WO2000042178A2 - Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central - Google Patents

Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central Download PDF

Info

Publication number
WO2000042178A2
WO2000042178A2 PCT/US2000/000819 US0000819W WO0042178A2 WO 2000042178 A2 WO2000042178 A2 WO 2000042178A2 US 0000819 W US0000819 W US 0000819W WO 0042178 A2 WO0042178 A2 WO 0042178A2
Authority
WO
WIPO (PCT)
Prior art keywords
crf
phosphorothioate
receptor
antisense oligonucleotides
antisense
Prior art date
Application number
PCT/US2000/000819
Other languages
English (en)
Other versions
WO2000042178A3 (fr
Inventor
Siew Peng Ho
Original Assignee
Dupont Pharmaceuticals Company
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 Dupont Pharmaceuticals Company filed Critical Dupont Pharmaceuticals Company
Priority to AU27256/00A priority Critical patent/AU2725600A/en
Publication of WO2000042178A2 publication Critical patent/WO2000042178A2/fr
Publication of WO2000042178A3 publication Critical patent/WO2000042178A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • This invention relates to antisense oligonucleotides directed against the mRNA of the corticotropin releasing factor subtype-2 (CRF 2 ) receptor which substantially reduce expression of CRF 2 receptors in the rodent brain and the use of antisense oligonucleotides in in vivo CNS studies of gene function and to treat a wide range of psychiatric disorders including anxiety, obsessive-compulsive disorder, panic disorders, post-traumatic stress disorder, phobias and depression.
  • CRF 2 corticotropin releasing factor subtype-2
  • CRF corticotropin releasing factor
  • CRF-overexpressing transgenic mice have been reported to exhibit an increase in anxiogenic (anxiety-producing) behavior (Stenzel-Poore et al . , Overproduction of Corticotropin- Releasing Factor in Transgenic Mice: A Genetic Model of Anxiogenic Behavior. J. Neuroscience 14, 2579-2584, 1995). Of particular importance is the question of whether these anxiogenic responses are mediated through CRF action on CRF X receptors, CRF 2 receptors or both.
  • CRF 2 - selective peptides or small molecule ligands have yet to be identified. In the absence of specific agonists or antagonists to this receptor, antisense suppression of CRF 2 receptor expression may provide evidence for the role of the receptor in normal physiology.
  • Antisense oligonucleotides are short oligonucleotides (typically from about 15 to about 25 nucleotides in length) which are designed to be complementary to a portion of a mRNA molecule of interest. Hybridization of an antisense oligonucleotide to its mRNA target site through Watson-Crick base-pairing initiates a cascade of events which terminate in oligonucleotide-directed degradation of the targeted mRNA molecule. A direct consequence of this mRNA degradation is the suppression of synthesis of the encoded protein. Studies done in the presence of significantly reduced levels of the targeted protein may reveal its function.
  • antisense oligonucleotides can be extremely useful tools for protein functional studies. In addition, they can be used to distinguish between closely related members of a family of proteins (such as CRF X and CRF 2 ) in ways that are often not possible with small molecule ligands.
  • Antisense oligonucleotides vary widely and unpredictably in their activity because their mRNA targets have significant secondary and tertiary structure which render larger portions of a mRNA molecule inaccessible to hybridization. Only 20-35% of antisense sequences have significant inhibitory activity (50% or more) .
  • Using a molecular technique we developed Ho et al . , Potent Antisense Oligonucleotides to the Human Multidrug Resistance-1 mRNA are Rationally Selected by Mapping R ⁇ A-Accessible Sites With Oligonucleotide Libraries. Nucl. Acids Res. 24, 1901-1907, 1996; Ho et al .
  • This invention relates to antisense oligonucleotides directed against the mRNA of the CRF 2 receptor which substantially reduce expression of CRF 2 receptors in the rodent brain. Suppression of CRF 2 receptor function using these oligonucleotides produced significant anxiolytic (anxiety- reducing) effects in animals. These data provide the first functional evidence that CRF 2 receptors play an important role in mediating the anxiogenic (anxiety-producing) effects of corticotropin releasing factor.
  • CRF 2 receptor antagonists including small molecules, to be effective in the treatment of a wide range of psychiatric disorders including anxiety, obsessive-compulsive disorder, panic disorders, post -traumatic stress disorder, phobias and depression.
  • An object of the present invention relates to a method of treating psychiatric disorders including, but not limited to, anxiety, obsessive-compulsive disorder, panic disorders, post- traumatic stress disorder, phobias and depression in a mammal, by administering to the mammal requiring such treatment a therapeutically effective amount of a pharmaceutical composition comprising antisense oligonucleotide (s) comprised of chimeric oligonucleotide (s) where 10-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a pharmaceutical composition comprising antisense oligonucleotide (s) comprised of chimeric oligonucleotide (s) where 10-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • Another object of the invention relates to a method of screening compounds to determine activity for the treatment of psychiatric disorders including, but not limited to, anxiety, obsessive-compulsive disorder, panic disorders, post-traumatic stress disorder, phobias and depression.
  • Another object of the invention relates to antisense oligonucleotides composed of chimeric oligonucleotides wherein between 10-70% of the 2 ' -deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • Another object of the invention relates to a method of treating a mammal having a disease mediated by CRF, or other closely related peptide, comprising administering to the mammal a composition that effectively inhibits binding of CRF, or other closely related peptides, to the CRF 2 receptor.
  • Figure la Schematic for antisense sequence selection.
  • Figure lb Identity of chimeric, semi-random oligonucleotide libraries .
  • Figure 2a Structure of most commonly used nucleotide analogs in antisense studies; the phosphorothioate variation produces CNS toxic effects.
  • Figure 2b Structure of modified oligonucleotide analogs that maintain potency but eliminate toxicity when incorporated into oligonucleotides for CNS applications.
  • Figure 2c One of several possible configurations for chimeric oligonucleotides .
  • Figure 3a Effect of antisense oligonucleotides on freezing behavior in rats.
  • Figure 3b Inhibition of 125 I-sauvagine binding in the lateral septum of antisense treated rats in the freezing assay.
  • Figure 4a Effect of antisense treatment on rodent behavior in the elevated plus maze.
  • Figure 4b Inhibition of 125 I-sauvagine binding in the lateral septum of antisense treated rats in the elevated plus maze assay.
  • Figure 5 Effect of anti-sauvagine-30 on freezing behavior in rats .
  • a method was therefore devised for locating sites on an mRNA molecule that are most accessible to hybridization with antisense oligonucleotides (Ho et al . , 1996; Ho et al . , 1998). This was accomplished ( Figure la) by probing an RNA transcript with a library of chemically synthesized, semi-random oligonucleotides ( Figure lb) . When mixed together, the accessible regions of the RNA should hybridize with complementary sequences found within the library. These regions are subsequently identified using ribonuclease H (RNase H) , which catalyzes the hydrolytic cleavage of the phosphodiester backbone of only the RNA strand of a hybrid RNA-DNA duplex.
  • RNase H ribonuclease H
  • RNA fragments produced should allow identification of those regions in a particular mRNA sequence which can then serve as sites for targeting antisense oligonucleotides.
  • Application of this R ⁇ A-mapping method to the R ⁇ A transcript containing the entire coding region of the CRF 2 receptor mR ⁇ A led to the identification of multiple R ⁇ A sites which are accessible to hybridization with antisense oligonucleotides (Table 1) .
  • Table 1 Sites in the CRF 2 receptor mRNA that are accessible to oligonucleotide hybridization. Sequence information is with reference to RNU16253.GB_RO (GenBank sequence, accession number U16253) .
  • Antisense oligonucleotides 15 to 25 nucleotides in length can be designed by targeting the 5 ' -end of the antisense oligonucleotide to accessible sites defined by the data provided in Table 1.
  • the antisense oligonucleotide used in the studies described below was a 20 nucleotide sequence (TGA CGC AGC GGC ACC AGA CC) targeted to positions 758-777 of accessible site E.
  • Antisense sequences directed against several of these sites inhibited CRF receptor synthesis by at least 50% in cell -based assays. This was determined through a CRF ⁇ 125 radioligand-binding assay using I-sauvagine. The antisense inhibition was sequence specific as 4 -base mismatches of the antisense oligonucleotides produced only minimal reductions in
  • oligonucleotides most commonly used in CNS in vivo antisense experiments are 2 ' - deoxyribonucleotide phosphodiester oligonucleotides and 2'- deoxyribonucleotide phosphorothioate oligonucleotides ( Figure 2a) . While being identical in chemical structure to double stranded DNA in genes, single stranded phosphodiester oligonucleotides however are susceptible to exonucleolytic and endonucleolytic degradation, with a half-life in serum of 20 minutes.
  • phosphodiester oligonucleotides are degraded, albeit more slowly.
  • Phosphorothioate oligonucleotides where one of the non- bridging phosphate oxygen molecules is replaced with a sulfur, are far more resistant to degrading enzymes.
  • phosphorothioate oligonucleotides have a half-life of over 12 hours and analysis of phosphorothioates extracted from rat brain shows these oligonucleotides to be chemically intact for at least 24 hours.
  • administration of these oligonucleotides in the brain produces chemistry-related but not sequence-specific toxic effects.
  • CRF 2 antisense sequences containing the phosphorothioate chemistry produced large inhibitory effects on the CRF 2 receptor but caused significant weight loss (similar to the Heinrichs report) and a host of pathophysiological symptoms in the treated animals. These effects were observed with many different sequences, antisense as well as control sequences, precluding the possibility that they are target-related effects.
  • CRF antisense oligonucleotides were administered intracerebroventricularly to target the lateral septum, a brain region containing high levels of CRF receptor and mRNA.
  • the lateral septum is part of the limbic brain region known for its involvement in modulating fear and emotion.
  • Rats treated with saline, antisense and mismatch-control oligonucleotides were tested in two different behavioral models of anxiety. Rodents display a characteristic freezing behavior when experiencing fear and anxiety. In the freezing model of anxiety, such behavior is induced by exposure to brief electrical foot-shocks. When such rats are returned to the shock box after several intervening days, they exhibit freezing behavior even in the absence of further shock exposure.
  • anxiolytic drugs such as benzodiazepines and selective serotonin reuptake inhibitors reduces the duration of freezing when previously shocked animals are returned to the shock box.
  • dosing of oligonucleotides began after two consecutive days of foot-shock treatment. Two hours following the last oligonucleotide administration on day 8 of dosing, rats were returned to the shock box and observed for 10 minutes.
  • the antisense oligonucleotide but not its mismatch control, reduced the duration of freezing by 50% ( Figure 3a) .
  • the elevated plus maze (EPM) is widely used for the determination of anxiolytic or anxiogenic drug effects.
  • the apparatus consists of a +-shaped maze, elevated 50 cm above the floor. Two opposing arms are open and exposed to the environment while the other two arms are enclosed with black
  • drugs currently prescribed for the treatment of anxiety disorders are effective in producing anxiolytic responses in rodents tested in the EPM.
  • rats were dosed for 8 days and then tested in the EPM 2 hours after the last oligonucleotide injection. Rats treated with the antisense oligonucleotide spent significantly more time in the open, exposed arms of the maze ( Figure 4a) . Such behavior is indicative of a reduced state of anxiety. Mismatch oligonucleotide-treated rats were not statistically different
  • A,C,G, and T correspond to the 5 ' -monophosphate forms of the deoxyribonucleosides deoxyadenine , deoxycytidine, deoxyguanine , and deoxythimine respectively, when they occur in DNA molecules .
  • chimeric refers to oligonucleotides that comprise two or more classes of nucleotides with different chemical structures.
  • antisense oligonucleotides refers to short oligonucleotides (typically from about 15 to about 25 nucleotides in length) which are designed to be complementary to a portion of a mRNA molecule of interest. Hybridization of an antisense oligonucleotide to its mRNA target site through Watson-Crick base-pairing initiates a cascade of events which terminate in oligonucleotide-directed degradation of the targeted mRNA molecule.
  • CRF 2 receptor (s) refers to cell surface receptors as described in U.S. Patent Number 5,786,203, issued July 28, 1998, the contents of which are herein incorporated by reference .
  • defined accessible site refers to multiple sites in the CRF 2 receptor mRNA which are accessible to hybridization with antisense oligonucleotides. These sites are further delineated in Table 1 above.
  • modified nucleotide residue includes but is not limited to 2 ' -methoxyribonucleotide phosphodiesters, 2 ' -methoxy-ethoxyribonucleotide phosphodiesters, 2 ' -fluoro-ribonucleotide phosphodiesters, 5- (1-propynyl) cytosine phosphorothioate, 5- (1-propynyl) uracil phosphorothioate, 5-methyl cytosine phosphorothioate, 2'- deoxyribonucleotide-N3 ' -P5 ' phosphoramidate, and polyamide nucleic acids.
  • composition includes but is not limited to peptides, proteins, antibodies, small chemical entities with a molecular weight less than 900 g/mole, nucleic acids, polysaccharides, and antisense oligonucleotides.
  • therapeutically effective amount of a composition of this invention means an amount effective to inhibit or treat the symptoms, in a host, of a psychiatric disorder including, but not limited to, anxiety, obsessive- compulsive disorder, panic disorders, post-traumatic stress disorder, phobias and depression.
  • An embodiment of the invention provides a method of treating psychiatric disorders including, but not limited to, anxiety, obsessive-compulsive disorder, panic disorders, post- traumatic stress disorder, phobias and depression in a mammal, by administering to the mammal requiring such treatment a therapeutically effective amount of a pharmaceutical composition comprising antisense oligonucleotides comprised of chimeric oligonucleotides where 10-70% of the 2 ' - deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • the modified nucleotide residues of the antisense oligonucleotides are selected from the following group: 2 ' -methoxyribonucleotide phosphodiesters, 2 ' -methoxy-ethoxyribonucleotide phosphodiesters, 2 ' -fluoro-ribonucleotide phosphodiesters, 5- (1-propynyl) cytosine phosphorothioate, 5- (1-propynyl) uracil phosphorothioate, 5-methyl cytosine phosphorothioate, 2'- deoxyribonucleotide-N3 ' -P5 ' phosphoramidate, and polyamide nucleic acids.
  • a more preferred embodiment provides the antisense oligonucleotide is from about 15 to about 25 nucleotides in length.
  • Another embodiment provides a method of treating a mammal having a disease mediated by a protein, comprising:
  • Another embodiment of the present invention provides a method for treating a mammal having a disease mediated by CRF, comprising administering to the mammal a composition that effectively inhibits binding of CRF, or other closely related peptides, to the CRF 2 receptor.
  • Another embodiment of the present invention provides a method of designing an inhibitor of the CRF 2 receptor comprising the steps of determining the three-dimensional structure of such receptor, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predictive reactive site, and determining the receptor- inhibiting activity of the molecule.
  • Another embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 10-70% of the 2 ' - deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 15-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 20-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 25-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 30-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 35-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 40-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 45-70% of the 2 ' - deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 50-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 55-70% of the 2'- deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • An even more preferred embodiment of the present invention provides sequences of antisense oligonucleotides composed of chimeric oligonucleotides where between 60-70% of the 2 ' -deoxyribonucleotide phosphorothioate residues are replaced with modified nucleotide residues.
  • a further preferred embodiment of the present invention provides for antisense oligonucleotides having a target base located within a defined accessible site, having a starting point at any base located within the defined accessible site, and having a length from about 15 to about 25 bases.
  • Another embodiment of the present invention provides a screening assay for determining compounds useful in the treatment of psychiatric disorders including, but not limited to, anxiety, obsessive-compulsive disorder, panic disorders, post -traumatic stress disorder, phobias and depression utilizing antisense oligonucleotides.
  • Another embodiment of the present invention provides a method of determining the structure of the binding region of the CRF 2 receptor.
  • Beaucage reagent for the synthesis of phosphorothioate linkages and fluorescein phosphoramidite for 5' -labeling of oligonucleotides was purchased from Glen Research. These reagents were used according to manufacturer's instructions.
  • oligonucleotide mixtures were purified by reverse phase HPLC on a PRP-3 column (Hamilton Co.) using a gradient of acetonitrile and 0.1 M aqueous triethylammonium acetate. Fractions collected off the HPLC column were lyophilized twice to remove excess triethylammonium acetate . An aqueous solution of the oligonucleotide was then extracted several times with butanol . Cation exchange was accomplished using ethanol precipitation in the presence of 0.3 M sodium acetate. The pH of the oligonucleotide solution was then brought up to pH 7.0 by addition of 0.01 M sodium hydroxide.
  • oligonucleotide was further purified by size exclusion chromatography using NAP-25 columns (Pharmacia) to remove residual fluorescein phosphoramidite reagent. Sterilization was accomplished by filtration through a 0.2 ⁇ m cellulose acetate filter (Rainin) and quantitated by UV spectrometry . The purity of oligonucleotides was determined by capillary gel electrophoresis (PACE2100, Beckman Instruments). Stocks of oligonucleotide in distilled water were stored at -20°C.
  • Example 2 Animals and surgery Male Sprague Dawley rats (Charles River) weighing 320-360 g at the time of surgery were individually housed in stainless steel cages and provided free access to food and water. Following a 4 day adaptation period, rats were stereotaxically implanted bilaterally, under Rompun (100 mg/kg) and ketamine (9 mg/kg) anesthesia, with chronic 26-gauge guide cannulae aimed at the lateral ventricles. Stereotaxic co-ordinates were: incisor bar 3.3 mm below interaural line; 0.2 mm posterior to bregma; ⁇ 2.7 mm lateral to midline; 3.8 mm ventral to skull surface and a 24° angle. The injector (33 gauge) projected beyond the tip of the guide cannulae by 0.5 mm. The animals were adapted by daily handling beginning 2 days after surgery.
  • Oligonucleotide infusions were started on the 8th day following surgery when rats were about 20 g above surgery weights.
  • Fresh oligonucleotide solutions were prepared daily by dissolving lyophilized oligonucleotide pellets in sterile saline. Rats were weighed daily at 9:00 AM before oligonucleotide infusion.
  • a microprocessor controlled syringe pump (Stoelting) , 1 ⁇ L of solution was injected per ventricle over 2 minutes. The injector was left in the guide cannula for an additional minute. Separate injectors for each individual rat were rinsed with ethanol and sterile water, and dried between daily injections.
  • Example 4 Freezing assay of anxiety The shock box consisted of a black Plexiglas chamber with walls and cover. The doors of the box were constructed of clear Plexiglas over which one-way mirrors were attached for observation. The floor of the box contained a Coulbourn stainless steel shock grid with the bars of the grid spaced 1 cm apart.
  • rats On the 8th day following surgical implantation of the guide cannulae, rats were placed in the box and allowed to habituate for 2 minutes. A total of 3 scrambled, randomized non-escapable foot-shocks (1.0 mA, 1 second duration) were then delivered at 20 second intervals to the grid floor. The rat was observed for freezing behavior for 15 minutes before it was returned to its home cage. This shock treatment was repeated the following day.
  • Oligonucleotide treatment was initiated the day after the second shock treatment. Two hours after dosing on the 8th day of oligonucleotide treatment, the rats were returned to the shock box and observed for freezing behavior for 10 minutes. This was followed by the administration of 2 foot-shocks (1.0 mA, 1 second duration, 20 second interval) after which the rat was observed for freezing for another 10 minutes. Immediately following this last 10 minute period, the rat was euthanitized.
  • Example 5 Elevated plus maze assay Oligonucleotide treatment of rats was begun on the 8th day following surgery. Rats were tested in the EPM 2 hours following dosing on the 8th day of treatment. At the start of the test, the rat was placed in the center square of the maze and its exploratory behavior during the ensuing 10 minutes was recorded by video-camera. An observer situated outside the test room scored the time spent in the open and closed arms, as well as the number of entries into each arm of the maze. The rats were euthanitized immediately following the conclusion of the test.
  • Example 6 Tissue preparation Rats were sacrificed by exposure to C0 2 . Brains were removed and frozen in methylbutane cooled on dry ice before storage at -80°C. Twenty ⁇ m sections through the lateral septum were cut on a cryostat (Kopf Instruments) for receptor autoradiography .
  • Non-specific binding was determined using 1 ⁇ M a-helical CRF (American Peptide) . Incubations were performed in preincubation buffer containing radioligand and appropriate antagonists for 150 minutes. Tissue sections were then washed twice for 5 minutes each, in PBS containing 0.01% Triton X- 100. After a final water rinse, excess water was aspirated and the sections were air-dried overnight. The sections and 1 S I standard strips (A ersham) were exposed to Hyperfilm ⁇ -Max (Amersham) for 72 hours .
  • CRF2 specific binding was performed using the NIH ImageMG 1.44 program. Optical density readings were converted to nCi of ligand bound per mg of protein tissue using 125 I standard strips. Between 7 to 9 adjacent sections were quantitated per rat.

Abstract

La présente invention se rapporte à des oligonucléotides antisens dirigés contre l'ARNm du récepteur de sous-type 2 de la corticolibérine (CRF2) qui réduisent sensiblement l'expression des CRF2 dans le cerveau des rongeurs, et à l'utilisation d'oligonucléotides antisens dans les études in vivo de la fonction génique au niveau du système nerveux central. L'invention permet de traiter une vaste palette de troubles psychiatriques, y compris l'anxiété, les névroses obsessionnelles, les troubles paniques, les syndromes de stress post-traumatique, les phobies et la dépression.
PCT/US2000/000819 1999-01-13 2000-01-13 Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central WO2000042178A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU27256/00A AU2725600A (en) 1999-01-13 2000-01-13 Antagonist blockade of crf2 receptors for the treatment of psy chiatric disorders and the use of chimeric antisense oligonucleotides in (in vivo) cnsstudies of gene function

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11574899P 1999-01-13 1999-01-13
US60/115,748 1999-01-13

Publications (2)

Publication Number Publication Date
WO2000042178A2 true WO2000042178A2 (fr) 2000-07-20
WO2000042178A3 WO2000042178A3 (fr) 2000-11-30

Family

ID=22363191

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/000819 WO2000042178A2 (fr) 1999-01-13 2000-01-13 Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central

Country Status (2)

Country Link
AU (1) AU2725600A (fr)
WO (1) WO2000042178A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1165093A1 (fr) * 1999-03-05 2002-01-02 Epigenesis Pharmaceuticals, Inc. Procede de validation/d'invalidation de cibles et des voies
EP1558639A2 (fr) * 2002-10-31 2005-08-03 Janssen Pharmaceutica N.V. Genes dont l'expression augmente en reaction a une stimulation par l'hormone liberatrice de la corticotropine
WO2014059341A2 (fr) 2012-10-12 2014-04-17 Isis Pharmaceuticals, Inc. Composés anti-sens et leurs utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010332A2 (fr) * 1995-09-14 1997-03-20 Brax Genomics Limited Oligonucleotides chimeres et leurs utilisations dans l'identification de sites de liaison antisens

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010332A2 (fr) * 1995-09-14 1997-03-20 Brax Genomics Limited Oligonucleotides chimeres et leurs utilisations dans l'identification de sites de liaison antisens

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ALTMANN, K.-H. ET AL.: "Novel chemistry" STEIN, C.A. & KRIEG, A.M. 'APPLIED ANTISENSE OLIGONUCLEOTIDE TECHNOLOGY'. WILEY-LISS, NEW YORK, US; CHAPTER 4,1998, pages 73-107, XP002119324 *
DE SOUZA EB. ET AL.: "Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders." PSYCHONEUROENDOCRINOLOGY 1995;20(8):789-819, XP000907546 *
HEINRICHS SC. ET AL.: "Corticotropin-releasing factor CRF1, but not CRF2, receptors mediate anxiogenic-like behavior." REGUL PEPT 71(1):15-21, 23 July 1997 (1997-07-23), XP000907396 cited in the application *
HO S P ET AL: "POTENT ANTISENSE OLIGONUCLEOTIDES TO THE HUMAN MULTIDRUG RESISTANCE-1 MRNA ARE RATIONALLY SELECTED BY MAPPING RNA-ACCESSIBLE SITES WITH OLIGONUCLEOTIDE LIBRARIES" NUCLEIC ACIDS RESEARCH, vol. 24, no. 10, 15 May 1996 (1996-05-15), pages 1901-1907, XP002006548 ISSN: 0305-1048 cited in the application *
HO SP. ET AL.: "Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries." NAT BIOTECHNOL 16(1):59-63, January 1998 (1998-01), XP002139261 cited in the application *
PENG HO S. ET AL.: "Modification of phosphorothioate oligonucleotides yields potent analogs with minimal toxicity for antisense experiments in the CNS." BRAIN RES: MOL BRAIN RES 62(1):1-11, 12 November 1998 (1998-11-12), XP000907388 *
SMAGIN GN. ET AL.: "The role of CRF2 receptors in corticotropin-releasing factor- and urocortin-induced anorexia." NEUROREPORT 9(7):1601-6, 11 May 1998 (1998-05-11), XP000907403 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1165093A1 (fr) * 1999-03-05 2002-01-02 Epigenesis Pharmaceuticals, Inc. Procede de validation/d'invalidation de cibles et des voies
EP1165093A4 (fr) * 1999-03-05 2002-07-24 Epigenesis Pharmaceuticals Inc Procede de validation/d'invalidation de cibles et des voies
EP1558639A2 (fr) * 2002-10-31 2005-08-03 Janssen Pharmaceutica N.V. Genes dont l'expression augmente en reaction a une stimulation par l'hormone liberatrice de la corticotropine
WO2014059341A2 (fr) 2012-10-12 2014-04-17 Isis Pharmaceuticals, Inc. Composés anti-sens et leurs utilisations
EP2906255A4 (fr) * 2012-10-12 2016-07-27 Ionis Pharmaceuticals Inc Composés anti-sens et leurs utilisations
EP4144845A1 (fr) * 2012-10-12 2023-03-08 Ionis Pharmaceuticals, Inc. Composés antisens et leurs utilisations

Also Published As

Publication number Publication date
AU2725600A (en) 2000-08-01
WO2000042178A3 (fr) 2000-11-30

Similar Documents

Publication Publication Date Title
JP6250078B2 (ja) 真核細胞におけるエキソンスキッピングの誘導
Ho et al. Modification of phosphorothioate oligonucleotides yields potent analogs with minimal toxicity for antisense experiments in the CNS
WO2000042178A2 (fr) Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central
US20020035083A1 (en) CRF2 ligands in combination therapy
CA2457131A1 (fr) Oligonucleotides et autres modulateurs de la voie du recepteur nk-1 et utilisations therapeutiques de ces derniers
AU2012200761A1 (en) Induction of exon skipping in eukaryotic cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU BR CA CN CZ EE HU IL IN JP KR LT LV MX NO NZ PL RO SG SI SK TR UA VN ZA

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AU BR CA CN CZ EE HU IL IN JP KR LT LV MX NO NZ PL RO SG SI SK TR UA VN ZA

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

122 Ep: pct application non-entry in european phase