunoassay
  • HPLC reverse phase HPLC
  • FIG. 21 shows a graph of plasma concentrations in vivo of GPE and G-2-MePE after intravenous (i.v.) injection. Filled squares represent concentrations of GPE at each time point, and filled triangles represent concentrations of G-2-MePE at each time point.
  • Plasma concentrations of GPE and G-2-MePE were markedly increased within 1 min after injection. After injection of 30 mg/kg GPE, a eak concentration of 40.0 ⁇ 10.8 mg/ml was observed. Plasma concentrations of GPE then rapidly declined according to a first-order kinetic process.
  • the first order rate constant for GPE was found to be 0.15 ⁇ 0.014 ng/ml/min, the t 1/2 was found to be 4.95 ⁇ 0.43 min and the estimated clearance of GPE from plasma was found to be 137.5 ⁇ 12.3 ml/hr.
  • the peak concentration was found to be 191 ⁇ 1 .1 mg/ml.
  • Plasma concentrations of G-2-MePE then declined according to a first-order kinetic process.
  • the first order rate constant for G-2-MePE was found to be 0.033 ⁇ 0.001 ng/ml/min, the t 1/2 was found to be 20.7 ⁇ 0.35 min and the estimated clearance was found to be 30.1 + 0.5 ml/hr.
  • the maxima! plasma concentration of G-2-MePE was about 4.8 times greater than the maximal plasma concentration of GPE, in spite of the larger dose of GPE delivered (30 mg/kg) compared to the dose of G-2-MePE delivered (10 mg kg).
  • G-2-MePE is a potent agent capable of reversing many of the adverse effects of aging in the brains of animals, including humans.
  • GPE analogs, including G-2-MePE therefore, can produce desirable therapeutic effects, including neuroprotection, improved memory, increased neuroblast proliferation and reduction in astrocytosis, and can be valuable in reversing or mitigating adverse effects of aging in humans.
  • MeCP2(lIox) male mice To determine whether G-2-MePE treatment can impact the development and progression of Rett Syndrome in a murine model of the disorder, we used hemizygous MeCP2(lIox) male mice.
  • MeCP2 knock-out (MeCP2- O) mouse system is widely accepted in the art as closely mimicking the range and the severity of physiological and neurological abnormalities characteristic of the human disorder, Rett Syndrome.
  • MeCP2 deficient mutant mice develop RTT symptoms at about 4-6 weeks of age and die between 10-12 weeks (Chen et al., 2001. Nat Genet 27: 327-331 ).
  • MeCP2 deficient mice have been previously reported to suffer from functional and ultrastructurai synaptic dysfunction, significant impairment of hippocampus-dependent memory and hippocampal long-term potentiation (LTP) (Moretti et al. The Journal of Neuroscience. 2006. 26(l ):319-327).
  • LTP long-term potentiation
  • To test the effects of the G-2-MePE treatment on synaptic function in the RTT model we compared hippocampal LTP in both vehicle and G-2-MePE treated animals at 9 weeks of age. To do so, we measured the slope of the fEPSP as a % of baseline potential in neurons in slices of hippocampus from MeCP2 deficient mice treated with either saline or G-2-MePE (FIG. 23).
  • FIG. 22 shows that G-2-MePE treatment increased survival of MeCP2 deficient mice.
  • Wild-type mice top line are control animals, and therefore their survival was 100% at each time point.
  • MeCP2 deficient mice treated with saline only died much more rapidly (dotted line) than wild-type mice, such that by about 1 1 weeks, only 50% of the MeCP deficient mice survived.
  • MeCP2 deficient mice treated with G-2- MePE survived substantially longer than saline-treated mice. At about 15 weeks, 50% of the animals survived.
  • Data initially presented showed that MeCP2 mice were impacted in terms of survival such that 50 percent of animals had died by 11 weeks in the untreated case.
  • G-2-MePE treated animals showed improved survival, with 50 percent having died at 16 weeks.
  • G-2-MePE can substantially increase survival of MeCP2 deficient mice. Because MeCP2 deficient mice are predictive of the pathology and therapeutic efficacy in human beings with Rett Syndrome, we conclude that G-2-MePE can increase life span of human beings with Rett Syndrome.
  • FIG. 23 shows results of our studies to determine if G-2- ePE treatment increased hippocampal long-term potentiation (LTP) as measured by the fEPSP slope in MeCP2 deficient animals compared to saline-treated mutant mice.
  • LTP hippocampal long-term potentiation
  • G-2-MePE can be effective in treating MeCP2 deficient mice in vivo. Because MeCP2 deficient mice are predictive of the pathology and therapeutic efficacy in human beings with Rett Syndrome, we conclude that G-2-MePE can be an effective therapy for people with Rett Syndrome.
  • Example 9 G-2-MePE Improves Dendritic Arborization and Increases Dendritic Spine Length
  • FIG. 24 depicts results of this study. Dendritic length in ⁇ (vertical axis) is plotted against the distance (in ⁇ ; horizontal axis) from the soma of the cells. For cells with dendrites close to the somas, the dendrites were short. However, as the distance from the somas increased saline-treatment (open squares) produced dendritic lengths that increased to a maximum at a distance of 70 ⁇ from the soma and declined at distances further away from the somas. In contrast, treatment with G-2MePE (filled squares) produced longer dendrites over much of the range of distances from the somas.
  • MeCP2 germline null allele mice are used (Chen et al, 2001). Genotyping is performed as in Chen et al. (Chen et al., 2001 ).
  • G-2-MePE synthesised Albany Molecular Research Inc. (Albany, NY) and supplied by Neuren Pharmaceuticals Limited
  • the treatment starts at P15 and is maintained throughout the course of the experiments.
  • Coronal sections (300 ⁇ thick) at or near sensorimotor cortex are cut in ⁇ 4°C ACSF using a Vibratome. Slices are incubated at 37°C for 20 minutes after slicing, and at room temperature for the remainder of the experiment. Slices are transferred to a Warner chamber and recordings are taken from visually identified pyramidal neurons located in layer 5.
  • Artificial cerebral spinal fluid (ACSF) containing 126 mM NaCl, 25 mM NaHC03, 1 raM NaHP04, 3 mM KCl, 2 mM MgS04, 2 mM CaC12, and 14 mM dextrose, is adjusted to 315-320 mOsm and 7.4 pH, and bubbled with 95% 02/5% C02.
  • the intracellular pipette solution contained 100 mM potassium gluconate, 20 mM KCl, 10 mM HEPES, 4 mM MgATP, 0.3 mM NaGTP, and 10 mM Na-phosphocreatine.
  • Borosilicate pipettes (3-5 ⁇ , WPI) are pulled using a Sutter P-80 puller (Sutter Instruments). Cells are visualized with an Achroplan 40x water-immersion lens with infrared-DIC optics (Zeiss) and detected with an infrared camera (Hamamatsu) projecting to a video monitor. Experiments are driven by custom acquisition and real-time analysis software written in Matlab (Mathworks, Natick, Mass.) using a Multiclamp 700B amplifier (Axon Instruments) connected to a BNC-21 10 connector block and M-Series dual-channel acquisition card (National instruments). Gigaseal and rupture is achieved and whole-cell recordings are continuously verified for low levels of leak and series resistance.
  • a 5 mV test pulse is applied in voltage clamp -10 times to measure input and series resistance. Then in current clamp ⁇ 10 pulses (500 ms, 40-140 pA at 10 pA increments), are applied to quantify evoked firing rates and cellular excitability. Access resistance, leak, and cellular intrinsic excitability are verified to be consistent across groups. Finally, spontaneous EPSCs under voltage clamp at -60 mV are sampled at 10 kHz and low-pass filtered at 1 kHz. Analysis is performed using a custom software package written in Matlab, with all events detected according to automated thresholds and blindly verified for each event individually by the experimenter. Golgi Staining
  • the wild type control group is composed of both wild type littennates of MeCP2+/- females or wild type age matched SVEV females.
  • animals are anesthetized with Avertin (0.016 ml/g) and the eyelids of one eye is sutured for 4 days. Prior to imaging, the suture is removed and the deprived eye re-opened. Only animals in which the deprivation sutures are intact and the condition of the deprived eye appears healthy are used for the imaging session.
  • a solution containing G-2-MePE is injected intra-peritoneally (IP) daily for the entire period of deprivation.
  • IP intra-peritoneally
  • mice are anesthetized with urethane (1.5 g/kg; 20% of the full dosage is administered IP each 20- 30 minutes up to the final dosage, 0.02 ml of cloroprothixene 1 % is also injected together with the first administration).
  • the skull is exposed and a custom-made plate is glued on the head to minimize movement.
  • the skull is thinned over VI with a dremel drill and covered with an agarose solution in saline (1 .5%) and a glass coverslip.
  • the animal is constantly oxygenated, its temperature maintained with a heating blanket and the eyes periodically treated with silicone oil; physiological conditions are constantly monitored.
  • the anesthetized mouse is placed in front of a monitor displaying a periodic stimulus presented to either eye, monocularly; the stimulus consisted of a drifting vertical or horizontal white bar of dimensions 9°x72°, drifting at 9 sec/cycle, over a uniformly gray background.
  • the skull surface is illuminated with a red light (630 nm) and the change of luminance is captured by a CCD camera (Cascade 512B, Roper Scientific) at the rate of 15 frames/sec during each stimulus session of 25 minutes.
  • a temporal high pass filter (135 frames) is employed to remove the slow signal noise, after which the signal is computer processed in order to extract, at each pixel, the temporal Fast Fourier Transform (FFT) component corresponding to the stimulus frequency.
  • the FFT amplitude is used to measure the strength of the visual evoked response to each eye.
  • the binocular zone is defined as the region activated by the stimulation of the eye ipsilateral to the imaged hemisphere.
  • Real time cardiac pulse rate is measured using a tail clip sensor (Mouse OX Oximeter—
  • mice are not anesthetized but physically restrained in a fitted open plastic tube. Prior to the recording session the tube is placed overnight in the cages housing the experimental animals to allow habituation. Body temperature is maintained at ⁇ 82-84°F throughout the recording time. We record 3 trials of 15 minutes for each mouse, mice are 8 weeks old and treated with vehicle or G-2-MePE from P 15.
  • Spontaneous motor activity is measured by using an infrared beam-activated movement- monitoring chamber (Opto-Varimax-MiniA; Columbus instruments, Columbus, Ohio), For each experiment, a mouse is placed in the chamber at least 3 h before recordings started. Movement is monitored during the normal 12-h dark cycle (7 p.m. to 7 a.m.). One dark cycle per animal per time point is collected.
  • Opto-Varimax-MiniA Columbus instruments, Columbus, Ohio
  • IGF-1 pathway and administration of ( 1-3)IGF-1 can reduce OD plasticity in wild type young mice (Tropea et al., 2006).
  • G-2-MePE treatment could stabilize the prolonged OD plasticity observed in adult MeCP2 mutants.
  • G-2-MePE treatment reduces the OD plasticity in the adult Mecp2+/- mice, indicating that indeed G-2-MePE can rapidly induce synapse stabilization or maturation.
  • MeCP2-/y mice develop Rett-like symptoms beginning at 4-6 weeks of age when they progressively become lethargic, develop gait ataxia and die between 1 and 12 weeks of age (Chen et al., 2001). Baseline locomotor activity is also recorded in mice after 6 weeks by counting nocturnal infrared beam crossing events within a caged area. MeCP2 knockout mice (KO) exhibits markedly reduced locomotor activity levels compared to wild-type mice (WT), but treatment with G-2-MePE (KO-T) elevates these levels.
  • MeCP2-/y mice treated with G-2-MePE also show a -50% increase in life expectancy (an increase in the 0.5 probability survival rate).
  • G-2-MePE treatment we also measure the effect of G-2-MePE treatment on neuron soma size in the hippocampus. Mice are treated with G-2-MePE as described above for locomotor activity. Soma size in neurons in the CA3 region of the hippocampus is significantly impaired in MeCP2 KO animals relative to wild-type animals. G-2-MePE treatment increases average soma size in KO animals, but has little or no effect on soma size in wild type animals.
  • Example I 1 Effect of Oral G-2-JVIePE on Survival in Rett Syndrome in Mice
  • Rett Syndrome is a chronic, debilitating disorder involving loss of motor skills
  • we can take advantage of unexpectedly beneficial therapeutic and pharmacokinetic properties of G-2- MePE and related compounds (U.S. Pat. Nos. 7,041 ,314, 7,605,177, 7,714, 070, 7,863,309 and U.S. Appl. Nos. 11/315,784 and 12/903,844).
  • survival In wild-type animals, survival is defined to be 100% at each time point. In MeCP2 deficient animals, survival is decreased substantially. However, after oral administration of G-2- MePE to MeCP2 deficient mice, survival is increased substanti lly.
  • G-2MePE can be effective in treating seizure activity in animals with neurodegenerative disease (U.S. Pat. No. 7,714,020). Therefore, we carry out experiments to determine whether G-2-MePE can also treat seizure activity in MeCP2 deficient mice.
  • Electroencephalograpic recordings of wild-type mice and MeCP2 deficient mice treated with either saline or G-2-MePE are obtained using methods described in U.S. Pat. No. 7,714,020.
  • G-2MePE can be effective in decreasing both motor seizures and non- convulsive seizures.
  • G-2-MePE can be an effective therapy for treating human beings with Rett Syndrome. Moreover, because G-2-MePE has unexpectedly longer half life than a naturally occurring compound ((1-3) IGF-1 ; Glycyl-Prolyl-Glutamate or GPE) (FIG. 21), we conclude that use of G-2-MePE has distinct and substantial advantages over other pharmacological agents, including GPE.
  • G-2-MePE is not degraded by gastrointestinal cells, is taken up by gastrointestinal cells, and is active in the central nervous system after oral administration (Wen et al., U.S. Appl. No. 12/283,684; U.S. 2009/0074865, U.S. Pat. No. 7,887,839, incorporated herein fully by reference), Therefore, G-2MePE need not be delivered intravenously, subcutaneously, intraventricularly, or parenterally.
  • oral formulations comprising micro-emulsions, coarse emulsions, liquid crystal preparations, nanocapsules and hydrogels can be used in manufacture of orally administered preparations such as tablets, capsules and gels that can improve neurological function and treat neurodegenerative conditions (U.S. Pat. No. 7,887,839).
  • Compounds of this invention can be used in situations in which a patient's motor functioning is below that needed to swallow a table or capsule.
  • compounds of this invention can be useful in providing therapeutic benefit from animals having other ASD, and in humans with autism, Asperger Syndrome, Childhood Disintegrative Disorder, and Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS).
  • Shank3- deficient mice are used in the study as a model of 22ql3 deletion syndrome associated with ASD.
  • Shank3 22ql 3 deletion syndrome has been linked with deletions or mutations in Shank3 gene (Bonaglia et al, 2006).
  • the Shank3 gene codes for a master scaffolding protein which forms the framework in glutamatergic synapses (Boeckers et al, 2006), Shank3 is a crucial part of the core of the postsynaptic density (PSD) and recruits many key functional elements to the PSD and to the synapse, including components of the a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGIu), and N-methyl-D-asparfic acid (NMDA) glutamate receptors, as well as cytoskeletal elements.
  • AMPA a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid
  • mGIu metabotropic glutamate
  • NMDA N-methyl-D-asparfic acid
  • Shank3 can cause a monogenic form of ASD with a frequency of 0.5% to 1% of ASD cases (Durand et al, 2007; Moessner et al, 2007; Gauthier et al, 2008).
  • Shank3 mRNA A 50% reduction of full length Shank3 mRNA was confirmed in heterozygotes (qPCR) as well as a reduced expression of Shank3 protein (by immunoblotting with Shank3 antibody N69/46).
  • Heterozygous mice generated by crossing wild-type mice with heterozygotes are used in this example to best model the haploinsufficiency of Shank , responsible for 22ql3 deletion syndrome.
  • G-2-MePE treated groups The animals are given placebo (water) or G-2-MePE formulated in water administered orally, b.i.d for 14 days. G-2-MePE is administered at two doses: 15 or 60 mg/kg.
  • Behavioral assessments are made at several time points, and include analysis of social interactions and ultrasonic social communication, in line with the methodology described by Bozdagi et al. Briefly, male-female social interactions in each treatment group are evaluated. The subject males are group-housed and individually tested in clean cages with clean litter. Each testing session lasts 5 min. Each of the subject mice is paired with a different unfamiliar estrus C57BL/6J female.
  • a digital closed circuit television camera Panasonic, Secaucus, NJ, USA
  • An ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM15 ; Avisoft Bioacoustics, Berlin, Germany) is mounted 20 cm above the cage.
  • Sampling frequency for the microphone is 250 kHz, and the resolution is 16 bits. While the equipment used cannot distinguish between calls emitted by the male subject and female partner, the preponderance of calls during male-female interactions in mice is usually emitted by the male.
  • the entire apparatus is contained in a sound-attenuating environmental chamber (ENV-01 8V; Med Associates, St Albans, VT, USA) illuminated by a single 25- Watt red light. Videos from the male subjects are subsequently scored by an investigator uninformed of the subject's genotype and treatment group on measures of nose-to-nose sniffing, nose-to-anogenital sniffing and sniffing of other body regions, using Noldus Observer software (Noldus Information Technology, Leesburg, VA, USA). Ultrasonic vocalizations are identified manually by two highly trained investigators blinded to genotype/treatment group information, and summary statistics are calculated using the Avisoft package. Interrater reliability is 95%. Data are analysed using an unpaired Student's i-test.
  • Olfactory habituation/dishabituation testing is conducted in male and female mice for each group.
  • the methodology is as previously described (Silverman et al 2010, Yang et al 2009 and Silverman et al 2010).
  • Non-social and social odors are presented on a series of cotton swabs inserted into the home cage sequentially, each for 2 min, in the following order: water, water, water (distilled water); almond, almond, almond (1 : 100 dilution almond extract); banana, banana, banana (1 : 100 dilution artificial banana flavouring); social 1, social I , social 1 (swiped from the bottom of a cage housing unfamiliar sex-matched B6 mice); and social 2, social 2, social 2 (swiped from the bottom of a second cage housing a different group of unfamiliar sex-matched 129/SvImJ mice).
  • One-way repeated measures ANOVA is performed within each treatment group for each set of habituation events and each dishabituation event, followed by a Tukey post hoc test.
  • Post-mortem, acute hippocampal slices (350 pm) are prepared from mice using a tissue chopper. Slices are maintained and experiments are conducted at 32°C. Slices are perfused with Ringer's solution containing (in niM): NaCl, 125.0; C1, 2.5; MgS0 , 1.3; NaH 2 P0 4 , 1.0; NaHC0 3 , 26.2; CaCl 2 , 2.5; glucose, 1 1.0. The Ringer's solution is bubbled with 95% 02/5% C02, at 32°C, during extracellular recordings (electrode solution: 3 M NaCl).
  • fEPSPs field excitatory postsynaptic potentials
  • AMPA receptor-mediated and NMDA receptor-mediated I/O relationships are measured in the presence of ionotropic glutamate receptor antagonists: 2-amino-2-phosphonopentanoic acid APV (50 ⁇ ) and 6-cyano-7- nitroquinoxa!ine-2,3-dione CNQX ( ⁇ ⁇ ).
  • Paired-pulse responses are measured with interstimulus intervals of 10 to 200 ms, and are expressed as the ratio of the average responses to the second stimulation pulse to the first stimulation pulse.
  • LTP is induced either by a high-frequency stimulus (four trains of 100 Hz, 1 s stimulation separated by 5 min), or by theta-burst stimulation (TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms), or by a single 100 Hz stimulation, for control and genetically-modified mice.
  • TLS ta-burst stimulation
  • Data are expressed as means ⁇ SD, and statistical analyses are performed using analysis of variance (ANOVA) or student's t-test, with significance set at an a level of 0.05.
  • Cumulative duration of total social sniffing by the male test subjects is lower in placebo treated Shank3-deficient group than in placebo treated wild-type group.
  • fewer ultrasonic vocalizations are emitted by the placebo treated Shank3 -deficient group than by the wild-type controls during the male-female social interactions.
  • G-2-MePE treatment in the two 3 ⁇ 4a «H-deficient groups results in a significant increase in the cumulative duration of total social sniffing in comparison to the placebo treated Shank3- deficient group. Moreover, the G-2-MePE treated groups display an increased number of ultrasonic vocalizations than the placebo treated mutant group.
  • Plotting field excitatory postsynaptic potential (fEPSP) slope versus stimulus intensity demonstrates a reduction in the I/O curves in the placebo treated Shank3 -deficient group versus the control group.
  • fEPSP field excitatory postsynaptic potential
  • G-2-MePE treatment in both heterozygous groups normalizes the AMPA receptor- mediated field potentials and causes an increase in the average slope of I/O function compared to the placebo treated group.
  • G-2-MePE treatment increased hippocampal long-term potentiation (LTP) and its maintenance in both Shank3 -deficient group in comparison to the placebo treated 53 ⁇ 4i7 «H-deficient group.
  • LTP hippocampal long-term potentiation
  • the animal models described above have been accepted in the art as demonstrating similar symptoms to the clinical human conditions. All mutant models discussed above (NLGN3, NLGN4, CADM1, NRXN1 , FMR1 , shank3) exhibit impaired social skills or increased social anxiety. Decreased excitatory transmission into the hippocampus has been identified in NRXN1, shank3, MeCP2 and FMRI mutant animal models. At present no polygenetic or multifactorial models of ASD have been described. The animal models described above, based on genetic defects that are known to produce ASD in human population, provide the best opportunity to test the efficacy of ASD therapies.
  • Example 14 G-2-MePE Treatment Changes the Morphology of Neurons in an in
  • RTT fibroblasts (carrying 4 distinct MeCP2 mutations) and control fibroblasts are generated from explants of dermal biopsies.
  • the shRNA against target MeCP2 gene is cloned into the LentiLox3.7 lentivirus vector (as described in Marchetto et al.).
  • the fibroblasts are infected with retroviral reprogramming vectors (Sox2, Oct4, c-Myc and Klf4). Two days after infection, fibroblasts are plated on mitotically inactivated mouse embryonic fibroblasts with hESC medium.
  • iPSC colonies that emerge from the background of fibroblasts are manually picked and transferred to feeder-free conditions on matrigel-coated dishes (BD) using embryonic stem celi culture media mTeSR (Stem Cell Technologies) and passaged manually.
  • Gene expression profiles of the generated clones are measured using human genome Affymetrix Gene ChipTM arrays to confirm that reprogramming is successful.
  • NPCs neural progenitor cells
  • EBs embryoid bodies
  • DMEM/F12 plus N2 medium serum-free supplement for growth and expression of post- mitotic cells
  • Resulting rosettes are collected after 7 days and dissociated with accutase and plated onto coated dishes with NPC media (DMEM/F12; 0.5X N2; 0.5X B27 and FGF2).
  • DMEM/F12; 0.5X N2; 0.5X B27 and FGF2 DMEM/F12; 0.5X N2; 0.5X B27 and FGF2
  • Mature EBs are dissociated with papain and DNAse for l h at 37°C and plated in poly-ornithine/laminin-coated dishes in NPC media without FGF2.
  • RTT neuronal cultures are treated with G-2-MePE ( ⁇ - ⁇ ) for 1 week.
  • Cell soma size is measured using suitable software (e.g. ImageJ) after identification of neurons using the Syn: :EGFPTM.
  • Neuronal networks derived from human iPSCs are infected with the lentiviral vector carrying the Sy DsRed reporter construct.
  • Cell cultures are washed twice with sterile Krebs HEPES Buffer (KHB) and incubated with 2-5 ⁇ Fiuo-4AMTM (Molecular Probes/Invitrogen, Carlsbad, CA) in KHB for 40 minutes at room temperature. Excess dye is removed by washing twice with KHB, and an additional 20 minutes incubation is done to equilibrate intracellular dye concentration and allow de-esterification.
  • KHB sterile Krebs HEPES Buffer
  • Fiuo-4AMTM Molecular Probes/Invitrogen, Carlsbad, CA
  • Time-lapse image sequences (100X magnification) of 5000 frames are acquired at 28 Hz with a region of 336 ⁇ 256 pixels, using a Hamamatsu ORCA- ERTM digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC) filter on an Olympus 1X81 inverted fluorescence confocal microscope (Olympus Optical, Japan). Images are acquired with MetaMorph 7.7TM (MDS Analytical Technologies, Sunnyvale, CA). Images are subsequently processed using ImageJTM and custom written routines in Matlab 7.2TM (Mathworks, Natick, MA). Electrophysiology
  • Whole-cell patch clamp recordings are performed from cells co-cultured with astrocytes after 6 weeks of differentiation.
  • the bath is constantly perfused with fresh HEPES-buffered saline (see supplemental methods for recipe).
  • the recording micropipettes (tip resistance 3-6 ⁇ ) are filled with internal solution described in the Supplemental materials. Recordings are made using Axopatch 200BTM amplifier (Axon Instruments). Signals are filtered at 2 kHz and sampled at 5 kHz. The whole-cell capacitance is fully compensated.
  • the series resistance is uncompensated but monitored during the experiment by the amplitude of the capacitive current in response to a 10- mV pulse.
  • AH recordings are performed at room temperature and chemicals are purchased from Sigma.
  • RTT iPSC-derived neurons are characterized by decreased number of glutamatergic synapses, reduced spine density and smaller soma size. RTT neurons also show certain eiectophysiological defects, i.e. a significant decrease in frequency and amplitude of spontaneous synaptic currents when compared to controls. The RTT neurons show a decreased frequency of intracellular calcium transients.
  • G-2-MePE in the above model to test whether any of the pathologies of the RTT phenotype can be attenuated.
  • G-2-MePE treated RTT cells Treatment of the cell cultures with each drug concentration improves all of the morphological and physiological parameters of the treated RTT cell cultures in comparison to the non-treated RTT controls. Specifically, we observe a significant increase in glutamatergic synapse numbers in the G-2-MePE treated RTT cells. All concentrations of G-2-MePE treatment increase VGLUT1 puncta number in the RTT-derived neurons. G-2-MePE treatment normalizes the frequency and amplitude of spontaneous post-synaptic currents as well as the frequency of calcium transients generated by synaptic activity of the G-2-MePE treated RTT neurons.
  • the iPSCs derived from RTT patients and neurons differentiated from them are characterized by abnormalities in the MeCP2 expression.
  • the vast majority of RTT cases are associated with mutations of the MeCP2 gene. Therefore the efficacy of G-2- ePE in the present in vitro model of human RTT is reasonably predictive of its efficacy in a human subject suffering from RTT.
  • FFT Fast Fourier Transform
  • the study is a randomized double blind placebo controlled parallel study with three doses of either placebo, 30 mg kg T.I.D oral G-2-MePE for five days, or 30 mg/kg T.I.D. oral G-2- MePE.
  • Subjects are tested at baseline using the following instruments: The Rett Syndrome Natural History / Clinical Severity Scale, Aberrant Behavior Checklist Community Edition (ABC), Vinelands, Clinical Global Impression of Severity (CGI-S) and their carers completed the Caregiver Strain Questionnaire (CSQ).
  • ABS Aberrant Behavior Checklist Community Edition
  • CGI-S Clinical Global Impression of Severity
  • CSQ Caregiver Strain Questionnaire
  • Subjects are brought into clinic on an inpatient basis to enable initial baseline recordings of EEG, ECG and respiratory rate continuously for 24 hours using polysomnography technology. Hand movements are also recorded using the Q-SensorTM. Derived EEG measures include: spikes per unit time in the EEG, overall power of frequency bands of the EEG, QTc and heart rate variability (HRV), and respiratory irregularities.
  • Derived EEG measures include: spikes per unit time in the EEG, overall power of frequency bands of the EEG, QTc and heart rate variability (HRV), and respiratory irregularities.
  • ANCOVA repeated analysis of covariance
  • G-2-MePE Treatment with G-2-MePE produces no more adverse events than are present during treatment with placebo, with all adverse events being of short duration and mild severity. No Serious Adverse Events are reported. No instances of increases in QTC are reported.
  • G-2-MePE produces a significant overall reduction of spikes per unit time in the EEG.
  • Treatment with 30 mg/kg T.I.D. oral G-2-MePE decreases spike activity compared to placebo. This dose of G-2-MePE also decreases the power of the delta band of the EEG compared to placebo.
  • Treatment with G-2-MePE also reduces total hand movements per twenty-four hour period as counted using the Q-SensorTM device. This effect is significant for the 30 mg/kg T.I.D. dose compared to placebo.
  • G-2-MePE Central Nervous System function
  • Dose dependent effects are also seen on hand use, as assessed by an objective counting device and subjective rating. This is of interest because purposeless hand wringing is both characteristic to the Rett Syndrome clinical phenotype and is unique to this disorder.
  • Severity Scale is improved by treatment. This measure primarily assesses eye contact. This raises the prospect that longer term treatment with G-2-MePE may improve social relatedness in the population.
  • G-2-MePE is well tolerated in this population. No effects are seen in either standard measures or areas of specific concern in the patient population, such as QTc interval prolongation or apnea.
  • G-2-MePE can treat symptoms of ASD.
  • the study is a double blind placebo-controlled crossover study with three phases. Subjects enter each phase of the crossover in a randomized order. In the test phases, subjects receive either placebo, 10 mg/kg T.I.D oral G-2-MePE for five days, or 30 mg kg T.I.D. oral G-2- MePE. Each phase of the crossover is separated by a washout period of fourteen days.
  • Subjects are administered two tasks - the Reading the Mind in the Eyes Test- Revised (RMET) and an Eye Tracking (ET) task, as well as Clinical Global Impression of Improvement (CGI-I). Tasks commence two hours following administration of placebo or either dose of G-2- MePE.
  • the RMET is a computer based task that assesses one's ability to read emotions from the eyes of subtle affective facial expressions and is a widely used test of emotion recognition in patients with autism (2001), Importantly, the RMET is capable of detecting improvement with even a single dose of a pharmacological agent (Guastella et al., 2010). Eye tracking issues are characteristic of patients with autism who spend less time looking at the eyes of photographs of human faces. Again, a single administration of a pharmacologic intervention can ameliorate eye tracking deficits in autism (Andari et al, 2010).
  • ANCOVA repeated analysis of covariance
  • G-2-MePE Treatment with G-2-MePE produces a significant overall improvement in performance of the RMET test. Treatment with 30 mg/kg T.I.D. oral G-2-MePE increases the percent correct responses on the RMET.
  • G-2-MePE Treatment with G-2-MePE produces significant improvements in performance in the Reading the Mind in the Eyes Test - Revised, and in performance of an Eye Tracking task. This effect is dose dependent, seen after treatment 30 mg/kg T.I.D. oral G-2-MePE.
  • G-2-MePE also produces an overall improvement in function as indexed by the Clinical Global Impression of Improvement. Free text annotation of the Case Report Forms from the study indicate this effect related to an improvement in social relatedness. This implies that the changes seen in the RMET and ET task may have relevance to social activity in daily life.
  • G-2-MePE is well tolerated in this population.
  • Example 17 Animal Models for Determining Effects of G-2-MePE on Autism Spectrum Disorders
  • G-2-MePE Effects of G-2-MePE are further tested in the following genetic models of ASD: the Tbx l heterozygous mouse, the Cntnap2 knockout mouse and the Slc9a6 knockout mouse.
  • G-2-MePE is also tested in the fmrl knockout mouse model of Fragile X Syndrome.
  • Tbxl Mutations of the TBX1 gene are associated with Autism Spectrum Disorders (Paylor et al., 2006). Transgenic Tbxl mice are selectively impaired in social interaction, ultrasonic vocalization, repetitive behaviors and working memory (Hiramoto et a!. : 201 1 ).
  • Cntnap2 knockout mice exhibit ASD-related phenotypes in social behavior, ultrasonic vocalization and repetitive behaviors (Penagarikano et al., 201 1).
  • Slc9a6 This gene has been implicated in syndromic ASD and encodes the sodim- hydrigen exchanger 6 (NHE6). Mutations in SLC9A6 are associated with intellectual disability (Gilfillan et al., 2008) and autistic behavior (Garbern et al distract 2010). On Slc9a6 KO mice exhibit motor hyper-activity and cerebellar dysfunction (Stromme et al., 2011).
  • Fmrl Silencing of the FMR1 gene produces Fragile X Syndrome, the phenotype of which includes autism; two thirds of patients with Fragile X Syndrome meet screening criteria for an Autism Spectrum Disorder (Harris et al, 2008). Pediatric patients with Fragile X Syndrome also show lowered seizure threshold. The fmrl knockout mouse replicates much of the phenotype of Fragile X Syndrome, including juvenile seizure susceptibility (Yan et al, 2004), Methods
  • the treatments are administered intraperitoneal ly: placebo (saline) or 20 mg/kg/day of G-2- MePE.
  • G-2-MePE treatment significantly improves all measures associated with the ASD phenotype.
  • Example 18 The Effects of G-2-MePE in Fragile-X Syndrome
  • FXS Fragile X Syndrome
  • Martin-Bell Syndrome also known as Martin-Bell Syndrome
  • Marker X Syndrome is the most common monogenetic cause of autism and the most common inherited cause of mental retardation. It is characterised by a range of intellectual disabilities, physical characteristics such as elongated face, large ears and enlarged testes (macro-orchidism), and a neurobehavioral phenotype that includes stereotypic movements, social anxiety and attention- deficit hyperactivity disorder (ADHD).
  • ADHD attention- deficit hyperactivity disorder
  • Symptoms and signs of FXS may include delays in crawling, walking or twisting, hand clapping or hand biting, hyperactive or impulsive behavior, mental retardation, delays in development of speech and language, tendency to avoid eye contact, stereotypic movements, social anxiety, and attention-deficit hyperactivity disorder (ADHD).
  • ADHD attention-deficit hyperactivity disorder
  • FXS The genetic basis underlying FXS is expansion of the CGG-repeat of the fragile X mental retardation I gene (FMRI) on the X chromosome.
  • the FMRI gene produces a protein called fragile X mental retardation protein, or FMRP.
  • the FMRI gene in normal people contains a trinucleotide segment (CGG) that is repeated from fewer than 10 times to about 40 times. These repeated CGG segments are interspersed with other trinucleotide segments (AGG), which may stabilize the oligonucleotide encoding FMRP.
  • CGG trinucleotide segment
  • the CGG trinucleotide may be repeated from about 200 times to over 1000 times, which makes the oligonucleotide encoding FMRP (i.e., FMRI RNA) unstable. This results in failure to express sufficient amounts of the fragile X mental retardation protein (FMRP), which is required for normal neural development. There is currently no drug treatment that has shown benefit specifically for Fragile X Syndrome.
  • FMRP fragile X mental retardation protein
  • Fmrl gene-knockout mutant mice are available, and show many of the features of clinical FXS, including macro-orchidism, learning deficits and hyperactivity.
  • the finrl knockout ⁇ finrl KO) model displays failure in neuronal pruning, showing dendritic supernumeracy and ERK and Akt hyperphosphorylation. Consequently, fmrl knockout mice represent a valuable tool for testing potential drug treatments of FXS in human subjects.
  • finrl KO mice treatment with G-2-MePE is effective in ameliorating many of the signs and symptoms of FXS.
  • this anxiety reduces upon repeated exposure. This reduction in anxiety is reflected as increased ongoing behavior and therefore either decreased freezing or increased locomotor activity.
  • This decrease in anxiety can be described as a habituation to the experimental situation.
  • This habituation reflects a decrease in perception of novelty and relies upon memory of prior experience. That is, locomotor activity will change during repeated exposure to a test environment as a rodent becomes familiar with that environment. This change will not occur if the rodent does not form memory of prior exposure to the environment. Therefore, habituation to the open field may be altered if cognitive ability is impaired in rodents. Rodents will also learn to associate an environment with presentation of punishment, and upon exposure to an environment associated with punishment will show freezing behavior. This response is known as contextual fear conditioning, which also relies on the ability to code environmental information into memory (Garcia et al (1997) J Neurophysiol. 78:76-81 ).
  • ERK Activation of ERK is reflected in phosphorylation of this intraneuronal signaling protein.
  • ERK is not normally revealed as activated by immunohistochemistry in human brain (Perry et al (1999) Neuroreport. 30:241 1 -2415). However, in disorders affecting CNS function, ERK may be aberrantly activated. This occurs in Alzheimer disease (Perry et al (1999) and in autism (Zou et al (201 1 ) Genes Brain Behav. 10:615-624). Phosphorylation of ERK also occurs in the brain in Fragile X Syndrome and in the finrl knockout mouse model of this disorder (Wang et al (2012) J Neurochem. 121 :672-679).
  • Akt Akt Activation of Akt is also seen in pathological states in the brain, such as Alzheimer disease (Griffm et al (2005) J Neurochem. 93: 105-117) and suicide (Karege et al (2007) Biol Psychiatry. 61 :240-245).
  • Fragile X Syndrome abnormal phosphorylation of the Akt - mToR pathway is also seen in the brain (Hoeffer et al (2012) Genes Brain Behav. 11 :332-341).
  • Fmrl knockout mice and wild-type littermates were generated on a C57BL/6J background and repeatedly backcrossed onto a C57BL/6J background for more than eight generations. Mice were group housed (4-6 per cage) and all animals were provided with ad libitum food and water unless otherwise stated. Mice were maintained on a 12 h light/dark cycle (lights off 19:00 to 7:00) in a temperature-controlled environment (21 ⁇ 1 °C),
  • Tasks were performed in the order described with no more than one task performed per day.
  • mice were tested once in the same apparatus and non- experimental mice were placed in the apparatus for some minutes before the experiment.
  • the apparatus was then cleaned with moist and dry tissues before testing each mouse.
  • the aim was to create a low but constant background mouse odor for all experimental subjects.
  • the animals were divided into the following treatment groups:
  • G-2-MePE was administered at the dose of 100 mg kg i.p. in 0.1 ml saline, once daily for
  • G-2-MePE G-2-MePE
  • NZ N66
  • NZ-2566 N66
  • 2566 the terms "G-2-MePE” was shown to have an unexpectedly long half-life in vivo compared to IGF(1 -3) ("GPE") and thus was considered to be a good candidate for therapy of animals including human beings with Fragile X Syndrome.
  • Hippocampal cell cultures were prepared from wild-type and fmrl KO foetal mice ( 14 - 16 d of gestation). After 3d in vitro, green fluorescent protein (GFP) was used to monitor dendritic spine density during time-course of culture (Ethell and Yamaguchi, 1999; Ethell et al., 2001 , Henkemeyer et al., 2003). Dendritic spines are usually formed between 7 and 14 days in vitro (DIV). By 14 DIV most dendritic protrusions are spines.
  • GFP green fluorescent protein
  • G-2- MePE a positive control (the mGluR.5 antagonist 2-methyl-6-(phenylethynyl)-pyridine or MPEP) and vehicle controls on dendritic spine density in fmrl KO and WT hippocampal primary cell cultures.
  • Fmrl KO and wild-type cultures were treated at 17 DIV.
  • compartmentalized culture system a microfluidic chamber (see FIG. 31A) which opens the possibility for fast drug testing with the capacity to detect in vitro drug effects such as spine morphology, neurite outgrowth and synapse formation.
  • the treated and untreated neurons in the compartmentalized chambers can be also being use for western blot analysis.
  • the compartmentalised culture is a new approach to evaluate the effect of drugs on anterograde and retrograde transport of messengers and proteins relevant to FXS. Other new possibility that offers this approach its access into exclusive axon compartment, in this area immunodetection and treatments are also possible.
  • ERK Phosphorylated Extracellular Signal-Regulated Protein Kinase
  • Akt Protein Kinase B expression was measured by Western blot analysis in lymphocytes from fmrl KO and wild-type animals administered ether vehicle or G-2MePE (100 mg kg, i.p.; 28 days) treated in the behavior experiments as previously described (Lopez Verrilli et al. 2009).
  • ERK is a classical MAPK signal transduction protein, responsible for growth factor transduction, proliferation, cytokine response to stress and apoptosis.
  • Akt is a key component in the PI3K/Akt/mTOR signalling pathway and regulates cellular survival and metabolism by binding and regulating many downstream effectors, such as Nuclear Factor- ⁇ (NficB) and Bcl-2 family proteins.
  • Akt and ERK 1/2 protein content were evaluated by blotting membranes with antiphospho-Akt (1 /1000) and antiphospho-ERK antibodies( 1/2000) (Cell Signaling).
  • 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-blotting membranes with b-actin antibody (1/1000) (Sigma Chemical Co.).
  • the open field is a grey PVC enclosed arena 50 x 30 cm divided into 10 cm squares. Mice were brought to the experimental room 5-20 min before testing. A mouse was placed into a corner square facing the corner and observed for 3 min. The number of squares entered (whole body) and rears (both front paws off the ground, but not as part of grooming) were counted. The latency to the first rearing (rear) was also noted. The movement of the mouse around the field was recorded with a video tracking device for 300s (vNT4.0, Viewpoint).
  • mice habituated to the environment and thus explored less, decreasing the amount movement shown over time. Movement and rearing were recorded during the 3 exposures. Failures to reduce locomotion or rearing at 10 minutes and 24 hours indicated deficits in short and long term memory, respectively.
  • Vehicle-treated fmrl KO mice moved a greater distance in the open field in comparison to the vehicle-treated wild-type group, demonstrating hyperactivity in the fmrl KO animals.
  • the hyperactivity was reversed in the fmrl KO animals by administration of G-2-MePE.
  • G-2-MePE was observed to significantly reduce movement in the 2 nd and 3 rd trials in fmrl knockout mice, suggesting improved memory retention of the test environment, as well as reducing rearing overall in these animals (i.e. attenuating hyperactivity) (FIG. 25).
  • Vehicle-treated fmrl KO mice showed increased rearing activity in comparison to the vehicle-treated wiid-type controls.
  • G-2-MePE had no effect in wild-type mice, but significantly reduced rearing activity in fmrl KO mice.
  • the fmrl KO mice did not show habituation at 10 minutes, which is an indication of a short term memory deficit.
  • G-2-MePE treated fmrl KO mice did not differ from wild-type + vehicle controls. (FIG. 26).
  • the successive alleys test was used to assess the effects of G-2-MePE on anxiety in the fmrl KO mice.
  • the apparatus consists of four successive, increasingly anxiogenic, linearly connected alleys (each succeeding alley is painted a lighter colour, has lower walls and/or is narrower than the previous alley). Animals were placed at the closed end of alley 1, facing the end wall. The latency to first enter each alley, the amount of time spent in each alley, and the number of entries into each alley was recorded during a total test time of 300 s. Results
  • the Elevated Plus Maze is a further test system for examining anxiety and locomotor activity.
  • the EPM was built according to the description of Lister (Lister 1987). Briefly, the maze consisted of two open and two closed arms opposite each other in a plus shape, raised above floor height. The open arms were more exposed and therefore more anxiogenic. Wild-type mice therefore spent more time in the closed arms and visited them more. Mice were tested for 5 min and their behaviour was recorded. Measures taken included time spent in the arms and the center of the maze, and number of arm entries.
  • fmrl knockout mice Compared to wild type mice, fmrl knockout mice showed a pattern of behavior that combined hyperactivity and cognitive impainnent. Surprisingly, fmrl knockout mice showed a slight reduction in the total number of arm entries (indicative of lower activity) (FIG. 28A), and a clear increase in the ratio of entries made into the open arms as a percentage of the total arm entries (FIG. 28B). This behavioral phenotype was not observed in fmrl knockout animals given G-2-MePE (FIGs. 28A and 28B). Indeed, the 'percent open arm entries' measure was completely normalised by drug treatment.
  • Time spent in the center (where open or closed arm entry choice is made), is known to be reduced by treatment with anxiolytic compounds, such as benzodiazepines.
  • Vehicle-treated fmrl knockout mice showed a very marked increase in the time spent in the center (FIG. 28C). This may therefore reflect indecision over which arm to enter, and be indicative of cognitive deficits in the mutant animals.
  • the extra time spent in the centre space (FIG. 28C) presumably underlies the reduced number of total arm entries. Time spent in the centre was normalised by 28 -days G-2-MePE treatment.
  • Contextual fear conditioning is the most basic of the conditioning (learning) procedures. It involves placing the animal in a novel environment (dark chamber), providing an aversive stimulus (a 1-sec electric shock at 0.2 mA to the paw), and then removing it. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the aversive stimulus. Freezing is a species- specific response to fear and is defined as absence of movement except for respiration. The clinical manifestation of Fragile X Syndrome is mental retardation in which learning and memory may be profoundly impaired. We tested G-2-MePE and vehicle treated finr 1 KO and wild-type animals in a fear-conditioning task in which we measured freezing behavior in response to context.
  • a foot shock an aversive experience
  • environmental cues a dark chamber
  • Mice are a social species, which engage in easily scored social behaviors including approaching, following, sniffing, allogrooming, aggressive encounters, sexual interactions, parental behaviors, nesting and sleeping in a group huddle.
  • social recognition and social memory in mice were evaluated by assessing the amount of time spent sniffing a novel mouse upon repeated exposures, to induce familiarity, and reinstatement of high levels of sniffing when a novel stimulus animal is introduced.
  • Fmrl knockout mice displayed heightened sniffing of the presented mouse (*** p ⁇ 0.001 versus vehicle-treated wild-type controls) (FIG. 30A), suggesting a dysfunction in social behavior. This effect was nullified by 28-day treatment with G-2-MePE (100 mg/kg, i.p.). G-2- MePE had no significant effect in the wild-type group. Sniffing behavior of G-2-MePE treated fmrl KO animals was not significantly different to G-2-MePE-treated wild-type group. We conclude that G-2MePE normalized the abnormalities in social behavior seen in fmrl KO mice.
  • G-2-MePE treated fmrl KO animals showed substantially and statistically significantly less sniffing than vehicle treated j 7 KO animals. These effects of G-2-MePE were substantial, statistically significant, and completely unexpected based on the prior art (FIG. 30A).
  • mice spontaneously dig in many substrates in the laboratory. This behavior comes from their ancestry in the wild, where they would forage for seeds, grain, insects, and other food to be found buried in the soil or leaf litter in their natural habitat. It exploits a common natural rodent behavior, provides quantitative data under controlled laboratory conditions, and has proved extremely sensitive to prion disease, Fragile X, strain differences, and brain lesions. We tested the effects of G-2-MePE on both wild-type and the fmrl KO group's nesting and marble burying behavior.
  • FIG. 30B and 30C show that fmrl KO mice engaged in significantly less marble burying (FIG, 30A) and nest building (FIG. 30B) than wild-type mice.
  • G-2-MePE had no significant effect in wild-type mice. After G-2-MePE administration to fmrl KO mice, this group was not significantly different to the G-2-MePE - treated wild-type group.
  • G-2-MePE treatment normalized marble burying behavior in fmrl KO animals. The same profile of effect was seen in nest building behavior.
  • G-2-MePE increased both marble burying and nest building behaviors.
  • Dendritic spine numbers are increased in fmrl KO mice compared to normal, wild-type animals.
  • G-2-MePE G-2-MePE
  • Fmrl KO mouse neurons were cultured in microfluidic chambers and GFP was used to visualize morphology.
  • GFP was used to visualize morphology.
  • We observed that neurons from fmr 1 KO animals, when treated with G-2-MePE at 0.5 nM, did not show a significant improvement (mean ⁇ SD, of n 3 independent experiments: 0.33 ⁇ 0.07), and nor at 5 nM (0.29 ⁇ 0.05) when compared to control cultures (0.26 ⁇ 0.08).
  • Fmrl knockout mice showed an increase in testis size and weight, as do human Fragile X Syndrome patients (macro-orchidism).
  • G-2-MePE had little effect on testis weight in wild-type mice. However, G-2-MePE administration significantly reversed the increase in testis weight seen in fmrl knockout mice (FIG. 32) This effect of G-2-MePE was substantial, statistically significant, and completely unexpected based on the prior art.
  • Neurons are critically influenced by Fragile X Mental Retardation Protein, which regulates local dendritic translation through phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (mTOR) and Ras-ERK signalling cascades and implicated in the mGluR5 signalling cascade.
  • mTOR phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin
  • Ras-ERK signalling cascades implicated in the mGluR5 signalling cascade.
  • ERK activation was increased in vehicle-treated finrl knockout mice compared to wild-type controls (FIGs. 33A and 33B).
  • pERK phosphorylated ERK
  • FIG. 33A and 33B Western blots were normalized to the amounts of GAPDH protein present.
  • Elevated ERK 1 /2 phosphoryl tion (Thr202/Tyr204) in finrl KO-vehicle treated mice was significantly reduced by treatment with G- 2-MePE (p ⁇ 0.05).
  • - fmrl KO mice showed a deficit in habituation (short term memory) which was reversed by G-2-MePE treatment.
  • fmrl KO mice showed a learning deficit in contextual fear conditioning, which was reversed by G-2-MePE.
  • mice showed abnormalities in marble burying and next building, which were reversed by G-2-MePE.
  • G-2-MePE can be an effective treatment for human beings suffering from Fragile X Syndrome and other autism spectrum disorders.
  • Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999 23 : 185-188
  • Gauthier J, Bonnel A, St-Onge J, Karemera L, Laurent S, Mottron L, Fombonne E, Joober R, Rouleau GA. (2005) NLGN3/NLGN4 gene mutations are not responsible for autism in the Quebec population.
  • Hiramoto T Kang G, Suzuki G, Satoh Y, Kucherlapati R, Watanabe Y, Hiroi N.
  • (201 1 ) Tbxl identification of a 22ql 1 .2 gene as a risk factor for autism spectrum disorder in a mouse model.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Psychiatry (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des composés, des compositions et des méthodes de traitement de troubles du spectre autistique (ASD) à l'aide de l'acide glycyl-2-méthylprolyl-glutamique (G-2-MePE) et des analogues de celui-ci. Les troubles du spectre autistique comprennent l'autisme, un trouble autistique, le syndrome d'Asperger, les troubles désintégratifs de l'enfance, le trouble envahissant du développement-non spécifié (PDD-NOS), le syndrome de l'X fragile et le syndrome de Rett. L'invention concerne des compositions contenant des composés comprenant des formulations hydrosolubles, des microémulsions eau-dans-l'huile, des émulsions grossières d'eau-dans-l'huile, des cristaux liquides eau-dans-l'huile, des nanocapsules, des comprimés et des gels administrés oralement. Les composés et compositions de cette invention peuvent être administrés de façon intraveineuse, intraventriculaire, parentérale ou orale, et peuvent être efficaces dans le traitement d'une neurodégénérescence, dans la promotion de la fonction neurologique, dans le traitement de l'activité épileptique et d'autres symptômes d'ASD, et peuvent prolonger la vie chez des animaux comprenant des être humains ayant des troubles du spectre autistique.
PCT/US2013/072049 2012-11-28 2013-11-26 Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique WO2014085480A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA2929286A CA2929286A1 (fr) 2012-11-28 2013-11-26 Traitement de troubles du spectre autistique a l'aide de l'acide glycyl-l-2-methylprolyl-l-glutamique
AU2013352294A AU2013352294A1 (en) 2012-11-28 2013-11-26 Treatment of Autism Spectrum Disorders using glycyl-l-2-methylprolyl-l-glutamic acid
EP13858943.7A EP2928300A4 (fr) 2012-11-28 2013-11-26 Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique
JP2015545191A JP2016506380A (ja) 2012-11-28 2013-11-26 グリシル−ｌ−２−メチルプロリル−ｌ−グルタミン酸を使用する自閉症スペクトラム障害の処置
BR112015012506A BR112015012506A2 (pt) 2012-11-28 2013-11-26 tratamento de transtornos do espectro do autismo usando ácido glicil-l-2-metilprolil-l-glutâmico
US14/602,600 US20150224164A1 (en) 2013-11-26 2015-02-09 Treatment of autism spectrum disorders using glycyl-l-2-methylprolyl-l-glumatic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261730829P 2012-11-28 2012-11-28
US61/730,829 2012-11-28

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/602,600 Continuation US20150224164A1 (en) 2013-11-26 2015-02-09 Treatment of autism spectrum disorders using glycyl-l-2-methylprolyl-l-glumatic acid

Publications (1)

Publication Number Publication Date
WO2014085480A1 true WO2014085480A1 (fr) 2014-06-05

Family

ID=50828422

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/072049 WO2014085480A1 (fr) 2012-11-28 2013-11-26 Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique

Country Status (6)

Country Link
EP (1) EP2928300A4 (fr)
JP (1) JP2016506380A (fr)
AU (1) AU2013352294A1 (fr)
BR (1) BR112015012506A2 (fr)
CA (1) CA2929286A1 (fr)
WO (1) WO2014085480A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016004117A1 (fr) * 2014-06-30 2016-01-07 Cerora, Inc. Système et signatures pour évaluation de biomarqueur périodique physiologique multimodale
EP3833358A4 (fr) * 2018-08-10 2022-04-13 New York University Utilisation de ligands agonistes du récepteur de l'igf-2 pour le traitement du syndrome d'angelman et de l'autisme

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8178125B2 (en) * 2006-03-14 2012-05-15 Neuron Pharmaceuticals Limited Oral formulations of glycyl-2-methylprolyl-glutamate
WO2012102832A1 (fr) * 2011-01-27 2012-08-02 Neuren Pharmaceuticals Limited Traitement de troubles du spectre autistique en utilisant l'acide glycyl-l-2-méthylprolyl-l-glutamique
US8278276B2 (en) * 2006-11-22 2012-10-02 Seaside Therapeutics, Inc. Methods of treating autism

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60232880D1 (de) * 2001-05-24 2009-08-20 Neuren Pharmaceuticals Ltd Gpe-analoga und peptidomimetika
US8637567B2 (en) * 2001-05-24 2014-01-28 Neuren Pharmaceuticals Ltd. Cognitive enhancement and cognitive therapy using glycyl-L-2-methylprolyl-L-glutamic acid
ES2367868T3 (es) * 2004-09-20 2011-11-10 Mount Sinai School Of Medicine Uso de memantina (namenda) para tratar el autismo, la compulsividad y la impulsividad.
EP1888618A4 (fr) * 2005-05-23 2009-07-15 Neuren Pharmaceuticals Ltd Analogues de glycyl-prolyl-glutamate
US7994127B2 (en) * 2007-06-08 2011-08-09 Massachusetts Institute Of Technology Treatment of rett syndrome
WO2011146109A2 (fr) * 2010-05-17 2011-11-24 Mount Sinai School Of Medicine Procédés et tests pour le traitement de sujets présentant une délétion, une mutation ou une expression réduite de shank3

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8178125B2 (en) * 2006-03-14 2012-05-15 Neuron Pharmaceuticals Limited Oral formulations of glycyl-2-methylprolyl-glutamate
US8278276B2 (en) * 2006-11-22 2012-10-02 Seaside Therapeutics, Inc. Methods of treating autism
WO2012102832A1 (fr) * 2011-01-27 2012-08-02 Neuren Pharmaceuticals Limited Traitement de troubles du spectre autistique en utilisant l'acide glycyl-l-2-méthylprolyl-l-glutamique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2928300A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016004117A1 (fr) * 2014-06-30 2016-01-07 Cerora, Inc. Système et signatures pour évaluation de biomarqueur périodique physiologique multimodale
US10254785B2 (en) 2014-06-30 2019-04-09 Cerora, Inc. System and methods for the synchronization of a non-real time operating system PC to a remote real-time data collecting microcontroller
EP3833358A4 (fr) * 2018-08-10 2022-04-13 New York University Utilisation de ligands agonistes du récepteur de l'igf-2 pour le traitement du syndrome d'angelman et de l'autisme

Also Published As

Publication number Publication date
EP2928300A1 (fr) 2015-10-14
AU2013352294A1 (en) 2015-07-09
JP2016506380A (ja) 2016-03-03
BR112015012506A2 (pt) 2017-07-11
CA2929286A1 (fr) 2014-06-05
EP2928300A4 (fr) 2016-07-13

Similar Documents

Publication Publication Date Title
JP6640275B2 (ja) グリシル−ｌ−２−メチルプロリル−ｌ−グルタミン酸を用いる自閉症スペクトラム障害の治療
US11197856B2 (en) Bicyclic compounds and methods for their use in treating autism
US20150224164A1 (en) Treatment of autism spectrum disorders using glycyl-l-2-methylprolyl-l-glumatic acid
US20210169964A1 (en) Methods of treating alzheimer's disease, huntington's disease, autism, and other disorders
WO2014085480A1 (fr) Traitement de troubles du spectre autistique à l'aide de l'acide glycyl-l-2-méthylprolyl-l-glutamique
US20210206714A1 (en) Compositions and methods for reducing tactile dysfunction, anxiety, and social impairment
US20190091284A1 (en) Amidated Dopamine Neuron Stimulating Peptides for CNS Dopaminergic Upregulation
US20180117113A1 (en) Amidated Dopamine Neuron Stimulating Peptide Restoration of Mitochondrial Activity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13858943

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2015545191

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112015012506

Country of ref document: BR

REEP Request for entry into the european phase

Ref document number: 2013858943

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2013858943

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2013352294

Country of ref document: AU

Date of ref document: 20131126

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2929286

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 112015012506

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20150528