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WO2017066434A1 - Inhibition de tropomyosine kinase b (trkb) de facteur neurotrophique dérivé du cerveau (bdnf) pour améliorer des déficits cognitifs - Google Patents

Inhibition de tropomyosine kinase b (trkb) de facteur neurotrophique dérivé du cerveau (bdnf) pour améliorer des déficits cognitifs Download PDF

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WO2017066434A1
WO2017066434A1 PCT/US2016/056822 US2016056822W WO2017066434A1 WO 2017066434 A1 WO2017066434 A1 WO 2017066434A1 US 2016056822 W US2016056822 W US 2016056822W WO 2017066434 A1 WO2017066434 A1 WO 2017066434A1
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mice
ims
cognitive
patient
disorder
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Claudia SCHMAUSS
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • 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
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • BDMF BRAIN-DERIVED NEUROTROPHIC FACTOR
  • TRKB TROPOMYOSINE KINASE B INHIBITION FOR IMPROVING COGNITIVE DEFICITS
  • the present invention relates to methods and treatments for improving cognitive functioning in patients in need thereof, in certain, aspects, the methods comprise administering at. least one brain-derived neurotrophic factor (Bdnfj-troporayosine kinase B (TrkB) inhibitor,
  • H ' DAC histone deacetylase
  • the present invention relates to a method of treating cognitive defects in a mood disorder in a patient in need thereof, .comprising administering a therapeutically effective amount of at ieast one brain-deri ved neurotrophic factor (Bdni)- tropomyosme kinase B (TrkB) inhibitor to the patient.
  • Bdni brain-deri ved neurotrophic factor
  • TrkB tropomyosme kinase B
  • the present invention relates to a method of improving cognitive functioning in a patient in need thereof comprising administering a therapeutically effective amount of at least one brain-derived neurotrophic factor (Bdrd)4:roporo.yosine kinase B (TrkB) inhibitor to the patient.
  • Bdrd brain-derived neurotrophic factor
  • TrkB tumor necrosis factor
  • the treating comprises at least a 10% improvement of cognitive functioning, compared with a baseline cognitive functioning reference or control.
  • the cognitive functioning improvement is greater than, about 50%.
  • the TrkB inhibitor is a TkrB antagonist
  • the TrkB inhibitor is administered by mouth, topically, rectally, or intravenously.
  • the mood disorder is an anxiety disorder or stress- induced disorder including early life stress, or a combination thereof.
  • the mood disorder is a panic disorder, an obsessive compulsive disorder, a post-traumatic stress disorder, or any combination thereof.
  • the patient is an adolescent or young adult.
  • the TkrB inhibitor comprises ANA- 12.
  • the method further comprises administering at least one selective serotonin reuptake inhibitor (SSRt) selected from the group consisting of
  • Citalopram (Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat);
  • Fluoxetine Prozac
  • Fluvosamine Livox
  • Sertraline Zaloft, Lusirai
  • the patient exhibits a mood disorder comprising an anxiety disorder or stress-induced disorder including early life stress, or a combination thereof.
  • a mood disorder comprising an anxiety disorder or stress-induced disorder including early life stress, or a combination thereof.
  • the patient is a mammal. In additional embodiments, the patient is a human. In additional embodiments, the patient exhibits a cognitive deficit in one or more of the fo.Howi.ng features: working memory or attention set-shifting.
  • Figs, 1A-C are diagram ' s showing the experimental design and dosing timeline for the model mice.
  • Fig, LA is a timeline showing that mice exclusively used for HDACl and HDAC3 CMPs were exposed to the IMS from P2 and Pi 5, weaned at P28 and raised to adulthood (P60). At P60, their brains were removed, SFR mice raised in paraiiei served as controls.
  • Fig, IB is a timeline showing how other groups of mice were chronically treated with either Ana.- 12 (IMS Balb/c mice) or with MS -275 (SFR Ba!b/c mice) from postnatal age P35 until P59. At the end of treatment, mice were tested in tire ASST followed by training and testing in the WM test.
  • WM test brains were collected for real-time RT-PCR and CMP experiments.
  • Age-arid sex-matched vehicle-treated mice served as controls.
  • Fig, 1 C shows a timeline of the Forster IMS C57BI/6 or foster IMS Balb/c mice that were raised by Balb/c mothers during IMS exposure, weaned ai P2S, aid tested in the ASST in adulthood (at P66).
  • One hour post completion of the IDS phase of the ASST brain were collected for real-time PCR and ChJP experiments. Age- and sex-matched non-tested mice served as controls.
  • Figs. 2A-D are graphs showing reduced HDACl levels at promoter ill of the Bdnf gene in IMS Balb/c mice.
  • Fig. 2 A is a graph illustrating comparison of the levels of HDACl and H.DAC3 at promoters ill and IV of the Bdnf gum and the promoters of the Bgr2 and fos genes in SFR and IMS Balb/c mice.
  • Data are mean ⁇ sent (n ⁇ 8/group for the HDAC3 Bdnf Pill ChIP (4 males and 4 females) and n ::: 6/group for ail other groups (3 males and 3 females)) and were compared by Student's t-tesi.
  • FIG. 2B shows Bdnf transcript variant IK expression in non-tested and WM-tested (20 s delay) SFR and IMS Balb/c mice. Data (mean ⁇ sem; n ⁇ ( S/group) were compared by 2-way ANOVA (see Results) and statistical differences were resolved post hoc (Takey HSD test) as indicated.
  • Fig. 2C shows a comparison of the levels of Pol II association at Mff/promotor III between non- tested and WM-tested IMS Balb/c mice. Data (mean * sem; n - 6/group) were compared using two-tailed Student's t test.
  • Fig, 2D shows Egr2 mRNA expression levels in non-tested and WM tested SFR and IMS Bato/c mice.
  • Rfk 3A-B are gnphs showing the effects of adokftcent ANA-12 treatment of IMS and SFRBalh/c imce on cognitive task perfonnauce in adnltibood.
  • Fig.3 A is a graph showing perJfonnauce of SFR, IMS and Ana-12-treated SFR and IMS BaJb/c mice in the ASST. Dan are mean ⁇ sem of 8 animab per group (4 males and 4 females) and woe coopered by 2-way ANOVA (lee Remits). Statistical dilfcrences were resolved post hoc (Tukey HSO tests) as indicated.
  • FIG. 3B is a graph illustrating (he performance of me same groups of mice in the WM test Data were compared by 2-way ANOVA (see Results) and significant differences resolved post hoc (Tnkey HSD tests) are iixbcaied. When tested at 20 s inter-trial delays, the percentages of correct arm entries of IMS Balb/c and Ana-12-treated SFR mice were signiflcantiy lower conmaiiedtoiioii-treatedSFRan ⁇
  • Figs.4A-D are graphs iBmtratmg the effect of adolescent MS-275 treatment of SFR BabVc mice on cognitive task perfixmance and expression of B
  • Fig.4B is a graph Castrating the performance of die same group of mice shown in Fig.4A in the spatial WM test At 20 s delay, significant differences were found between the two groups (Sbjdenfs t test).
  • Fig. 4C is a graph of the real-time RT-PCR measures of Bdnf transcript variant Dl expression. Date
  • Fig.4D is a graph for the same group of mice shown in Fig. 4C, quantifying the real-time RT-PCR measures of Egr2 mRNA and revealed no significant differences between the three groups (ANOVA, F(2,16) - 0.5; p- 0.62).
  • 5B is a graph coatwmsj the levels of HDACl aid HD AC3 et promoters 1 ⁇ and IV of the Bdnfgaa* and the pomotort of tibe £gr2 and For geoei in SFR and IMS C57B1/6 mice raised by their biological mothea and foster IMS C571/6 mice raised by Balb/c mothers. Data are mean ⁇ sen (n-6/group).
  • One- way ANOVA revealed sigmficant diflerences between groups only for the levels of HDAC1 at the -%r2 gene promoter (F(2,14)-9.13 ⁇ 4 p-0.005).
  • Poet hoc Tokey-Kramer multiple compariaoos resolved these differences for foster IMS C57BI/5 mice that had significantly lower levels of HDAC1 cocnpared to SFR and IMS C57BI/6 mice.
  • Figs.4LA-D are graphs showing that Fooler IMS C57BI/6 mice exhibit increased Egr2 mRNA expression after ASST testing and increased histone H3 acetylation at the Egr2 promotor.
  • Fig. 6A shows the consparison of the levels of Pol ⁇ aaMciation with the Egr2 promotor between non-tested and ASST-tested foster IMS C57B1/5 mice.
  • FIG. 6D shows die ratio of acH3K9 over H3K9me3 levels associated with the Egr2 promoter of SFR and IMS C57BI/6 and Balb/c mice compared to foster IMS C57B1/6 mice.
  • Data are mean ⁇ sem (n - 5/group).
  • One-way ANOVA revealed iigniftcant diflerences between groups (F(4,19) « 3.9; p - 0.02) that were resolved post hoc for foster IMS C57B1/6 mice that exhibited a significantly higher acH3KMDK9me3 ratio.
  • *p ⁇ 0.05 compared to all other groups.
  • mice show pronounced cognitive deficits in adulthood, namely deficits, in -wciiing .memory and, attention set-shifflng.
  • the present data i-tosteste mat these mice also exlubh reduced association of HDACl wim promotor ⁇ of 4e brsMerived neurotiophic factor ( ⁇ gene, and mat cognitive testing leads to abnormally increased Bdnf mRNA expression.
  • promoter HI are unaltered in such C57B1/6 mice, although they exhibit decreased levels of HDAC1 at the nromotor of the earty-growtb response gene 2 (Egr2) and atmonnaOy increased Egi2 mRNA expression after cognitive testing.
  • Egr2 earty-growtb response gene 2
  • atmonnaOy increased Egi2 mRNA expression after cognitive testing.
  • mice have been utilized as co-relating with human developmental learning, and models of human emotional behavior for many years (See MiOan, M. J. 2008 and Sdimtuss et al., 2014).
  • P1-P28 moose pup day 1- mouse pup day 28 mice correspond developmentaDy to earty human childhood, with P21- P28 as the typical weaning time for ow mouse pups.
  • Mice beyond P28 to P60 correspond to human adolescence (Set MiHan, M. J.2008). Mice mat are beyond P60(iiicwpi-p day 60), and m particular beyond P66 are coniidered to correspond to human adults.
  • IMS Bafo/c mice are found in the fcrebrain neocortex, and they are attributable to a lustone-based epigeDetic response to IMS exposure, namely increased acetylabon of histone H4 protein that is due to deuesaed activity of several dare ⁇ / ⁇ HDACs, inclnding class I HDACs 1 and 3 (Levine et si, 2012).
  • This epigenetic phenotype emerges during adolescent development and persists into adulthood. While it ameliorates the severity of foe emotional phenotype in adulthood (ticvine et aL.
  • the data provided herein illustrate mat both IMS Balb/c mice and foster IMS C57B1/6 mice exhibit reduced HDACI association with promoters of distinct plasticity-associated genes, and that cognitive test exposure triggers an. abnormally increased, expression of these genes.
  • the affected genes differed between the two strains;
  • IMS Baib/c mice reduced HDAC I levels axe found on promoter HI of the Aft/gene, and increased expression Bdnf and hence, increased Bdnf-iropomyosine kinase B (TrkB) receptor signaling can explain the cognitive phenotype of IMS Balb/c mice.
  • Cii compound duciiminitKin
  • ELS- early life stress for example everts during childhood that exceed 0» child's coping resources inchiding physical, sexual. emotional tad vertMl abuse, lfttghm. deprivation, disaster, household dysAmctions such at witnessing violence, criminal activity, parental separation parental death or illness, poverty, and gubitancc abuse. See, Pechtri and Pizzagalli 2011);
  • TrkB ncn-coiijpetiUve antegonist of TrkB (Kd ⁇ » ⁇ 10 nM and 12 uM for the high- and km-aflmity sites, respectively), the tnrin receptor of breixh derived neurotrophic factor (BDNF/Bdnf) (Cazorla 2011).
  • BDNF/Bdnf breixh derived neurotrophic factor
  • the compound crosses the Wood- braiit-berrier and exerts central TrkB blockade, producing effects as early at 30 ramattt (-400 nM) and at long at 6 hours (-10 nM) fofowing hffltperitoneal injection in mice (Cazorla 2011).
  • ANA-12 produces rapid antidepressant- and anxwrytie-like eflects in animal models, the former of which have been ducidtted to be mediated by blockade of BI ⁇ signahng m Ae nticleu
  • MS-275 is also known as Entinostat, or SNDX-275 and is a benzamide bjstonc deacetylaae inhibitor undergoing clinical trials for treatment of various cancers.
  • Fjirmottat inhibits class I HDACl and HDAC3 win IC M of 0.51 uM and 1.7 ⁇ , respectively . It has the formula; Q»H*N-*1 ⁇ 4.
  • TrkB ioJnbiior conspounds include LOXO-lOl; N- T04; AZ623 and Enlrectinib. It it expected (hat any TrkB nrtbitor mat can penetrate the blood brain barrier will be suitable for die methods described herein.
  • LOXO- 101 is expected to be a suitable inhibitor and is described in: Cancer Discovery 5:1049-1057, 2015. Robert C. Doebefe el al. An Oncogenic NTRK Fusion in a Patient with SoA-Tissue Sarcoma with RMpoue to the TroponiyoeiR-Rilettxi Kinase Fusion tahmiior LOXQ-101.
  • Activation ''stimulation/* and “treatment,'* as it applies to ceils or to receptors, may have the same meaning, e.g., activation, sthmnation, or treatment of acefl or receptor with a tigand, unless indicated otherwise by me context or expboitjy.
  • Ligand*' encompasses natnml and synthetic Kgm-fa cytokines, cytokine variants, analogues, »** sod binding compounds derived from amibodies.
  • Ligand also encc3 ⁇ 4npasses small molecules, e ⁇ ., peptide mimetic* of cytokines and peptide nmnetics of antibodies
  • Activation can reifar to ccfl activation as regulated by internal mechanisms as well as by external or environmental factors.
  • Response,' * eg., of a cell, tissue, organ, or otganitni encompasses a change in biochemical or physiological behavior, e.g., concentration, density, adhftion, or migration within a biological conyartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms sixdh as genetic programming.
  • Activity of a molecule may describe or refer to the landing of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stmailatn gene expression or cefl signaling, differentiation, or tnatnrstion; to antigenic activity, to the modulation of activities of other molecules, and the hike. "Activity'' of a molecule may also refer to activity in modulating or maintaining cetHo-ccfl mteractiora, e.g.. adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton.
  • Activity can also mean specific activity, e*, [catalytic actrvity]/[mg protein], or [immunological activity)/[mg protein], cot-ceotEstion in a biological ttxi-partment, or me like. “Activity” may refer to modulation of cemponerrtsofthemnateof t ⁇
  • *3 ⁇ 4omology refers to sequence similarity between two rwrynucleotide seqoenoes or between two polypeptide snnjuffnr** when they are optimally aligned.
  • a position in both of die two compared sequences is occupied, by die seme base or amino acid monomer sobuntt, e ⁇ , if a position in each of two DNA n»lecules is occupied by adenine, men the molecules are homologous at (hat position.
  • the percent of homology is the number of honiologoos positions shared by die two sequences divided by the total number of positions compared *100.
  • isolated nucleic acid molecule means a DNA or RNA of genomic, raRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
  • nucleic acid molecule comprising a particular nucleotide sequence does not encompass intact chromosomes
  • isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified, sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequeoces that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences thai are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequenee or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates hi the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • trans ⁇ formants*' and “transformed cells” include the primary subject ceil and cultures derived therefrom without regard for the number of transfers, it is also understood that not ail progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have fee same function or biological activity as screened for in the originally tr afisfoaned cell are included. Where disimct designations ate ratended, it will be clear from the context
  • PCR refers to a procedure or technique in which specific nucleic acid sequences, RNA and/or DNA, are amplified as described in, e,g., U.S. Pal No.4,683,195.
  • sequence inJbniietioa from the ends of the region of interest or beyond is used to design oligonucleotide primers.
  • These primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • the 5" terminal nocleotides of me two primers can coincide with the ends of die amplified material.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Muliis et at. (1987) Cold Spring Harbor Symp. Quant. BtoL 51.263; Erich, etL, (1989) PCR TECHNOLOGY (Stockton Press, N.
  • PCR is comridered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample axqmsing the use of a known nuclek acid as a primer and a nucleic acid polymerase to amplify or generate a - ⁇
  • isolated refers to a cell mat has been isolated from its natural environment (e.g., from a tissue or subject).
  • the term "ceD line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are donal populations derived from a single progenitor cefJ. It is further known in the art mat spontaneous or induced changes can occur in karyotype during storage or transfer of such cionelpci»lalions.71)m
  • the terns "recombmsjit c*U” refers tt DNA segment, such as DNA segment mat leads to the transcription of a otologicalfy-active polypeptide or production of a biologkalhr active nucleic acid such as an RNA, has been introduced.
  • vector* includes any genetic castnsnt, such as it plasrnsi, phage, uaiisposon, coemid, chromosome, artificial chiornosome, virus, virion, etc, which Is capable of replication when wwr iatrrrt with the proper control mff0mr ⁇ m and which can transfer gene. yfnnm ⁇ fff 3 ⁇ 4p ⁇ Y
  • useful vectors are contemplated to be those vectors in which me nucleic acid segment to be transcribed is positioned under the ttssacriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or. introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrases “operativeiy positioned,” “operativeiy linked,” “under control,” or “under transcriptional control” means that the promoter Is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shKNA, miRNA) from a transcribed gene.
  • a biologically-active polypeptide product or inhibitory RNA e.g., shKNA, miRNA
  • “Inhibitors” and “antagonists,” or “activators” and ''agonists,” refer to inhibitory or activating molecules, respectively, e.g., for the activation of, e.g., a iigand, receptor, cofactor, a gene, cell, tissue, or organ.
  • a modulator of, e.g., a gene, a receptor, a Iigand, or a cell is a molecule that alters an activity of the gene, receptor, iigand, or cell, where activity can be activated, inhibited, or altered in its regulator ⁇ 1 properties.
  • the modulator may act alone, or it may use a cofactor, e.g., a protein, metal ion, or small molecule.
  • Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g., a gene, protein, Iigand, receptor, or cell.
  • Activators are compounds that increase, activate, facilitate, enhance activation, sensitize, or up regulate, e.g., a gene, protein, Iigand, receptor, or cell.
  • An inhibitor may also be defined as a compound that reduces, blocks, or inactivates a constitutive activity.
  • An "agonist” is a compound that interacts with a target to cause or promote an increase in the activation of the target.
  • An "antagonist” is a compound that opposes the actions of an agonist.
  • An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist.
  • An antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist.
  • samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activator or inhibitor and are compared to control samples without the inhibitor.
  • Control samples i.e., samples not treated with antagonist, are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 25%.
  • Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, iMro patently*
  • Bndpoints in actuation or iidubitiaa can be momtorcd as follows. Activation, inhibition, and response to treatment, &g., of a cell, physiological find, tisane, organ, and predetermined qiiantity or percentage of, e.g., indicia of inflammation, oncogenicity, or cell degnmularionorse
  • the cndpoim may cop.p.ise, eg., a predetennined quantity of ion fan, or transport; ccfl migration; ceo adhrsion; cell proliffe-etion; potential for tnctattsttr, cell differentiation; and change in pbenotype, e.g., change in expression of gene relating to tnflammarion, apoptosia, transfonnation, cell cycle, or metastasis (see, e.g ⁇ Knight (2000) Ann.
  • An endpoint of inmuftkn is generally 75% of the control or lest, preferably 50% of the control or less, more preferably 25% of the control or less, and most preferably 10% of the control or leas.
  • an endpoint of actrvation is at least 150% the control, preferably at least two times me control, more preferably at least four times me control, and most preferably at least ten times the controL
  • Small molecule is defined as a molecule with a molecular weight mat is km man 10 kOa, typically less than 2 kDe, preferably less than 1 kDa, and most preferably less than about 500 Da.
  • Small molecules include, but are not timited to, inorganic molecule*, organic molecules, organic molecules containing an aorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide nrimetics, and antibody nmnerks.
  • a small molecule may be mora permeable to cells, less susceptible to degradation, and less apt to elicit an immune response than large molecules SmallinoJecnlee.
  • TrkB inhibitor e.g., ANA- 12
  • SSRI inhibitor e.g., Citalopiam (Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil Seroxat); Fluoxetine (Prozac); Fluvoxamine (Luvox); Sertraline (Zoloft, Lustral), or similar compositions described herein, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease or being at elevated at risk of acquiring a disease, for which the agent has therapeutic activity.
  • TrkB inhibitor e.g., ANA- 12
  • SSRI inhibitor e.g., Citalopiam (Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil Seroxat); Fluoxetine (Prozac); Fluvoxamine (Luvox); Sertraline (Zoloft, Lustral), or similar compositions described here
  • the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such sympiomt ' s) by any clinically measurable degree.
  • the amount of a therapeutic agent that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.
  • an embodiment of the present invention may not be effective in alleviating the target disease sympiom(s) in every subject, it should alleviate the target disease symptom(s) in a statistically significant number of subjects as determined, by any statistical test known in the art such as the Student's t-test, the chr-test, the U-test according to Mann and Whitney, the Kmska!-Waliis test (B-test), Jonckheere-Terpstra-tesi and the Wilcoxon-test.
  • any statistical test known in the art such as the Student's t-test, the chr-test, the U-test according to Mann and Whitney, the Kmska!-Waliis test (B-test), Jonckheere-Terpstra-tesi and the Wilcoxon-test.
  • Treatment refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications.
  • Treatment as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses combination treatments including: one or more TrkB inhibitors, optionally in. combination with one or more SSR.T inhibitor (described herein)., or related methods described herein as applied to a human or animal subject, a cell, tissue, physiological compartment, or physiological fluid.
  • Formulations of therapeutic and diagnostic -agents may be prepared by mixing with acceptable carriers, exctpients, or stabilizers in the form of, e.g., iyophjfeed powders, slurries, aqueous solutions or suspensions (see, e.g., Hardmaa, et al.
  • Toxicity and therapeutic efficacy of the therapeutic compositions, administered alone or in combination with another agent can be determined by standard pharmaceutical procedures to cell cultures or experimental animals, e.g., for determining the LD S0 (the dose lethal to 50% of the population) and the EDse (the dose therapeutically effective in. 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index (LD 5 0/ EDse).
  • therapeutic compositions exhibiting high therapeutic indices are desirable.
  • the data obtained from these ceil culture assays and animal studies can be used in formulating a range of dosage for use in human..
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration.
  • composition of the invention is administered to a subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).
  • Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal., or Inira-arterial
  • the composition or therapeutic can be administered by an invasive route such as by injection (see above).
  • the composition, therapeutic, or pharmaceutical composition thereof is administered intravenously, subcuianeously, intramuscularly, mtraarterial!y, ulcerra-attieularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery.
  • Administration by non- invasive routes e,g ., orally; for example, in a pill, capsule or tablet
  • non- invasive routes e,g ., orally; for example, in a pill, capsule or tablet
  • compositions can be administered with medical devices known in the art.
  • a pharmaceutical composition of the invention can. be administered by injection with a hypodermic needle, including, e.g., a prefil!ed syringe or autoinjector.
  • compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; S,312,335; : 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; S,312,335; : 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556.
  • inhibit or “treat” or “treatment” includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the sympioms of such disorder.
  • the terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the terms denote that a beneficial result has been conferred on a vertebrate subject with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.
  • the present methods are suitable for treating adolescent or young adult patients (with typical age ranges from 30-22; but including those as young as 5- 10), in certain embodiments, patients in need of treatment will include those that have experienced ELS (early life stress, as described for example in PechteJ. and Piz/agalli 201. 1). In certain embodiments, it is expected that the patient will exhibit a cognitive deficit in one or more of the following features; working memory and attention set-shifting.
  • the patient will exhibit improvement in at least one of these features including working memory and attention set-shifting, during and following treatment with an effective amount of at least one TrkB inhibitor, when compared to normal baseline or a control patient value. Additionally, in certain instances and based on the present results in the model data, treatment should continue until improvements arc observed, at which time treatment can be stopped. The benefits of the treatment are expected to endure following treatment, as shown in the model, data provided herein.
  • SSRI's selective serotonin reuptake iiiiibitor
  • the TrkB inhibitor e.g. ANA-12
  • desired SSRI may be combined into one formulation for ease of patient delivery and compliance.
  • the pharmaceutical compositions may also contain a pharmaceutically acceptable excipient
  • excipients include any phannaceutieal agent that does not itself induce the production of antibodies .harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable satis can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromid.es, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pil buffering substances, and the like., may be present hi such vehicles.
  • auxiliary substances such as wetting or emulsifying agents, pil buffering substances, and the like.
  • kits comprising the components of the combinations of the invention in kit form.
  • a kit of the present invention includes one or more components including, but not limited to, one or more TrkB inhibiting compounds (such as Ana-12) as discussed herein, in association with one or more additional components including, but not limited to a -pharmaceuticalfy acceptable earner and/or an SSRI agent, as discussed herein.
  • Kits may also include primers, buffers, and probes along with instructions for determining elevated levels of nucleic acid, proteins., or protein fragments of a desired target.
  • a kit in one embodiment, includes additional compoimds/c-omposition of the invention or a pharmaceutical composition thereof in one container ⁇ e.g., in a sterile glass or plastic vial) and a second pharmaceutical composition in another container (e.g., in a sterile glass or plastic vial).
  • the kit comprises a combination of the invention, including one or more TrkB inhibitors (e.g. ANA-12) in combination with one or more SSRI inhibitors (as described herein) along with a pharmaceutically acceptable carrier, optionally in combination with one or more additional therapeutic agent components formulated together, optionally, in a pharmaceutical composition, in a single, common container.
  • TrkB inhibitors e.g. ANA-12
  • SSRI inhibitors as described herein
  • the kit can include a device for performing such administration.
  • the kit can include one or more hypodermic needles or other injection devices as discussed above.
  • the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
  • the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
  • Example L Reduced levels of HDACl at Bdnf promoter 111 and increased Bdnf mRNA expression in IMS Balb/e mice exposed to cognitive tests.
  • Egr2 One of the targeted genes is Egr2, It was previously shown with immunocytochemicai studies on C57B1/6 mice performing an attention-set shifting task (ASST) that Egr2 immunoreactiyity was specifically increased in the ventrolateral orbital frontal cortex and the pre- and infmlimbic subregions of the medial prefrontal cortex (mPFC) during the associative lea ning phases of the ASST, and that infraiirabie Egr2 expression farther increased when mice performed the set-shifting phases of the ASST (DeSteno and Schmauss, 2008). In contrast to ASST-tesied mice, .rake that performed.
  • ASST attention-set shifting task
  • ASST-tesied mice in contrast to Egr2 gene induction, ASST-tesied mice also exhibit Fos gene induction in other brain regions, including orbital, motor and somatosensory cortices, hippocampus, dorsal striatum, nucleus aecmnhens, thalamus, and hypothalamus (Glickstein et al, 2005).
  • forebrain neocortical tissue that includes the entire mPFC was used to determine the levels of HDACI and H.D.AC3 on pmmoiors of the Bdnf, Egr2 f and Fm genes.
  • Fig, 2A in SFR Balb/e mice the highest levels of HDACI were found at Bdnf promotor III, and HDAC i association with the same promoior was significantly reduced in IMS Balb/c mice.
  • the levels of HDACI at Bdnf promotor IV as well as the HDACI levels at the promoters of the Egr2 and Fos genes were indistinguishable between SFR and IMS mice.
  • no significant differences were found for HDAC3 associations with any of the promoters examined.
  • a 2 -way AN0VA revealed a significant main effect of rearing groups (SFR and IMS; F( 1,20) ⁇ 21 ,9; p ⁇ 0,0001), a significant main effect of treatment (tested versus non tested; F(1 ,20) ::: 30.4; p ::: 0.0001 ), and a significant interaction between these main effects (F(l,20)- 18.5, p ⁇ 0.0004).
  • Post hoc Tukey BSD tests resolved these differences for WM-tesied IMS Balb/c mice thai expressed significantly more Bdnf mRNA compared with non-tested IMS mice as well as tested and nonnested SFR mice.
  • Example 2 Reducing Bdnf-TrfcB signaling during adolescent development improves the cognitive functions of IMS Balb/c.
  • Fig, 3A summarizes results obtained from the ASST.
  • mice proceed through.4 consecutive test phases, beginning with a simple discrimination (SD) between (wo different odors or textures to find a food reward, and followed by a compound discrimination (CD) to which an additional stimulus property (one new odor or texture) is added that does not guide correct response selection. Then, a completely new set of odors and textures is presented, but the stimulus property (odor or texture) that associated with correct response selection in the CD still guides correct response selection. This is the intxadiraeasional set shifting phase (IDS) of the ASST. Finally, another set.
  • SD simple discrimination
  • CD compound discrimination
  • IDS intxadiraeasional set shifting phase
  • MS-275-treated SFR mice exhibit deficits ia the EDS phase of the ASST that are similar to those found in IMS mice. Moreover, they exhibit a significant deficit in the spatial WM test at 20 s inter-trial delay (Fig. 4B).
  • Fig. 4B Next we compared the levels of mRNA transcribed from Bdnf promotor III and the Egr2 promotor between SFR Balb/c mice and non-tested and WM-iested SFR Balb/c mice that were treated with MS-275 using real-time RT-PCR. The tested MS-275-treated mice completed the spatial WM test (20s delay) one hour prior to brain collection (see Fig. IB).
  • FIG. 4C illustrates that although the levels of Bdnf inRNA did not differ between SFR and non-tested MS-275-treated Baib/c mice., tested MS «275*treated Balb/c mice expressed significantly more Bdnf transcript variant III mRNA. in contrast, Egr2 mRNA levels did not significantly differ between the three groups of mice (Fig. 4D).
  • MS-275 treatment during adolescent development of SFR mice leads to the same cognitive deficits detected in IMS Balb/c mice and the same test- induced increase of Bdnf transcript variant ill mRNA expression.
  • Example 4 Cognitive deficits of C57BI/6 mice fostered by Balb/c mothers during IMS exposure.
  • HDACl bm not HDAC3
  • HDACl levels at the Egr2 promoter of ASST-tested foster IMS C57B1/6 mice did not differ from corresponding baseline levels nieasured at P60 (1/2 40 : 0.42 ⁇ 0.09 (foster IMS C57B1/6 at P60) and 0.37 ⁇ 0.03 (ASST-tested tbsuy IMS C37BV6 at P66)X IiisumniiKy.
  • HDAC1 am a common denominator for BaJb/c and C57B1/6 mice raised by BaBVc mothers during IMS exposure
  • the genes affected and the histone modification profiles at die promotonoftheafiecfirf genes are also influenced by the genetic backgnnmds of die pups.
  • the IbJhiwing an adolescent (typically 10-18) or young adult patiem (18-24) with a mood/emotional disorder which can be an anxiety disorder or stress- induced disorder, inchiding early life stress, or a cornbmation thereof.
  • the mood disorder can also be a panic disorder, an obsessive ccmpfilsrve disorder, a post-tFenmtic stress disorder, or any ty ⁇ ft ⁇ ftw .”flwoA In. certain einbcdnnontt ⁇ it is. wrpgcteii mat adnhs could also benefit ftpin twtsjhT-cnts described herein.
  • the patiax win be treated whn an erre ⁇
  • TrfcB inhibitor such at ⁇ -12. It is also expected thai tbe treated human patient will exhibit an impravement in one or more of die typical indicators of cognitive fiucaVxiing inchiding wotting timmmy mtfuftimi set*sbifttng. The treatnieat will be continued for » period of time nntiJ these inyOvements are observed and mamtained and this time can vary fiom several week ⁇ to several monms. h u expected that these trealed patients will be less likely to experience a relapse.
  • SSRI serotonin renptake inhibitor
  • U is expected that Jbr patients treated with both a TrkB inhibitor and al least one SSIR, that both their cognitive and emotional phenotypes will exhibit improvement and will be less likely to experience a relapse.
  • the treatment will be continued for a period of tune until these inaxwemenls are observed and maintained and Pus time can vary Scorn several weeks, to several months.
  • Qptinial cognitive task perfanjance' depends upon fineljMuned babjace between neuronal activation and uthibmon, a that is by tightly regulated gene induction and repression (Rao et al, 2000; Millan et aL, 2012; Pezze et aL, 2014).
  • HDACs are in a strong position to modulate the expression of plasticity-related genes nnpKcatad in the control of cognitive Junctions
  • the present study asked whether die cognitive deficits found in nuce exposed to early Efe stress can., be expstined by reduced o3 ⁇ 4 pi ration of class I HDACs 1 and/or 3 that we previously detected in IMS BaUWc mice (Uvine et aL, 2012).
  • IMS Baftvc mice exhibit reduced association of HDAC1 (but not HDAC3) specifically with Bdnf promoter IU, and mat cognitive task exposure leads to increased density of the tmnacrinuonafly active form of Pol ⁇ at this premolar, a raiding consistent with the large increase in Bdnf transcript variant III expression found after cognitive testing.
  • non-stressed Balb/c mice were treated during adolescence with the HD AC] -preferring inhibitor MS-275, they exhibit the same cognitive deficits found in IMS mice as well as the same enhancement of test-induced Bdnf gene expression.
  • the results of these pharmacological studies indicate clearly that the cognitive deficits and the associated change in Bdnf gene expression are due to reduced HDAC1 activity.
  • IMS-triggered alteration in maternal care Huot et al., 2004; Schmauss et al, 2014
  • IMS-triggered cognitive deficits as well as the reduced HDAC1 association with promoters of distinct plasticity-related genes are influenced by the genetic backgrounds of the pups.
  • IMS C57B1/6 mice raised by Balb/c mothers during IMS exposure exhibit deficits in attention set-shifting, but no WM deficits.
  • HDACl at Bdnf promoter ill exhibit decreased HDACl levels at the promotor of the Egr2 gene, a gene selectively induced in the ventrolateral orbital frontal cortex and the pre- and infralimhic suhregions of the rnPFC during ASST (but not WM) exposure (DeSteno and Schmauss, 2008), Indeed, only foster IMS C57B1/6 mice exhibit a robust (-5 fold) increase of tnmscription of the Egr2 gene upon ASST exposure.
  • IMS Balb/c mke foster IMS C57BV6 mice also exhibit a different hwtone ipodificinon phenotype at file E&2 proniotor that is characterized by an incraased ratio of acetylated H3K9 (a histone mark of open chromatin and active gene transcription) over H3K9me3 (a madrar of gene represtjon).
  • IMS Balb/c mice exhibit mcreased acetylation of bJstone H4 protein, especially histone H4K12 acetylatm atJ- ⁇ promotor HI (Schmauss, 2015), increased aoetyiation of histone H3 protein was not detected in these mice (Levine et aL, 2012).
  • IMS Balb/c and foster IMS C57BI/6 mke do not only exhibit reduced HDACl levels at promoters of different genes, (he pnunotors of the affected genes also difler in their histone modification profiles. Nevertheless, despite vie cognitive task- induced enhanced ttnnsuiption of diflerenl plasticiry-ielated genes in bom strains of mice, reduced HDACl association with the respective promoters is a common epigenetic phenotype. myonantty.
  • HDAC2 knockout mice with increased histone H4 and H2B acetylation in the hippocampus exhibit increased associative learning and improved WM, and they have increased hippocampal spine density and synaptic terminals (Guan et al., 2009).
  • HDAC2 deficient mice also exhibit increased acetylation of histone H3 and H4 at Bdnf promoter MI, Egr2, and Fox, and they are refractory to the effect of SAHA on synaptic plasticity and learning (Guan et at, 2009),
  • HDAC2 A specific role of HDAC2 in memory impairment is former supported by findings of increased HDAC2 expression in the hippocampal CA1 subfiekl and the PFC in an animal model of severe neurodegeneration. In this model, shRNA-mediaied. HDAC2 knockdown lead to increased acetylation of histone H4K12 at promotors of plasticity-associated genes along with increased expression of these genes, re-instated morphological and synaptic plasticity of surviving neurons, and improved associative and spatial memory (Graff ei al., 2012).
  • HDACl and HDAC3 were identified specific roles for HDACl and HDAC3 in distinct hippocampal-dependent cognitive processes.
  • TrkB inhibitor such as ANA- 12
  • TrkB inhibitor such as ANA- S 2
  • one or more SSRi including any combination of: Citaloprara (Celexa); Bscitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxatnine (Luvox); Sertraline (Zoloft, Lustra! ⁇ would result in improvement to both the cognitive deficits and emotional p ' henotype- e.g., depressive condition/s.
  • SSRi including any combination of: Citaloprara (Celexa); Bscitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxatnine (Luvox); Sertraline (Zoloft, Lustra! ⁇
  • the present results demonstrate for the first time that an epigenetk response to early life stress exposure contributes the emergence of working memory and attention set-shifting deficits, i.e. deficits that often accompany mental disorders and that are largely resistant to treatment.
  • the results show that reduced histone deacetylase 1 levels at promotors of distinct plasticity-related genes allow- for abnormally increased, expression of these genes upon, cognitive testing, an effect that influences cognitive task performance negatively.
  • the genes affected by reduced HDACI activity differ in genetically different strains of mice, indicating that genomic sequence variations and their differential interaction with the stress-modulated epigenome contribute to and determine the outcome of early-life stress-triggered cognitive impairment.
  • Balb/cJ and C57BV6J mice were housed in a temperature- and light controlled barrier facility with free access to food and water. All experiments involving the animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committees at Columbia University and the New York State .Psychiatric Institute,
  • mice either raised their own pups or fostered Balb/c or C57B1/6 pups from other litters from the clay of birth.
  • pups exposed to the IMS paradigm were separated from their mothers daily for three hours (from 1 :00 to 4:00 PM), starting at postnatal age P2 and ending at P15, They were weaned at P28 and group housed by sex (see Fig. 1A).
  • Other pups of Balb/c or C57B1/6 mothers were left undisturbed with their mothers and were also weaned at P28. These mice are referred to as standard-facility reared (SFR). Housing and. husbandry conditions were identical for SFR and IMS mice.
  • Drug treatments were identical for SFR and IMS mice.
  • MS-275 Sigma Aidrich, St. Louise, MO
  • 24 h Ana- 12; Calbiochem; BiMeriea, MA
  • MS-275 is also known as Entinostat, or SNDX-275 and is a benzaraide histone deacelylase inhibitor undergoing clinical trials for treatment of various cancers.
  • Entinostat inhibits class 1 HDACl and HDAC3 with ICso of 0.51 ⁇ and 1.7 uM, respectively, and is capable of penetrating the blood brain barrier weakly (See, Hooker J.M., et af, Histone deacetylase inhibitor, MS-275, exhibits poor brain penetration: PK studies of [C]MS-275 ttsing Positron Emission Tomography. ACS Chem Neurosci. 2010;I( l):65-73). It has the formula: C21H2ON4Q3.
  • ANA- 12 is a selective, small-molecule non-competitive antagonist of TrkB (Kd ::: 10 sM and 12 uM for the high- and low-affinity sites, respectively), the mam receptor of brain- derived neurotrophic factor (BDNF/Bdnf) (Cazorla 2011).
  • the compound crosses the blood- brain-barrier and exerts central TrkB blockade, producing effects as early as 30 minutes (-400 nM) and as long as 6 hours (- 10 .oM) following iiUraperitoneal injection in mice (Cazorla 201 .1). It blocks the neurotrophic actions of BDNF without compromising neuron survival (Cazorla 201 1).
  • ANA- 12 produces rapid antidepressant- and anxiolytic- like effects in animal models, the former of which have been elucidated to be mediated by blockade of B ' DNF signaling in the nucleus accumbens. (Zhang JC 2015 and Shirayarna Y 2015). It has also been found to alleviate methamphetamine-induced depression-like behavior (including anhedonia), behavioral sensitization, and nucleus accumbens aeuropiasticity changes with subchronic (1.4-day) administration, in mice, whereas the TrkB agonist 7,8-dihydroxyflavone was ineffective in doing so (Ren Q 2015).
  • IMS Batb/dmce fhst received Ana-12 consiimeri 1 iiu/kg/cley, and SFR mice that received MS-275 nomwiwl 15 uM/day. We previously shewed thai iieimer trettraent attend the emotional behavior of these mice when tested at the end of this treatment (Schmeass, 2015).
  • Ana-12 effectively blocked the antidepressant efiixts of fluoxetine in IMS Balb/c mice (Schmanss, 2015) and, in SFR BaJb/c mice, MS-275 increased the levels of acH4K12 and Pol ⁇ at Bdnf promoter III only when coHKhnmktefed with fluoxetine (Schmauss, 2015).
  • mice were mod restricted such that they gmdually (over the period of 4 to 5 days) lost 10-15% of their free-feeding body weights. Prior to ASST testing, mice tailed to dig for food buried dee
  • mice did not roaimam spatial WM at 30 s imeHriei delay. Only correct arm entries were rewarded with food, and the percentage of correct arm entries in the total number of 10 trials per deary period was taken as a measure of response accuracy.
  • fcrabaan ofocortk-jrt tissue was dissected from male and femak mice using the mesodkncerjbalk junction as the anatomic landmark for me caudal border of the forebram.
  • Real time PGR was performed using die iQ Real Time PCR detection System (Bio-Rad, Hercules, CA) and SYBR Green (Bio-RadX Bdnf mRNA was amplified using me primers targeting Bdnf transcript variant m reported by Tsankova et al. (2006). Egr2 mRNAwas ampliiSed using the primer
  • CbJP was performed on forebnun neocorucal tissue of groups of male and female mke (selected from different titters as described above) either at P60 (baseline) or alter cognitive testing wim and without drug treatment (see Fig. 1). Dissected tissue was fixed with disrupter (Misomx, Fanningdak, NY) to an average DNA length of 200 to 400 base pairs.
  • Beads were treated according to me mstruction of flw mamiftctnrer. manunourei: iphtted DNA and « serial dilation of IX input were analyzed by SYBR-Greea real-tone PCR nsing primers targeting Bdnf promoter ⁇ (original nomenclature) adopted from Tsankova et al (2006) and. the promoters of the Eg.r2 md Pos genes adopted from Bahari-Javao et al, (2012).
  • HDAC3 is a critical negative regulator of long-term memory formation. J. Neurosci . 31, 764-774.
  • Atncaoti an integrated review of hmnan literature. Pgydxyharuacology 214, 55-77.
  • mice with agtHiepeudent memory iiupaiiineul in mice. Science 328, 753-756.
  • Trrvadi MJHL, Greer, TX., 2Q14.Cogiutrve ⁇ h/3 ⁇ 4fpia3 ⁇ 4iw inyKcaiions 25 for treatment J. Affect Disord. 152, 19-27.
  • the invention can be embodied in other specific forms without douarhiig front die spirit or essential characteristics thereof.
  • the fuiegoing efnbodiroents are to be considered in all respects illustrative rather than titrating on the invention described herein. Further, it should be ojideratood that the order of steps or order for perforating certain actions is immaterial so long as die invention remains operable. Moreover, two or more steps or actions

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  • Rheumatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des procédés et des traitements permettant d'améliorer le fonctionnement cognitif chez des patients qui en ont besoin. Selon certains aspects, les procédés consistent à administrer au moins un inhibiteur de tropomyosine kinase B (TrkB) de facteur neurotrophique dérivé du cerveau (Bdnf).
PCT/US2016/056822 2015-10-14 2016-10-13 Inhibition de tropomyosine kinase b (trkb) de facteur neurotrophique dérivé du cerveau (bdnf) pour améliorer des déficits cognitifs Ceased WO2017066434A1 (fr)

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CN110664992A (zh) * 2019-11-13 2020-01-10 山东大学第二医院 Bdnf在制备治疗母婴分离抑郁症药物中的应用
WO2020154434A1 (fr) * 2019-01-24 2020-07-30 Pharmatrophix, Inc. Compositions et méthodes de traitement et de prévention d'un dysfonctionnement cognitif
WO2020154941A1 (fr) * 2019-01-30 2020-08-06 Nippon Zoki Pharmaceutical Co., Ltd. Agent d'inhibition ou d'atténuation d'une inflammation dans le cerveau
CN112913776A (zh) * 2021-01-22 2021-06-08 复旦大学附属中山医院 一种改善急性心肌梗死预后的小鼠模型构建方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020154434A1 (fr) * 2019-01-24 2020-07-30 Pharmatrophix, Inc. Compositions et méthodes de traitement et de prévention d'un dysfonctionnement cognitif
WO2020154941A1 (fr) * 2019-01-30 2020-08-06 Nippon Zoki Pharmaceutical Co., Ltd. Agent d'inhibition ou d'atténuation d'une inflammation dans le cerveau
CN110664992A (zh) * 2019-11-13 2020-01-10 山东大学第二医院 Bdnf在制备治疗母婴分离抑郁症药物中的应用
CN110664992B (zh) * 2019-11-13 2022-11-04 山东大学第二医院 Bdnf在制备治疗母婴分离抑郁症药物中的应用
CN112913776A (zh) * 2021-01-22 2021-06-08 复旦大学附属中山医院 一种改善急性心肌梗死预后的小鼠模型构建方法
CN112913776B (zh) * 2021-01-22 2023-03-10 复旦大学附属中山医院 一种改善急性心肌梗死预后的小鼠模型构建方法

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