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WO1997028189A1 - Procedes d'accroissement des niveaux endogenes du facteur de liberation des corticotropines - Google Patents

Procedes d'accroissement des niveaux endogenes du facteur de liberation des corticotropines Download PDF

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Publication number
WO1997028189A1
WO1997028189A1 PCT/US1997/001572 US9701572W WO9728189A1 WO 1997028189 A1 WO1997028189 A1 WO 1997028189A1 US 9701572 W US9701572 W US 9701572W WO 9728189 A1 WO9728189 A1 WO 9728189A1
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WIPO (PCT)
Prior art keywords
crf
ligand inhibitor
related peptide
binding protein
peptide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US1997/001572
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English (en)
Inventor
Dominic P. Behan
Stephen C. Heinrichs
Steven W. Sutton
Phillip J. Lowry
Jean E. F. Rivier
Errol B. Desouza
Wylie W. Vale, Jr.
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University of Reading
Neurocrine Biosciences Inc
Salk Institute for Biological Studies
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University of Reading
Neurocrine Biosciences Inc
Salk Institute for Biological Studies
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Priority to JP9527840A priority Critical patent/JP2000505798A/ja
Priority to EP97905690A priority patent/EP0877759A1/fr
Priority to CA 2244864 priority patent/CA2244864A1/fr
Priority to AU22520/97A priority patent/AU2252097A/en
Publication of WO1997028189A1 publication Critical patent/WO1997028189A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine 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
    • 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
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to methods of increasing endogenous levels of neuropeptides, and specifically to increasing corticotropin- releasing factor levels in the brain
  • CRF Alzheimer's disease
  • AD Alzheimer's disease
  • CRF content Alterations in brain CRF content have also been found in Parkinson's disease and progressive supranuclear palsy, neurological disorders that share certain clinical and pathological features with AD.
  • CRF content is decreased and shows a staining pattern similar to cases of AD (Whitehouse et al , 1987, DeSouza, 1988)
  • CRF is decreased to approximately 50% of control values in frontal, temporal, and occipital lobes (Whitehouse et al , 1987, DeSouza, 1988)
  • Some depressive disorders are also associated with decreased levels of CRF Patients in the depressive state of seasonal depression and in the period of fatigue in chronic fatigue syndrome demonstrate lower levels of CRF in the cerebrospinal fluid (Vanderpool et al., J. Cl . Endocrinol. Metab.
  • hypoactivation ofthe stress system as manifested by low CRF levels may play a role in other disorders as well
  • some forms of obesity are characterized by a hypoactive hypothalamic-pituitary-adrenal axis (Kopelman et al , Clm. Endocrinol (Oxford) 28 15, 1988, Bernini et al , Horn?. Res. 31 133, 1989)
  • some patients with post-traumatic stress syndrome have low cortisol excretion (Mason et al , J. Neu. Men. Dis.
  • the present invention exploits the correlation of reduced levels of CRF with various neuro-physiologically based disorders and diseases to effectively treat such diseases by increasing levels of free CRF, and further provides other related advantages.
  • CRF in the brain by administering to a patient an effective amount of a ligand inhibitor of a CRF/CRF-binding protein (CRF/CRF-BP) complex
  • Administration of the ligand inhibitor causes release of CRF from the CRF-binding protein
  • the ligand inhibitor may be a peptide derived from CRF, homologous to CRF, or unrelated to CRF as long as it is capable of causing the "release" of CRF.
  • Ligand inhibitors may also be non-peptide compounds which are isolated from natural or synthetic chemical libraries
  • the ligand inhibitor is a peptide selected from the group consisting of h/rCRF (amino acids 6-33), h/rCRF (amino acids 9-33), and h/rCRF
  • therapeutic compositions comprising the ligand inhibitor in combination with a physiologically acceptable carrier or diluent.
  • methods for improving learning and/or memory, decreasing food intake (including food intake induced by neuropeptide Y), activating CRF neurocircuitry, treating diseases associated with low levels of CRF in the brain, treating symptoms associated with Alzheimer's disease, treating obesity, treating atypical depression, treating post-partum depression, treating age-related memory deficit, treating symptoms associated with dementia, reducing weight gain, and treating substance abuse withdrawal are provided within such methods.
  • a therapeutically effective amount of a ligand inhibitor of a CRF/CRF-binding protein is administered to a patient as treatment for these conditions Criteria for choosing candidates for therapy are presented, as well as methods for assessing efficacy of treatment.
  • methods are provided for increasing the level of free CRF-related peptide by administering to a patient an effective amount of a ligand inhibitor of a CRF-related peptide/CRF-BP complex
  • the CRF-related peptide is urocortin
  • agents having a high affinity to human CRF-BP can be administered which will effectively compete with human CRF in the formation of complexes with CRF-BP and will, in this manner, increase the effective /// vivo concentration in a mammal of endogenous hCRF, and/or the effective concentration of a CRF agonist or CRF antagonist optionally administered along with such agent, for the purpose of achieving a particular therapeutic purpose
  • these agents serve to block the effect of CRF-BP and thus to increase the concentration of endogenous CRF in those regions of the body where CRF-BP is present
  • peptides between about 19 and 28 residues in length have been discovered which have a high affinity to hCRF-BP but, which themselves exhibit relatively little propensity to bind to the CRF receptor
  • such peptides can be administered to prevent the clearance of endogenous CRF from pa ⁇ icular regions and thereby stimulate the biological effect of CRF m vivo, and in certain instances, it may be advantageous to administer such
  • methods are provided for screening for particularly effective CRF antagonists by carrying out dual screening assays of such potential antagonists to determine their potential for blocking the ability of CRF to bind to native CRF receptors and to also determine the binding affinity between such a candidate CRF antagonist and hCRF-BP.
  • this method comprises contacting CRF with CRF- BP in the presence of a ligand inhibitor in an aqueous solution, further mixing in a nonionic detergent, separating the nonionic detergent and the aqueous solution, and detecting the amount of CRF in the aqueous solution, thereby determining whether the ligand inhibitor disrupts the CRF/CRF-binding protein complex
  • the mixing is performed at a temperature above the cloud point of the nonionic detergent and octylphenoxypolyethoxyethanol is a preferred nonionic detergent
  • Figure 1 presents the amino acid sequences of CRF from human (hCRF) (SEQ ID NO 1), sheep (oCRF) (SEQ ID NO 4), suckerfish urotensin I (sf UROT I)
  • Figure 2 is a graph showing the levels of CRF bound to CRF-BP and free CRF CRF levels were determined in brain tissue from Alzheimer's patients and normal age-matched controls Levels were established with or without the addition of a CRF-BP ligand inhibitor, ⁇ -helical ovine CRF(9-41 )
  • Figure 3 is a graph showing the levels of CRF (panel A) and CRF-BP
  • panel B in four areas of brain tissue taken from normal controls or Alzheimer's patients
  • Figure 4 presents the test results of the Morris water maze and the elevated plus-maze following intracerebroventricular (ICV) injection of vehicle or CRF(6-33) or CRF (1-41 ) Rats, in groups of 7-10 animals, received ICV injections 15 min prior to testing In the Morris water maze test, times to reach the platform were recorded In the elevated plus-maze test, percent time spent in the open arm was recorded An asterisk indicates a statistical significant difference
  • Figure 5 is a chart showing test results of rats in the Y-maze test Groups of 7-10 rats received either 0, 1, 5, or 25 ⁇ g CRF(6-33) in an ICV injection 15 min prior to testing Percent correct responses were determined An asterisk indicates a statistical significant difference
  • Figure 6 is a graph depicting the quantity of food intake by lean and obese rats following a 7 day infusion of either vehicle, oCRF, or h rCRF (6-33)
  • Figure 7 is a graph depicting body weight change in lean and obese rats following a 7 day infusion of either vehicle, oCRF, or h/rCRF (6-33)
  • Figure 8 is a graph depicting the effect of administration of CRF (6-33) ligand inhibitor in the performance of young and aged rates in a one-way active avoidance learning test
  • Figure 9 is a graph depicting the effect of administration of control vehicle or 25 ⁇ g r/hCRF (6-33) ICV in aged rats Following acquisition training, retention was tested at 1 day and 7 day intervals **, p ⁇ 0 05, + , p ⁇ 0 05
  • Figure 10 is a graph depicting the effect of administration of CRF (6-33) ligand inhibitor in a passive avoidance test Rats were either given vehicle alone or 25 ⁇ g ligand inhibitor ICV 15 min prior to training
  • Figure 1 1 are graphs depicting the effect of administration of CRF (6-33) on body weight change during nicotine withdrawal
  • CRF refers to a peptide that regulates the release of adrenocorticotropin (ACTH), ⁇ -endorphin, and other pro-opiomelanocortin (POMC)- derived peptides from the pituitary
  • ACTH adrenocorticotropin
  • POMC pro-opiomelanocortin
  • CRF-binding protein refers to a protein or proteins present either as a soluble factor in human plasma or associated with cell membranes and that has the ability to inhibit the function of CRF as measured by one of two methods ( 1 ) CRF-induced ACTH release from cultured pituitary cells or from a perfused rat anterior pituitary system, or (2) CRF-induced cAMP formation from cells possessing CRF receptors or from cells which have been transfected with cloned CRF receptors Examples of cDNA clones encoding CRF-BP have been isolated from human liver and rat brain (Potter et al , Nature 349.423, 1991 ) "CRF/CRF-BP" refers to the complex of CRF and CRF-BP Binding of
  • CRF and CRF-BP may be through hydrophobic, ionic, or covalent interactions
  • Human CRF binding protein refers to a 37 kDa serum protein that, by specifically binding hCRF, inactivates hCRF as an ACTH secretogogue in vitro and in vivo.
  • Human CRF-BP has a high affinity for hCRF and a low affinity for oCRF, suggesting hCRF-BP may expedite the elimination of peripheral plasma hCRF hCRF loses its ability to stimulate ACTH /// vitro and m vivo when bound to hCRF-BP
  • the first 8 amino acids of the CRFs are believed to be involved in receptor activation while the C-terminus is primarily responsible for receptor affinity hCRF-BP appears to prevent hCRF from stimulating corticotrophs by binding the central domain and thus preventing the ligand from interacting with the receptor and causing ACTH release
  • CRF-related peptide refers to a peptide having 30% or greater identity to CRF and/or is active in binding to one or a combination of hCRF-BP (assay described herein) or CRF receptors Rl and R2 (alpha and beta), or in activating the accumulation of cAMP from cells expressing CRF Rl and CRF R2 (alpha or beta) Briefly, binding to CRF receptors is assayed by incubating adherent cells transfected with the CRF receptor gene with or without unlabeled CRF-related peptide, as described in U.S Serial No.
  • r/hCRF is added (e.g., 125 1- CRF) and the reaction incubated for 2 hours at room temperature
  • the fluid is aspirated, and the cells are washed three times with PBS If non-adherent cells are used, the cells are washed by centrifugation A solution of 4M guanidine thiocyanate or other solubilizer is added to the cells to solubilize the tissue An aliquot of solubilized sample is counted.
  • a peptide that demonstrates > 50% inhibition at 1 ⁇ M or less is considered to be a CRF-related peptide
  • the accumulation of cAMP is an alternative assay Briefly, in this assay, a peptide is added to cells expressing a CRF receptor A 1 mM solution of isobutylmethylxanthine is also added to inhibit the breakdown of cAMP by phosphodiest erases. Cells are incubated for 1 hr at 37°C and then washed A solution of 95% ethanol and 20 mM HCI is added for approximately 12-18 hr at -20°C to extract cAMP.
  • the EtOH/HCl is removed to a tube and dried by centrifugation m vacuuo
  • the cAMP is reconstituted with NaOAc buffer, pH 7 5 and assayed for cAMP with a radioimmunoassay kit (Biomedical Technologies, Inc , Stoughton, MA) or equivalent
  • a radioimmunoassay kit Biomedical Technologies, Inc , Stoughton, MA
  • a peptide that demonstrates 50% maximal cAMP stimulation (as determined by stimulation with h/rCRF) at 1 ⁇ M or less is considered to be a CRF-related peptide
  • the present invention presents a method for increasing the level of free CRF in the brain by administering an effective amount of a ligand inhibitor of a CRF/CRF-BP complex, such that CRF is released from CRF-BP
  • the "ligand inhibitor of the CRF/CRF-BP complex” displaces CRF either in a reversible or irreversible fashion. Displacement may occur by causing a bound CRF molecule to become free CRF.
  • the binding of the ligand inhibitor may inhibit binding of free CRF to CRF-BP because of a high affinity to human CRF-BP, thus competing with endogenous CRF for binding to hCRF-BP
  • Reversible or irreversible displacement of CRF may be mediated by the ligand inhibitor binding directly to the CRF binding site or alternatively by the ligand inhibitor binding to a site that is not the CRF binding site and causing allosteric displacement of the bound CRF
  • Ligand inhibitors may be peptides derived from CRF or CRF-related sequences, or random peptides which are designed to cause displacement of bound CRF Additionally, ligand inhibitors may be non-peptide molecules derived from natural libraries of small molecules, synthesized analogues of natural molecules, specifically designed small molecules
  • the ligand inhibitors must be accessible to the brain, either administered through the CNS or systemically
  • the characteristics of the ligand inhibitor are such that it is a low affinity antagonist at the CRF receptor (Kj > 1 ⁇ M) or has a 100-fold selectivity to the CRF binding protein (Kj ⁇ l O nM)
  • the CRF-BP ligand inhibitor may also exhibit some moderate agonist activity at the CRF receptor (Kj > 50 nM)
  • Peptide sequences which bind to CRF-BP and displace endogenous CRF may be derived from CRF peptide sequences, CRF-related peptide sequences, or unrelated peptide sequences It has been found that relatively short peptides between about 19 and 28 residues in length are effective to competitively bind to hCRF-BP while at the same time exhibiting very low binding affinity to CRF receptors As a result, this particular group of peptides bind to the hCRF-BP while not substantially binding to and/or interacting with the CRF receptor When given alone, these peptides have the effect of increasing the amount of endogenous free CRF, which would remain available to interact with the CRF receptors Thus, these peptides can be used to assure or increase the effect of endogenous CRF in a particular region or body organ These peptides may similarly be used to increase the efficacy of CRF receptor agonists or antagonists if given together with these substances in a cocktail, however, administration with a CRF antagonist would be
  • Suitable peptides include fragments of human CRF, i.e., hCRF( 1-41 ), which has the sequence
  • hCRF(6-33) having the sequence Ile-Ser-Leu-Asp-Leu-ThrPhe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala- Arg-Ala-GluGln-Leu-Ala-Gln-Gln-Ala-His-Ser (SEQ ID NO 1 )
  • This fragment can be shortened by up to 4 residues at the N-terminus and/or up to 5 residues at the C- terminus by elimination of residues in sequence, e.g., hCRF (9-33), hCRF (6-30) and hCRF (8-29)
  • Other examples of peptides that may alternatively be used include analogues of hCRF (6-33) such as [Nle 21 ]-hCRF (6-33), [Nle l ]-hCRF (9-33), [Ile 24 ]- hCRF (6-33), [Asn 26 ] -h
  • peptides which may be employed for this purpose are defined by the following sequence (SEQ ID NO.2). Xaa 4 -Xaa 5 ,-Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa I0 - Xaa, r Xaa 12 -Xaa 1 -Xaa 14 -Xaa
  • Another group of preferred peptides are defined by the following sequence (SEQ ID NO.2) Xaa -Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa, 0 -Xaa 1 ,-Xaa, 2 - Xaa, 3 -Xaa 14 -Xaa 15 -Xaa 16 -Xaa ] ] 7 -Xaa 18 -Xaa ] 9 -Xaa 2 o-Xaa 2 i-AJa-Xaa 23 -Xaa 24 -Glu- Xaa 26 -Xaa 27 -Xaa 2 g-Xaa 29 -Xaa 0 -Xaa,
  • particularly preferred peptides include the following analogues [He 8 - 19 - 24 , Asn 17 - 26 , Met 18 ,, Glu 27 - 29 , Arg 28 , Gly 32 , Leu 3 ]-hCRF(6-33) [Asn 17 - 26 , Nle 18 , He 19 - 24 , Glu 27 - 29 , Arg 28 , Gly 32 , Leu 33 ]-hCRF(9-33) [He 8 - l9 - 24 , Asn 17 - 26 , Met 18 , Glu 27 , Arg 28 ]-hCRF(4-28) [He 19 - 24 , Asn 17 - 26 , Nle 18 , Glu 27 , Arg 28 ]-hCRF (7-31 ) [He 8 - 19 , Asn 17 - 24 - 26 , Met 18 , Glu 27 , Arg 28 ]-hCRF (7-31 ) [He 8 - 19 ,
  • Peptides such as hCRF (6-33) and hCRF (9-33) and the other analogues specifically mentioned hereinbefore are synthesized manually using solid phase methodology or automated with a Beckman Model 990 peptide synthesizer, as described in U.S. Patent No 4,489,163, the disclosure of which is incorporated herein by reference.
  • tertbutoxycarbonyl (Boc) is used for ⁇ -amino protection, and TFA- CH 2 C1 2 (3:2) is used for deprotection Standard couplings are mediated by 1,3- diisopropylcarbodimide (DIC), while difficult couplings are accomplished using 2-(lH- benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU)
  • DIC 1,3- diisopropylcarbodimide
  • HBTU 2-(lH- benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
  • HF hydrous hydrofluoric acid
  • Crude peptides are purified using multiple-step, reversed-phase HPLC
  • a polypeptide analogue includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been substitute
  • Conservative substitutions may be made with a residue having a functionally similar side chain, as long as the polypeptide displays the ability to cause an increase in free CRF, such as by binding strongly to CRF-BP.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, alanine, glycine, leucine or methionine, for another; the substitution of one polar (hydrophilic) residue for another, such as arginine for lysine, glutamine for asparagine, threonine for serine; the substitution of one basic residue such as lysine, arginine or histidine for another, and the substitution of one acidic residue, such as aspartic acid or glutamic acid for the other
  • conservative substitution also includes the use of a chemically derivatized residue in place of a non- derivatized residue provided that such polypeptide displays the desired binding activity. As previously mentioned, such conservative substitutions can be made for one or
  • “Chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine
  • Chemical derivatives also include those peptides which contain one or more naturally occurring amino acid derivatives of the standard amino acids
  • 4-hydroxyproline may be substituted for proline
  • 5-hydroxylysine may be substituted for lysine
  • 3-methylhistidine may be substituted for histidine
  • homoserine may be substituted for serine
  • ornithine may be substituted for lysine
  • random peptides may be synthesized and subsequently screened to identify peptides that meet the functional criteria of displacement of CRF from the CRF/CRF-BP complex, discussed in more detail below Random peptides may be generated by biological methods, or by combinatorial chemical technologies (see Gallop et al., J. Med. Chem. 37 1234, 1994)
  • Biological methods of generating random peptides include at least four different methods
  • random nucleotides may be inserted into a host gene for display of the peptide on the surface of microorganisms (Charbit et al , Em bo. Journal 5:3029, 1986; Agterberg et al , Gene 88 37, 1990; Fuchs et al , B o Tech 9 1369, 1991 , Thery et al , Appl. Environ. Microbiol.
  • various cell surface proteins of bacteria are used as fusion partners in which oligonucleotides are inserted to produce peptides fused into one of the extracellular loops of the protein LamB, O pA, PhoE, PAL, and pilin proteins have all served as vehicles for peptide display on bacteria.
  • the vectors employed are derived from filamentous phages, such as Ml 3, fl, and fd Typically, coat proteins, such as pill or pVIII, serve as peptide expression vehicles (Smith, Science 228 1315, 1985, Parmley and Smith, Gene 73 305, 1988, Cwirla et al , Proc. Natl. Acad. Sci.
  • peptides may be attached to plasmids (Cull et al , Proc. Natl. Acad. Sci.
  • peptides may be displayed on polysomes after stalling translation By causing accumulation of RNA and polysomes containing nascent peptides still linked to their encoding RNA (Gallop et al , J. Medicinal Chem. 37.
  • Small, non-peptide molecules, natural or synthesized may also be used as ligand inhibitors.
  • Libraries of small molecules to be screened for ligand inhibitors of the CRF/CRF-BP complex are obtained from soil samples, plant extracts, marine microorganisms, fermentation broth, fungal broth, pharmaceutical chemical libraries, combinatorial libraries (both chemical and biological) and the like
  • Such libraries may be obtained from a variety of sources, both commercial and proprietary Molecules, which have a similar, but not identical, structure to the candidate compound may be synthesized and tested for ligand inhibition as described below.
  • a small peptide sequence can be modeled around the solution NMR structure of CRF.
  • a peptide sequence derived from residues 20-27 can be modeled from a reference solution structure of human CRF, which has been determined by lH NMR and distance geometry with restrained molecular dynamics (Protein Engineering 6. 149, 1993)
  • the intact CRF molecule is used as a basis for modeling because it is possible that the alpha helical structure of CRF is lost as bigger deletions are made.
  • the 3-D structure of the small peptide, which spans the important contact residues, can then be used to search a computer bank for non-peptide molecules which mimic the peptide's structure Molecules with a lower IC-50 value than the starting molecule are selected.
  • Further molecules are synthesized based upon the physical and biochemical characteristics of the initial compounds and their molecules A particularly preferred candidate will have an IC-50 value of 10 nM or less
  • the determination of whether any of the inhibitors discussed above will have the requisite properties can be made by assaying the ability of the inhibitor to displace CRF from CRF/CRF-BP binding complex and secondly, by evaluating its ability to bind the CRF receptor.
  • Candidate ligand inhibitors may be screened for their ability to displace CRF from the CRF/CRF-BP complex by biological assay or by /// vitro assay
  • One suitable biological assay is the measurement of ACTH release from cultured pituitary cells This assay is performed in the following manner Anterior pituitary glands from rats are washed six times with sterile HEPES buffer and transferred to a solution containing collagenase After subsequent transfer to a 25 ml Bellco dispersion flask, the pituitaries are stirred for 30 min at 37°C, triturated, incubated for a further 30 min, and again triturated The partially dispersed cells are then collected by centrifugation The cell pellet is resuspended in 10 ml of neuraminidase and again collected by centrifugation The pellet is reconstituted in 25 ml of BBM-P (BBM (Irvine Scientific) plus 100 ⁇ g/L, cortisol, 1 ⁇ g/L insulin
  • a preferred mode of screening candidate ligand inhibitors is by an vitro ligand immunoradiometric assay (LIRMA)
  • LIRMA vitro ligand immunoradiometric assay
  • CRF-BP may be isolated from brain tissue, serum or cells expressing a recombinant form
  • Recombinant hCRF- BP may be produced in Chinese Hamster Ovary (CHO) cells bearing the pSG5-hA3 and RSV-neo plasmids Stable CHO transfectants are cloned by dilution under G418 (Sigma Chemical, St.
  • hCRF-BP transfected CHO cells are inoculated into a 10,000 MWCO bioreactor (Cell Pharm Micro Mouse, Unisyn Technologies, Tustin, CA) Enriched medium is harvested from the bioreactor daily and stored at -20°C until purification Closed roller bottles containing recombinant cells and tissue culture medium, which are slowly rotated in a 37°C environment, may alternatively be used hCRF-BP may be purified by a 3-step process, with fractions from each step being evaluated using the assay described below First, enriched medium is affinity- purified using a Bio Pilot chromatography device (Pharmacia LKB Biotechnology, Uppsala, Sweden).
  • Affi-Prep 10 Human CRF is coupled to Affi-Prep 10 (Bio Rad Laboratories, Richmond, CA) via primary amino groups using N-hydroxysuccinimide After coupling, the affinity gel is packed into an XK16 or equivalent column (Pharmacia LKB Biotechnology, Uppsala, Sweden)
  • Affinity purification consists of percolating enriched medium through the column at 2 ml/min, washing with 10 bed volumes of 100 mM HEPES HCI (pH 7 5) at 5 ml/min and eluting 1 bed volume fractions using 80 mM triethylammonium formate (pH 3.0) containing 20% acetonitrile at 5 ml/min Elution under mildly basic conditions, e.g., pH about 10.5, may alternatively be used.
  • Secondary purification utilizes gel chromatography Affinity-pure hCRF-
  • BP is lyophilized and reconstituted in 6M guanidine-HCl buffered with 0 IM ammonium acetate (pH 4.75)
  • An FPLC device is used in conjunction with two Superose 12 HR 10/30 columns (Pharmacia LKB Biotechnology, Uppsala, Sweden) connected in series for this purification step.
  • the affinity-pure hCRF-BP is loaded in I ml and subsequently eluted with 6M guanidine HCl/0 IM ammonium acetate (pH 4 75) at 0 4 ml/min, collecting fractions every minute
  • the HPLC device consists of 2 model 100A pumps (Beckman, Palo Alto, CA), an Axxiom HPLC controller (Cole Scientific, Calabasas, CA), a Spectroflow 773 absorbance detector set to 214 nm (Kratos Analytical, Ramsey, NJ), and a Pharmacia, model 482 chart recorder (Pharmacia LKB Biotechnology, Uppsala, Sweden).
  • Buffer A is 0 1% trifluoroacetic acid (TFA)/5% acetonitrile
  • buffer B is 0.1% TF A/80% acetonitrile.
  • LIRMA CRF-BP isolated from brain tissue, serum, or cells expressing a recombinant form is added to wells of a 96-well plate, to small polypropylene microfuge tubes, or to glass borosilicate tubes in a binding buffer (0 02% NP-40 in 50 mM phosphate-buffered saline) 125 I-h/rCRF (New England Nuclear) and the candidate ligand inhibitor at 10 ⁇ M are added and the reaction is incubated for one hr at room temperature
  • An appropriately diluted anti-CRF-BP antibody such as a rabbit anti-hCRF-BP (Potter et al , Proc. Natl Acad.
  • the candidate ligand inhibitor displaces CRF from CRF-BP, the pellets will contain less radioactivity in comparison to controls in which no candidate peptide is added Maximum inhibition (/ e , 100%) of the binding of , 25 I-h/rCRF to the CRF-BP is defined by the amount of radioactivity left in the pellets after incubation with 10 ⁇ M of the CRF-BP peptide ligand h/rCRF (6-33)
  • the binding potency of the candidate ligand inhibitor will be measured relative to the potency of the standard h/rCRF (6-33)
  • K is important
  • Ligand inhibitors may also be screened by an /// vitro assay in which bound and free CRF are separated by detergent phase separation.
  • CRF-BP isolated as described above is incubated with 1-h/rCRF and the candidate ligand inhibitor at lO ⁇ M in a binding buffer (0 02% NP-40 in 50 mM phosphate-buffered saline)
  • a detergent such as octylphenoxypolyethoxyethanol, sold as Triton X- l 14TM
  • Triton X-l 14TM is added and mixed by vortexing Triton X-l 14TM and other nonionic detergents are insoluble in water above their cloud point temperature At this temperature, there occurs a microscopic phase separation Below this temperature, the detergents form clear micellar solutions and above this temperature, two clear phases, one depleted and one enriched in detergent, are formed
  • the cloud point temperature of Triton X-l 14TM is 20°C.
  • Triton X-l 14TM is preferred CRF, which has amphiphilic alpha helices, is more soluble in Triton X-l 14TM and thus partitions to the detergent phase
  • CRF bound to CRF-BP is more soluble in an aqueous solution
  • Phase separation is conveniently accomplished by centrifugation
  • the aqueous phase (on top) may be removed and the amount of I-h/rCRF determined
  • a reduction of radioactivity relative to that obtained in the absence of ligand inhibitor means that the ligand inhibitor displaced CRF from CRF-BP.
  • NPY or other neuropeptides of interest will generally partition into the Triton X-l 14TM detergent phase
  • the method described above may be generally employed to screen for neuropeptide-binding proteins Briefly, by way of example, tissue from various organs is homogenized in 1 % NP-40/PBS solubilization buffer Particulate matter is removed by centrifugation for 10 min at 3000 x g A 50 ⁇ l aliquot from the supernatant is incubated with 500 pM of the ' 1-labeled neuropeptide and the assay is performed as above Serum or plasma may also be used as a potential source of neuropeptide-binding proteins A range of concentrations (0 1 - 1000 nM) of unlabeled neuropeptide is coincubated with the radiolabeled neuropeptide to assess whether the putative binding protein specifically binds the radiolabeled neuropeptide When binding is specific, the radioactivity remaining in the aqueous phase after Triton X-l 14TM separation is decreased Using this method, an
  • this method may be employed to screen for ligand inhibitors of the neuropeptide to its neuropeptide-binding protein Briefly, radiolabeled neuropeptide is incubated with the neuropeptide-binding protein or soluble receptor and the reaction performed as described above For these assays, either recombinant neuropeptide-binding protein or receptor or crude neuropeptide-binding protein isolated from tissue sample may be used
  • a preferred ligand inhibitor either has a low affinity antagonist effect at the CRF receptor or has a 100-fold selectivity to the CRF binding protein Therefore, compounds with an IC-50 value in the range of 10-100 ⁇ M and a specific inhibition of the CRF/CRF-BP complex are further tested for binding to the CRF receptors
  • the ability of the ligand inhibitor to antagonize the CRF receptor is assessed in a cAMP production assay
  • the assay compares the potency of the ligand inhibitor to increase levels of free CRF which thereby bind the CRF receptor, and stimulate cAMP production
  • the test cell lines express the CRF receptor as stable transfectants The assay is performed according to Battaglia et al (Synapse 7:572, 1987) with minor modifications Test cells are incubated for 1 hr with various concentrations of CRF and ligand inhibitors The cells are washed, and intracellular cAMP is released upon incubation of the cells for 16-18 hr and is subsequently extracted in 20 mM
  • the ligand inhibitor can be evaluated in a binding assay with the human CRF receptor.
  • the human CRF receptor and a binding assay for such receptor and human CRF are described in Chen et al , Proc. Natl. Acad. Sci.
  • the agent may be evaluated with radioactively labeled [Nle 21 , Tyr 32 ] oCRF to compute an inhibitory binding affinity constant (K,)
  • K inhibitory binding affinity constant
  • the agent has a receptor K, of at least about 100 nM and more preferably greater than 1000 nM It may altematively be satisfactory to use the rat CRF receptor because human CRF and rat CRF have the identical amino acid sequence.
  • An additional assay using rat anterior pituitary cells to measure ACTH secretion can be carried out to determine whether a ligand inhibitor functions as a CRF agonist of hCRF receptors.
  • the procedure which is used is that as generally set forth above except that only ligand inhibitor is added to the cells Antagonistic action may be determined by performing the assay in the presence of a challenge dose of CRF The performance of the ligand inhibitor is compared to the performance of what has become a standard antagonist for this purpose, such as CD-Phe 12 , Nl e 21 ]-rCRF( 12-41 ) or a fragment of alphahelical CRF( AHC), such as AHC (9-41 )
  • hCRF (6-33) is shown to have a CRF agonist bioactivity much less than the standard oCRF, which is arbitrarily considered as 1.0.
  • This peptide does not exhibit substantial CRF antagonist activity. Because this peptide has less than about 0 1% of the CRF agonist activity ofthe standard peptide, it is acceptable from this standpoint
  • the peptide hCRF (9-33) is even a weaker CRF agonist, having substantially less than 0 01% ofthe activity of oCRF.
  • an agent should have less than about 25% of the CRF agonist activity of oCRF and that it should not exhibit substantial CRF competitive antagonist activity. Preferably, it should have less than 5% of the antagonist activity of the present standard peptide [DPhe 12 , Nle 21 - 38 ]-hCRF( 12-41 )
  • the lower its value in such an assay the better it should function in this method because its potential blocking effect as a result of binding to CRF receptors will be minimized.
  • the invention also provides methods for screening peptides or other agents to select more effective CRF antagonists for in vivo administration to mammals
  • a candidate peptide is first evaluated in the well-known assay described hereinbefore for determining its biological effectiveness to inhibit a test dosage of CRF from stimulating the secretion of ACTH from a culture of rat interior pituitary cells
  • the candidate peptide is then evaluated in the hCRF-BP competitive binding assay described hereinbefore in order to determine its K, which, as explained hereinbefore, is indicative of its affinity for binding to hCRF-BP, which has the tendency to clear the injected peptide from the target cell sites to which it is directed
  • a particularly effective CRF antagonist can be chosen which has a high value in the CRF antagonism assay and which also has a high (CRF-BP) K strongly indicating that it exhibits a relatively low affinity for binding to hCRF-BP
  • CRF-BP CRF-BP
  • the present invention provides methods for increasing the level of free CRF in the brain through the administration of a ligand inhibitor of a CRF/CRF-BP complex.
  • the increase in level of free CRF may be measured by in vitro assays, such as ELISA, stimulation of ACTH release, or stimulation of cAMP production
  • an increase in free CRF due to administration of the ligand inhibitor is measured relative to a reference ligand inhibitor, in this case h/rCRF (6-33)
  • a minimal acceptable value of increase is 10% of the value for h rCRF (6-33), a moderate value is 50%, a preferred value is 80%, and a particularly preferred value is 100%
  • the level of free CRF may be measured by two-site ELISA on homogenates of brain samples, cerebrospinal fluid, or on other bodily tissues and fluids Total CRF is first quantitated as follows Wells of an ELISA plate are coated with an anti-CRF antibody, such as protein G purified-sheep anti
  • RC-70 a rabbit anti-human CRF antibody
  • an enzyme-conjugated antibody that detects RC-70, or the equivalent, is added Altematively, RC-70 antibody may be enzyme-conjugated Preferred conjugates are horseradish peroxidase and alkaline phosphatase, but one skilled in the art will recognize that many different acceptable alternatives are available, including a radiolabel instead of an enzyme Enzyme substrate is added, and color development proceeds After termination of the reaction, absorbance measurements are used to quantify the amount of total C
  • bound CRF may be quantitated by ELISA by coating plates with an anti-CRF-BP antibody followed by detection of the bound CRF with an anti-CRF antibody.
  • Bound CRF can be specifically displaced by the CRF-BP ligand ⁇ - helical oCRF(9-41) resulting in a decrease in the signal detected Alpha helical oCRF(9-41) is used for the displacement as it does not crossreact with RC-70, anti-CRF antibody.
  • the displaced CRF present in the supematants may then be assayed by two-site ELISA, as described Free CRF may then be determined by calculation ofthe difference between total CRF and bound CRF or by a direct assay In a direct assay, following capture of the bound complex by the anti-CRF-BP monoclonal antibody, the supematants are removed and the free CRF measured in a two-site ELISA, as described.
  • a ligand inhibitor, ⁇ -helical ovine CRF is shown herein to increase free CRF levels in brain tissue of both normal individuals and Alzheimer's disease patients
  • a two-site ELISA was used to determine the amount of total and bound CRF Addition of ⁇ -helical ovine CRF resulted in release of all bound CRF (see Figure 2 and Example 5)
  • other procedures may be performed in vivo These include MRI, PETSCAN, spectscanning or other similar imaging techniques, some of which use a radiolabeled ligand to CRF-BP or to CRF receptors
  • a preferred method is image analysis using PET position-emitting ligands (e.g., ⁇ C, 18 F) of single photon-emitting ligands (e.g.,2 I-labeled ligand to CRF-BP or to CRF receptors) Free CRF levels are
  • administering may be used to treat diseases or syndromes in which there are decreased levels of CRF CRF levels may be measured directly in cerebrospinal fluid or in the brain by imaging or other methods (e.g.
  • Such diseases or syndromes include symptoms of dementia or learning and memory loss, obesity, chronic fatigue syndrome, atypical depression, post-partum depression, seasonal depression, hypothyroidism, post- traumatic stress syndrome, nicotine withdrawal, vulnerability to inflammatory disease Definitions of these syndromes (except for obesity, chronic fatigue syndrome, and vulnerability to inflammatory diseases) are provided in Diagnosis and Statistical Manual of Mental Disorders (4th ed ), American Psychiatric Association, Washington, D C , 1994 (hereinafter DSM-IV)
  • CRF-BP may be a modulator of a family of CRF-related peptides
  • in vitro experiments show that rat urocortin can disrupt the CRF/CRF-BP complexes normally found in human brain tissue.
  • ligand inhibitors of the present invention may also be used to elevate free levels of urocortin and other members of the CRF family in mammals
  • Assays for measuring the increase of urocortin (as well as other CRF-related peptides) are performed essentially as described herein for CRF, except that the appropriate detection molecules are employed (e.g., antibody to urocortin)
  • urocortin, sauvagine, urotensin, and the like and their analogues may be used to inhibit CRF/CRF-BP complexes and raise free CRF levels or inhibit urocortin/CRF-BP complexes and raise free urocortin levels
  • ligand inhibitors may be useful in treating hypertension.
  • urocortin appears to significantly suppress feeding behavior in animals, and therefore, such lig
  • the present invention provides methods for improving learning and memory through the administration to a patient of a therapeutically effective amount of a ligand inhibitor of a CRF/CRF-BP complex
  • a patient may be identified through a clinical diagnosis based on symptoms of dementia or learning and memory loss
  • Individuals with an amnestic disorder are impaired in their ability to learn new information or are unable to recall previously learned information or past events
  • the memory deficit is most apparent on tasks to require spontaneous recall and may also be evident when the examiner provides stimuli for the person to recall at a later time
  • the memory disturbance must be sufficiently severe to cause marked impairment in social or occupational functioning and must represent a significant decline from a previous level of functioning
  • the memory deficit may be age-related or the result of disease or other cause
  • Dementia is characterized by multiple clinically significant deficits in cognition that represent a significant change from a previous level of functioning Memory impairment involving inability to learn new material or forgetting of previously learned material is required to make the diagnosis of a dementia Memory can be formally tested by asking the person to register, retain, recall and recognize information
  • the diagnosis of dementia also requires at least one of the following cognitive disturbances: aphasia, apraxia, agnosia or a disturbance in executive functioning
  • aphasia, apraxia, agnosia or a disturbance in executive functioning
  • the level of free CRF in the cerebrospinal fluid may be measured by ELISA or RIA
  • brain imaging as described with a labeled ligand specific to the CRF-BP or CRF receptor may be used to quantitate free receptor or CRF-BP, thus allowing one to know that free CRF is decreased
  • imaging of the brain with a ligand specific to unbound CRF may be used to directly assay the amount of free CRF in the brain
  • Minimental Test for Learning and Memory a standard test used by clinicians to determine if a patient has impaired learning and memory (Folstein et al., J. Psychiatric Res. 72: 185, 1975).
  • Improvement in learning and memory constitutes either (a) a statistically significant difference between the performance of ligand-inhibitor treated patients as compared to members of a placebo group; or (b) a statistically significant change in performance in the direction of normality on measures pertinent to the disease model
  • This strategy has been successfully employed in identifying therapeutically useful cholinomimetics for memory improvement.
  • Animal models or clinical instances of disease exhibit symptoms which are by definition distinguishable from normal controls
  • the measure of effective pharmacotherapy will be a significant, but not necessarily complete, reversal of symptoms Improvement can be facilitated in both animal and human models of memory pathology by clinically effective "cognitive enhancing" drugs which serve to improve performance of a memory task.
  • cognitive enhancers which function as cholinomimetic replacement therapies in patients suffering from dementia and memory loss of the Alzheimer's type significantly improve short-term working memory in such paradigms as the paired-associate task (Davidson and Stern, 1991).
  • Another potential application for therapeutic interventions against memory impairment is suggested by age-related deficits in performance which are effectively modeled by the longitudinal study of recent memory in aging mice (Forster and Lai, 1992).
  • the cognitive enhancing effects are measured by the Morris maze (Stewart and Morris, in Behavioral Neuroscience, R. Saghal, Ed. (IRL Press, 1993) p.
  • the Morris water maze is one of the best validated models of learning and memory, and it is sensitive to the cognitive enhancing effects of a variety of pharmacological agents (McNamara and Skelton, Brain Res. Rev. 75 33, 1993)
  • the task performed in the maze is particularly sensitive to manipulations of the hippocampus in the brain, an area of the brain important for spatial learning in animals and memory consolidation in humans.
  • improvement in Morris water maze performance is predictive of clinical efficacy of a compound as a cognitive enhancer
  • treatment with cholinesterase inhibitors or selective muscarinic cholinergic agonists reverse learning deficits in the Morris maze animal model of learning and memory, as well as in clinical populations with dementia (McNamara and Skelton, 1993, Davidson and Stern, 1991 , McEntee and Crook, 1992, Dawson et al , 1992)
  • this animal paradigm accurately models the increasing degree of impairment with advancing age (Levy et al., 1994) and the increased vulnerability of the memory trace to pre-test delay or interference (Stewart and Morris, 1993) which is characteristic of amnesiac patients.
  • the test is a simple spatial learning task in which the animal is placed in tepid water, which is opaque due to the addition of powdered milk The animals learn the location of the platform relative to visual cues located within the maze and the testing room; this learning is referred to as place learning
  • 15 min prior to training on each of days 1-3 groups of animals receive ICV injections of control solution or 0.1, 1, 5, or 25 ⁇ g of the ligand inhibitor peptide h/rCRF (6-33) or h rCRF, which is additionally an agonist at the CRF receptor
  • a non-peptide inhibitor is used, amounts injected are adjusted accordingly
  • Control animals typically reach the platform within five to ten seconds after three days of training
  • the measure of the memory modulator effects of a ligand inhibitor is a shift of this time period
  • Administration of a ligand inhibitor results in a dose-dependent increase in availability of synaptic CRF and a behavioral dose-dependent increase in acquisition and memory retention.
  • the Y-maze test based on visual discrimination is another assay of learning and memory in animals.
  • two arms of the maze end in a translucent plastic panel behind which there is a 40-watt electric bulb.
  • the start box is separated from the third arm by a manually-activated guillotine door
  • all animals are allowed to explore the maze for five min, and food pellets are available in each arm
  • each animal is placed in the start box with the door closed
  • the animal is allowed to move down the arms and eat the pellets which are located in both arms
  • animals receive six trials in groups of three where one arm is closed at the choice point, no discriminative stimulus is present, and two food pellets are available in the open goal box
  • days 4-10 a light at the end of the arm with the food pellets is illuminated and ten trials are run, again in groups of three The time it takes for the animal to reach the food pellets is recorded
  • the one-way active avoidance test is another assay of learning and memory in animals It may be used to assess improvement in age-related memory deficits.
  • an animal is placed in a footshock compartment, an opening door to a safe compartment serves as a signal for avoidance
  • an animal is placed in a Skinner box enclosure that contains a grid floor composed of stainless steel bars
  • a seven watt light and tone generator at each end of the box serve as conditioned stimuli
  • a rat or mouse is initially trained by being placed in the footshock compartment facing away from the door
  • a shock is administered simultaneously with the door opening to the safe compartment
  • the test is repeated, only the shock is delayed for 10 seconds after the door is opened The time it takes the animal to leave the footshock compartment is recorded
  • the passive avoidance test is another assay of learning and memory within this test, an animal is placed in the safe compartment of the Skinner box and when it enters the footshock compartment, the door is closed and a mild shock is administered The latency time for entering the second compartment is recorded Memory is tested 1 to 7 days later. At this time, a shock is not administered
  • the elevated plus maze test measures anxiogenic responses in an approach-avoidance situation involving an exposed, lighted space versus a dark, enclosed space Both spaces are elevated and are set up as two runways intersecting in the form of a plus sign
  • This type of approach-avoidance situation is a classical test of "emotionality" and is very sensitive to treatments that produce disinhibition and stress Animals are placed in the center of the maze and are allowed free access to all four arms in a five minute testing period The time spent in each arm is recorded
  • Daily pre-test administration of doses of h/rCRF by ICV injection that produced increases in learning and memory also produced anxiety as evidenced by the results of the elevated plus maze test ( Figure 4, lower panel)
  • a dose-dependent suppression of exploration was observed
  • the usefulness of a CRF-receptor agonist i.e., Kj ⁇ 1 nM] for treatment of memory and learning deficits that are due to decreased levels of CRF is of dubious value because of the associated side effects
  • the Wechsler Memory Scale is a widely-used pencil-and-paper test of cognitive function and memory capacity. In the normal population, the standardized test yields a mean of 100 and a standard deviation of 15, so that a mild amnesia can be detected with a 10-15 point reduction in the score, a more severe amnesia with a 20-30 point reduction, and so forth (Squire, 1987).
  • a battery of tests including, but not limited to, the Minimental test, the Wechsler memory scale, or paired-associate learning are applied to diagnose symptomatic memory loss
  • These tests provide general sensitivity to both general cognitive impairment and specific loss of learning/memory capacity (Squire, 1987)
  • these clinical instruments also identify age-related cognitive decline which reflects an objective diminution in mental function consequent to the aging process that is within normal limits given the person's age (DSM IV, 1994)
  • “improvement” in learning and memory is present within the context of the present invention if there is a statistically significant difference in the direction of normality in the paired-associate test, for example, between the performance of ligand-inhibitor treated patients as compared to members of the placebo group or between subsequent tests given to the same patient
  • the present invention provides methods for decreasing food intake or reducing weight gain through the administration to a patient of a therapeutically effective amount of a ligand inhibitor of a CRF/CRF-BP complex CRF has been shown to be an important modulator of food intake and weight gain
  • administration of CRF agonists or conditions that elevate endogenous CRF levels diminish food intake (Appel et al , Endoc. 128 3237, 1991 , Krahn and Gosnell, Psychiat. Med. 7 235, 1989, McCarthy et al , Am. J. Physiol.
  • Patients may be identified by being obese An obese individual weighs more than a target weight considered normal for that person's age, gender and height and can be identified objectively by a body mass index (BMI - calculated as weight in kilograms/height in meters 2 ) at or higher than the 85th percentile of the same reference population (National Center for Health Statistics, "Obese and Overweight Adults in the United States " Series 1 1, No BO, U.S.
  • diets are specially formulated with differing proportions of macronutrients, such as carbohydrate, protein, and fat, so as to measure preference for specific nutrients based on sensory attractiveness or post-ingestive benefit. Diet selection is altered, in part, by a wide variety of neurochemical systems These tests are useful for detection of subtle changes in food intake regulation which impact phenomena, such as craving or binging, and are relevant for the diagnosis of eating disorders, such as anorexia nervosa and obesity Following establishment of a baseline for animals, 15 min prior to testing each animal receives an ICV injection of control solution or a dose of 1, 5, or 25 ⁇ g of a peptide ligand inhibitor, or appropriate doses for a non-peptide ligand inhibitor.
  • macronutrients such as carbohydrate, protein, and fat
  • Food intake is measured as described for the feeding test or the diet self-selection in the cafeteria environment, and test results are compared to baseline
  • overeating in an animal model of nicotine withdrawal and in genetically obese rats (Zucker strain) provide other models to test the effect of a ligand inhibitor on appetite regulation.
  • controls were examined over four (day 14-17) or seven (day 14-21 ) days following nicotine withdrawal, with concurrent administration of CRF (6-33) or saline, and during recovery following discontinuation of all treatments (day 22-24; day 22-28).
  • animals are administered nicotine in a chronic fashion These animals show inhibition of normal weight gain and reduction of food and water intake.
  • mice Upon cessation of nicotine treatment, animals significantly increase both body weight and intake of food and water. The effect of ligand inhibitors on appetite during nicotine withdrawal is assessed by administering the ligand inhibitor three days following nicotine cessation. A 25 ⁇ g dose of CRF (6-33) ICV diminished food intake in nicotine withdrawal subjects without suppressing feeding in untreated controls.
  • CRF 6-33
  • ICV diminished food intake in nicotine withdrawal subjects without suppressing feeding in untreated controls
  • mice e.g., ob/ob
  • rats Zucker strain; fa/fa
  • These animals offer other models of overeating to assess the efficacy of ligand inhibitors.
  • Zucker rats are used as subjects. Groups of rats are treated with vehicle or ligand inhibitor on a daily basis over a set time period, such as one week Subsequent weight gain or food intake is measured. Normal Zucker rats (not genetically obese) serve as controls.
  • obesity is related not only to overeating, but may also be related to consumption of nutritionally imbalanced diets such as a disproportionately large intake of sweet or fatty foods.
  • nutritionally imbalanced diets such as a disproportionately large intake of sweet or fatty foods.
  • the present invention provides methods for treating diseases associated with low levels of CRF through the administration to a patient of a therapeutically effective amount of a ligand inhibitor of a CRF/CRF-BP complex.
  • Such patients may be identified through diagnosis of eating disorders, neuroendocrine disorders, and cognitive disorders, such as Alzheimer's disease.
  • the essential feature of seasonal depression is the onset and remission of major depressive episodes at characteristic times of the year In most cases, the episodes begin in fall or winter and remit in spring Major depressive episodes that occur in a seasonal pattern are often characterized by prominent anergy, hypersomnia, overeating, weight gain, and a craving for carbohydrates and must persist for a period of at least two weeks during which there is either depressed mood or the loss of interest or pleasure in nearly all activities
  • post-traumatic stress disorder The essential feature of post-traumatic stress disorder is the development of characteristic symptoms following exposure to an extreme traumatic stressor involving direct personal experience of an event that involves actual or threatened death or serious injury to one's own or another's physical integrity
  • the person's response to the event must involve intense fear, helplessness, or horror
  • the traumatic event is reexperienced as intrusive recollections or nightmares which trigger intense psychological distress or physiological reactivity
  • the full symptom picture must be present for more than one month and cause clinically significant distress or impairment in social or occupational functioning.
  • the essential feature of nicotine withdrawal is the presence of a characteristic withdrawal syndrome that develops after the abrupt cessation of, or reduction in, the use of nicotine-containing products following a prolonged period (at least several weeks) of daily use
  • Diagnosis of nicotine withdrawal requires identification of four or more of the following dysphoric or depressed mood, insomnia, irritability or anger, anxiety, difficulty concentrating, restlessness or impatience, decreased heart rate and increased appetite or weight gain These symptoms must cause clinically significant distress or impairment in social, occupational functioning.
  • Improvement constitutes either (a) a statistically significant change in the symptomatic condition of a treated individual as compared to a baseline or pretreatment condition on measures pertinent to the disease model, or (b) a statistically significant difference in the symptomatic condition of ligand-inhibitor treated patients and members of a placebo group.
  • Clinical instances of disease exhibit symptoms which are, by definition, distinguishable from normal controls. For depression, several rating scales of depression are used.
  • the present invention provides methods for treating Alzheimer's disease through the administration to a patient of a therapeutically effective amount of a ligand inhibitor of a CRF/CRF-BP complex.
  • Such patients may be identified through clinical diagnosis based on symptoms of dementia or learning and memory loss which are not attributable to other causes
  • patients are also identified through diagnosis of brain atrophy as determined by magnetic resonance imaging.
  • CRF CRF-BP assay.
  • Formalin-fixed samples of the cerebral cortex and hippocampus were embedded in paraffin and subsequently sectioned and stained with hematoxylin/eosin and silver impregnation. Examination of stained sections from brains of AD patients showed abundant neuritic plaques and neurofibrillary tangles typical of AD, whereas control cases showed none.
  • CRF-BP has previously been identified and characterized in rat brain, sheep brain, and human plasma In the cerebral cortex of brains studied here, the majority (>85%) of CRF-BP was membrane associated Pharmacological characteristics of CRF-BP solubilized from human brain membranes from either controls or AD patients showed no differences in binding characteristics to CRF and ligand inhibitors when compared to a recombinant form of the soluble plasma CRF-BP.
  • CRF-BP ligand inhibitors increase the free concentration of CRF.
  • treatment with a CRF-BP ligand inhibitor raised free CRF levels in AD brains to the level found in normal brains
  • AD Alzheimer's disease
  • An animal model of Alzheimer's disease which focuses on cholinergic deficits is produced by the administration of scopolamine, a non-selective postsynaptic muscarinic receptor antagonist that blocks the stimulation of postsynaptic receptors by acetylcholine.
  • scopolamine a non-selective postsynaptic muscarinic receptor antagonist that blocks the stimulation of postsynaptic receptors by acetylcholine.
  • memory deficits are readily apparent as measured by passive avoidance or delayed-matching-to-position tests, which distinguish motor or perceptual deficits from amnesia or cognitive enhancing effects of experimental treatments.
  • the Morris maze and Y-maze tests following scopolamine-induced amnesia are utilized to test memory impairment and subsequent enhancement following administration of ligand inhibitor.
  • the design of the experiment is essentially as described above, but is modified to include treatment 30 min prior to training on each of days 1 to 3 with an ip injection of scopolamine hydrobromide (0.3 mg/kg).
  • This amnestic dose of scopolamine impairs acquisition and retention of spatial and avoidance learning paradigms in the rat
  • the anti-amnestic effects of 1, 5, or 25 ⁇ g of a peptide ligand inhibitor, or appropriate doses of a non-peptide ligand inhibitor, are measured relative to the concurrent control groups who receive or do not receive scopolamine.
  • the effect of the ligand inhibitors on reversal of scopolamine- induced amnesia using the Y-maze is performed similarly to the Y-maze test described above. Modification of this test includes treatment 30 min prior to training on days 5 to 10 with an ip injection of scopolamine hydrobromide (0 3 mg/kg)
  • the anti-amnestic effects of 1, 5, or 25 ⁇ g of a peptide ligand inhibitor administered ICV or equivalent doses of a non-peptide ligand inhibitor are administered centrally or systemically, are measured relative to concurrent control and scopolamine treated-control groups.
  • This test may be used to determine improvement following therapeutic treatment with ligand inhibition
  • "improvement" in Alzheimer's disease is present within the context of the present invention if there is a statistically significant difference in the direction of normality in the Weschler Memory Scale test, for example, between the performance of ligand-inhibitor treated patients as compared to members of the placebo group or between subsequent tests given to the same patient.
  • scopolamine-induced amnesia in humans can be used as a model system to test the efficacy of the ligand inhibitors
  • the terms "pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably.
  • the materials are capable of administration to a mammal without the production of undesirable physiological effects, such as nausea, dizziness, gastric upset and the like.
  • a ligand inhibitor of a CRF/CRF-BP complex is administered to a patient in a therapeutically effective amount.
  • a therapeutically effective amount is an amount calculated to achieve the desired effect, either increasing the level of free CRF in the brain, improving learning and memory, decreasing food intake, activating CRF neurocircuitry in the brain, treating diseases associated with low levels of CRF in the brain, treating the symptoms associated with Alzheimer's disease, treating obesity, treating atypical depression, treating substance abuse withdrawal, treating post-partum depression, or age-related memory loss.
  • the route of administration may vary with the particular treatment and also with whether a peptide or non-peptide ligand inhibitor is administered Routes of administration may be either non-invasive or invasive.
  • Non-invasive routes of administration include oral, buccal/sublingual, rectal, nasal, topical (including transdermal and ophthalmic), vaginal, intravesical, and pulmonary.
  • Invasive routes of administration include ICV, intraarterial, intravenous, intradermal, intramuscular, subcutaneous, intraperitoneal, intrathecal and intraocular.
  • Intracerebroventricular (ICV) injections are performed on animals as follows. Animals are anesthetized with halothane and secured in a KOPF stereotaxic instrument. A guide cannula aimed above the lateral ventricle is implanted and anchored to the skull with two stainless steel screws and dental cement. For injections, a 30 gauge stainless steel cannula attached to 60 cm of PE 10 tubing is inserted through the guide to 1 mm beyond its tip. Two microliters of ligand inhibitor are injected by gravity flow over a one minute period simply by raising the tubing above the head of the animal until flow begins. Procedures for the other routes of administration are well known in the art. The required dosage may vary with the particular treatment and route of administration.
  • dosages for peptide ligand inhibitors are given to achieve an end concentration approximately 50 to 125 ⁇ g per 1.5 g of brain tissue or 15 to 38 n oles per 1.5 g of tissue.
  • Treatments may need to be continuous for retention of therapeutic benefit
  • Patients are monitored by assessing CRF levels in cerebral spinal fluid or in the brain by imaging as described above
  • patients are monitored by assessing performance under various tests as described for each of the treatments.
  • Therapeutic administration is performed under the guidance of a physician, and pharmaceutical compositions contain the ligand inhibitor in a pharmaceutically acceptable carrier.
  • Such carriers are well known in the art and typically contain non-toxic salts and buffers
  • Such carriers may comprise buffers like physiologically-buffered saline, phosphate-buffered saline, carbohydrates such as glucose, mannose, sucrose, mannitol or dextrans, amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants and preservatives
  • Acceptable nontoxic salts include acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application)
  • acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like If the active ingredient is to be administered in tablet
  • the peptides being administered under the guidance of a physician will usually be in the form of pharmaceutical composition that contains the ligand inhibitor and a conventional, pharmaceutically-acceptable carrier Usually, the dosage will be from about 1 to about 1000 micrograms of the peptide per kilogram of the body weight of the host animal per day, frequently it will be between about 100 ⁇ g and about 1 mg but may vary up to about 10 mg
  • Treatment of subjects with these peptides can be carried out to alleviate symptoms and correct detrimental manifestations which are characteristic of the relatively low ambient CRF levels which occurs in Alzheimer's disease patients and chronic fatigue syndrome patients In the former case, treatment improves short and medium term memory
  • Treatment of subjects with these ligand inhibitors can also be carried out to boost the effective
  • CRF peptide When the agent is administered along with CRF or a CRF agonist, such CRF peptide may be administered as a daily dosage of from about 1 to about 200 micrograms of the CRF peptide per kilogram of body weight of the host animal
  • suitable CRF agonists include those described in U S Patents Nos 4,415,558, 4,489, 163, 4,594,329, 5, 1 12,809, 5,235,036 and 5,278, 146, the disclosures of which are incorporated herein by reference
  • the CRF antagonist When the agent is administered along with a CRF antagonist, the CRF antagonist may be administered in an amount between about 0 01 to about 10 mg of the peptide per kilogram of body weight of the host animal.
  • Preferred CRF antagonists include [D-Phe 12 , Nle 21 ,8 ]-hCRF( 12-41 ) and [D-Phe 12 , Nle 21 - 38 , CML 37 ]-hCRF( 12-41 )
  • Preferred CRF agonists include [His 20 , Nle 21 , Leu 38 ]- hCRF, [D-Phe 12 , Nle 21 - 8 , Leu 6 ]-hCRF, [D-Pro 4 , D-Phe 12 , Asp 25 , Nle 21 «]-hCRF and [D-Pro 4 , D-Phe 12 , Nle 21 - 38 , CML 37 ]-hCRF
  • Ligand - immunoradiometric assay to assay for ability of ligand inhibitor displacement of CRF from CRF-BP
  • the assay is performed in 600 ⁇ l polypropylene microfuge tubes or a 96-well plate.
  • 50 ⁇ l of a 250 ng/ml of purified recombinant CRF-BP is added to 150 ⁇ l of PBS binding buffer (50 mM sodium phosphate, 0.15 M NaCl, and 0.02% NP-40).
  • PBS binding buffer 50 mM sodium phosphate, 0.15 M NaCl, and 0.02% NP-40
  • 125 I-h/r CRF at a final concentration of 200 pM and 50 ⁇ l of a 10-100 ⁇ M concentration of the ligand inhibitor are added and incubated for 1 hr at room temperature.
  • the antibody-bound I25 I-CRF precipitate is then collected by centrifugation (3000 x g) at 4°C for 20 min in a Beckman GS-15R centrifuge Using a Beckman Biomer 1000 robotic workstation, the tubes are aspirated and washed once with 600 ⁇ l of PBS plus 0 02% NP-40 Tubes containing the pellets are then transferred to 12 x 74 mm counting tubes and counted in a gamma counter
  • K, Inhibitory binding affinity constant
  • Example 2 Detergent phase separation to assay for the ability of ligand inhibitor displacement of CRF from CRF-BP
  • Recombinant human CRF-BP at 200 ng/ml is incubated in binding buffer (phosphate buffered saline, pH 7 4/0 02% NP-40) with radiolabeled I 25 I-h/r CRF (80 pM) for 2 hr at room temperature Following incubation, bound and free CRF are separated by the addition of a 1 10 dilution of Triton X-l I 4 1 in assay buffer octylphenoxypolyethoxyethanol (SIGMA) Triton X- 1 14 I is insoluble in water at room temperature and in aqueous solution can be separated into a detergent phase After addition of the Triton X-l 14TM, the tube is vortexed and immediately centrifuged at room temperature at 12,000 x g for 5 min The detergent phase is found at the bottom of the tube while the aqueous phase remains at the top CRF, which as amphiphilic alpha helices, segregates to the detergent phase However, when CRF is bound
  • the anterior pituitary glands are washed 6 times with sterile HEPES buffer and transferred to 20 ml of collagenase solution (4 mg/ml) The pituitaries in collagenase are then transferred to a 25 ml Bellco dispersion flask and stirred for 30 min at 37°C After 30 min, the pituitary cell suspension is then triturated by drawing the pituitaries through a 10 ml pipette and incubated for 30 min more before more trituration.
  • the cell suspension is incubated for a further 45 min and the partially dispersed cells are transferred to a sterile 50 ml tube and centrifuged at 4000 rpm for 4 min.
  • the cell pellet is reconstituted in 10 ml of neuraminidase (8 ⁇ g/ml) and vortexed
  • the suspension is placed in a water bath for 9 min, vortexed again for 4 min and centrifuged again
  • the supernatant is poured off and the cell pellet is reconstituted in 25 ml of BBM-P (250 ml BBM-T plus 5 ml of 2% fetal calf serum) by vortexing
  • the cells are collected by centrifugation, and finally the suspension is reconstituted in 22 ml of BBM-P
  • the cells are then plated at a density of 50- 100,000/well in a 48 well plate and incubated in a humidified CO2 chamber for 2 days On the day of assay, the cells are washed once with
  • the cells are stimulated with a maximally stimulating dose of h/rCRF (1 nM) in the presence and absence of a blocking concentration of CRF-BP (5 nM)
  • This concentration of CRF-BP reduces the amount of ACTH released from the pituitary cells by binding to h/rCRF The reduction is expressed as a fraction of the amount of ACTH released by 1 nM CRF in the absence of CRF-BP
  • the CRF-BP (5 nM) which is bound to h/rCRF ( 1 nM), is incubated with a range of concentrations of ligand inhibitors (e.g., typical concentrations for the CRF-BP ligand h/rCRF (6-33) range from 0 1-1000 nM)
  • the ligand inhibitor binds to CRF-BP and displaces CRF from the complex resulting in a dose-dependent reversal of the inhibition of h/rCRF induced-ACTH secretion by CRF-BP
  • the standard assay mixture contains 2 mM L-glutamine, 20 mM HEPES, 1 mM IBMX
  • the lysate is transferred into 1 5 ml Eppendorf tubes, the wells are washed with an additional 200 ⁇ l of EtOH/HCl, and the wash is pooled with the lysate
  • the lysates are lyophylized and resuspended in 500 ⁇ l of sodium acetate buffer, pH 6 2 cAMP is measured with a single antibody kit from Biomedical Technologies Inc (Stoughton,
  • ELISA plates were coated for 2 hr at 37°C with protein G-purified sheep anti-CRF antibody (20 ⁇ g/ml) diluted in 50 mM sodium bicarbonate buffer, pH 9 5. Plates were washed once with TTBS and blocked with 1% casein in TBS for 1 hr at room temperature One hundred microliters of the samples or standard were added to each well and allowed to bind at room temperature Plates were washed five times with TTBS RC-70 rabbit anti-human CRF antibody (diluted 1 1000 in TTBS/1% BSA) was added Following incubation for 1 hr at room temperature, plates were washed five times with TTBS buffer and then exposed to horseradish peroxidase conjugated-goat anti-rabbit (GAR) second antibody for 1 hr at room temperature. Plates were finally washed five times with TTBS and developed by the addition of 100 ⁇ l of TMB microwell peroxidase substrate solution (Kikegaard and Perry Laboratories, Inc ) Ab
  • Bound CRF was determined by capture of the CRF/CRF-BP complex in wells which had been pre-coated with an anti-human CRF-BP monoclonal antibody (5 ⁇ g/ml of antibody in 50 mM sodium bicarbonate buffer, pH 9 5) followed by detection of the bound CRF with RC-70 anti-human CRF antibody essentially as described for the total CRF ELISA
  • Free CRF is measured in the supernatant following capture of the bound complex by the anti-CRF-BP monoclonal antibody Following binding of sample material, the supernatant is removed to a new ELISA plate coated with protein
  • Candidate ligand inhibitors may be screened for their ability to displace
  • CRF from CRF/CRF-BP complex A suitable assay, such as ACTH release from cultured pituitary cells (see Example 2) or two-site LIRMA (see Example 1), is used to measure free CRF and CRF-BP levels, respectively
  • Example 1 In the LIRMA assay, generally the procedure from Example 1 is followed.
  • the ligand inhibitor at a 10 ⁇ M concentration is added to the reaction along with the 125 h/r CRF If the candidate ligand inhibitor displaces CRF from CRF-BP, the pellets will contain less radioactivity in comparison to controls in which no candidate peptide is added
  • Candidate peptides are re-screened using a 6 point-dose curve IC-50 values are calculated
  • CRF 6-33
  • ovine CRF ovine CRF were screened in this manner against CRF/CRF-BP complex.
  • the affinities of these peptides for cloned human pituitary CRF receptor was determined in membrane preparations of stable transfectants of the receptor in Ltk" mouse fibroblast cells using a previously characterized radioligand binding assay (DeSouza, J. Neurosci. 7 88, 1987)
  • Example 4 Five cerebrocortical samples from Alzheimer's patients and five samples from age-matched, normal controls were prepared as in Example 4 and pooled Levels of total CRF, bound CRF, and free CRF were measured as described in Example 4 As can be seen in Figure 2, 40% of the total CRF was complexed to CRF-BP in brain extracts from normal individuals and 60% was complexed in brain extracts from Alzheimer's patients
  • Tissue was also incubated with 50 nM of the ligand inhibitor ⁇ -helical ovine CRF (9-41 ) The supernatant was then assayed for displaced CRF by two-site ELISA assay as described in Example 4.
  • the Morris Water Maze Test is a simple spatial learning task that requires a minimal amount of stress and experience No motivational constraints such as shock or food deprivation are necessary
  • the animal is placed in tepid water, which is opaque due to the addition of milk powder
  • the latency time to find a hidden platform is monitored.
  • the animals learn the location of the platform relative to visual cues located within the maze and the testing room; this learning is referred to as place learning.
  • This test is particularly sensitive to manipulations of the hippocampus, a critical brain area involved in spatial learning in animals and memory consolidation in humans
  • the apparatus used in this test is a pool (46 4 cm in diameter, 45 7 cm high) filled to a depth of 23 cm with opaque water (22°C-25°C)
  • the top of a weighted target platform, 10 cm in diameter, is located 1 -2 cm beneath the water surface
  • Four equal quadrants of the pool are distinguished by designs located on the inner surface.
  • the animal is placed into a designated quadrant ofthe tank and the time to approach and ascend the hidden platform is measured, the location of subject placement and platform remain constant throughout the experiment After climbing on top of the platform, the animal is allowed to rest for 20 sec Subjects that do not find the platform within 60 sec are placed onto the platform and allowed to rest for 20 sec
  • Rats were treated by ICV injection 15 min prior to testing with either the ligand inhibitor h/rCRF (6-33) or the CRF receptor agonist h/rCRF Doses of h rCRF (6-33) were 0, 1, 15, 25, 50, or 125 ⁇ g; doses of h/rCRF were 0, 0, 1 , 1 or 2 5 ⁇ g Seven to 10 rats per group were treated.
  • Statistical analysis confirmed significant improvement in performance following treatment with either h/rCRF (6-33) (p ⁇ 0 05) or CRF (p ⁇ 0.05) ( Figure 4) There was a significant improvement in performance over time as well (p ⁇ 0.0001 )
  • Example 9 Y-maze visual discrimination test
  • the Y-Maze visual discrimination test is a learning test using positive reinforcement to study learning with minimal stress to the animals Subjects are meal deprived and fed only after the training session, animals have the option of not responding, but do so in most cases because the positive reinforcing properties of the food pellets, which rats prefer to regular chow
  • the Y-maze contains three arms of equal length (61 cm long, 14 cm wide, 30 cm high) One arm is used as a start box and is separated from the other two goal arms by guillotine doors which are manually operated The vertical surface at the ends ofthe two distal arms is equipped with an eight watt electric bulb
  • rats which have been food-deprived to 80% body weight, were allowed to explore the maze for 5 min with two food pellets (45 mg Noyes) available at the end of each goal arm
  • each rat was allowed one trip down each of the goal arms which were baited with pellets
  • rats received six spaced trials in squads of three animals where one goal arm was closed at the choice point and two 45 mg pellets were available in the open goal box, but no discriminative visual stimulus was provided (light off)
  • the open arm alternated from left to right over the six trials, as well as from subject to subject
  • both goal arms were open and the light at the end of one goal arm was illuminated
  • Elevated plus-maze test predicts how animals respond to an approach-avoidance situation involving an exposed, lighted space versus a dark, enclosed area.
  • both spaces are elevated off the ground and constitute two runways intersecting in the form of a plus sign
  • This type of approach-avoidance situation is a classical test of "emotionality" and is very sensitive to treatments that produce disinhibition (such as sedative or hypnotic drugs) and stress No motivational constraints are necessary and the animal is free to remain in the dark or venture out onto the open arms
  • the elevated plus-maze apparatus has four arms (50 cm long, 10 cm wide) situated at right angles to each other and elevated from the floor (50 cm) Two of the arms are enclosed with walls (40 cm high) and two arms have no walls (open arms) Subjects were placed individually into the center of the maze and allowed free access to all four arms for a 5 minute testing period The time spent in each arm was recorded automatically by photocell beams and a computer interface Groups of 7-10 rats received ICV injections of the ligand inhibitors h/rCRF (6-33) or h/rCRF ( 1 -41 ) Rats received 0, 0 1 , 1 , or 25 ⁇ g of h/rCRF ( 1 -41 ) Doses of 1 and 25 ⁇ g of h/rCRF ( 1 - 1 ) produced statistically significant more time on the open arms, indicating increased anxiety (Figure 5) In marked contrast, memory- enhancing doses of CRF (6-33), as well as doses two- to five-fold higher (50-125 ⁇ g
  • the apparatus for both rats and mice is the same as used in passive avoidance testing It consists of a Skinner box enclosure 48 cm long by 16 cm high by 19 cm deep that contains a grid floor composed of 28 stainless steel bars, 6 3 mm in diameter and spaced 1 1 7 mm apart for a rat and 62 bars, 3 2 mm in diameter and spaced 5.2 mm apart for a mouse.
  • a 7 watt light and tone generator positioned at each end of the box serve as conditioned stimuli
  • the position of the animal is detected by breakage of an array of sixteen photobeams spaced at 3 3 cm intervals just above the grid floor.
  • a rat or mouse is placed at the end of the footshock compartment, facing away from the door On Trial 1 only, the door is left closed for 10 sec, then opened and footshock is administered simultaneously This makes the first trial an escape-only trial, ensuring that the animal does not avoid shock by entering the safe compartment through spontaneous exploration
  • the footshock continues until the animal escapes or until 20 sec (for mice) or 30 sec (for rats) have elapsed
  • the door is closed and the inter-trial interval is begun If an animal does not escape the shock before it is turned off, it is coaxed through the door into the safe compartment, then the door is closed and the inter-trial interval is begun
  • a 20 sec (for mice) or 30 sec (for rats) inter-trial interval separates the end of one trial from the beginning of the next During the inter-trial interval, the latency of the subject's response (escape or avoidance) and the number of avoidances made on all trials after the first are recorded Any response with a latency of less than 10 sec is considered an avoidance
  • the subject is placed back in the footshock compartment facing away from the door
  • the door is opened immediately after placing the subject in the footshock compartment, and the animal is allowed 10 sec to avoid footshock If an avoidance occurs, the door is shut and the inter-trial interval is immediately initiated If the subject fails to enter the safe compartment within 10 sec, it receives footshock, which continues until the animal escapes the shock or a maximum of 30 seconds have elapsed
  • the door is closed and the inter-trial interval begins If an animal does not escape the shock before it is turned off
  • the retention score is obtained by subtracting the number of avoidances made during training from those made during testing; higher difference scores are taken to reflect better retention
  • the apparatus for both rats and mice is the same as used in active avoidance testing (see example 1 1 )
  • the animal is placed individually in one compartment of the learning apparatus, which is separated from a second compartment by a guillotine door Following a three minute habituation period, the door is opened and the latency to enter the second compartment is recorded
  • the door is closed, and a 0 5 second AC footshock (Coulburn precision shocker) is delivered
  • the subject is removed and placed in its home cage
  • the animal is returned to the compartment in which it was initially placed for the learning trial, the door is opened and the latency to enter the second compartment is recorded, but the animal is not shocked
  • subjects are administered a single 0 5 second shock for the duration of the experiment
  • the apparatus control and response recording are computer automated (San Diego Instruments, Gemini Avoidance System)
  • the shock stimulator is research grade, precision-regulated equipment which is electrically isolated and overshoot limited for operator and subject safety For most strains of rats with weights between 150 and 350 grams, an effective footshock
  • Nicotine withdrawal is achieved by surgical removal of the pump after 14 days.

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Abstract

On augmente les niveaux du facteur de libération des corticotropines (CRF) ou du peptide associé au facteur CRF en administrant à un patient un inhibiteur ligand d'un complexe du facteur CRF/protéine de liaison du facteur CRF ou d'un complexe du facteur CRF-peptide associé/protéine de liaison du facteur CRF. Cet inhibiteur se fixe sur la protéine de liaison du facteur CFR, provoquant ainsi la libération du facteur CRF ou accroissant la concentration du peptide associé à ce facteur. L'administration de cet inhibiteur peut apporter une amélioration dans les domaines de l'apprentissage et de la mémoire, de la diminution de l'apport alimentaire ou dans le traitement des maladies associées à des concentrations basses du facteur CRF dans le cerveau, notamment dans la maladie d'Alzheimer. On décrit également un procédé de criblage de composés afin de choisir notamment des antagonistes efficaces du facteur CRF, en vue d'une administration in vivo de ceux-ci à des mammifères.
PCT/US1997/001572 1996-02-01 1997-01-29 Procedes d'accroissement des niveaux endogenes du facteur de liberation des corticotropines Ceased WO1997028189A1 (fr)

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JP9527840A JP2000505798A (ja) 1996-02-01 1997-01-29 コルチコトロピン放出因子の内因性レベルを上昇させる方法
EP97905690A EP0877759A1 (fr) 1996-02-01 1997-01-29 Procedes d'accroissement des niveaux endogenes du facteur de liberation des corticotropines
CA 2244864 CA2244864A1 (fr) 1996-02-01 1997-01-29 Procedes d'accroissement des niveaux endogenes du facteur de liberation des corticotropines
AU22520/97A AU2252097A (en) 1996-02-01 1997-01-29 Methods for increasing endogenous levels of corticotropin-relea sing factor

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EP3008465B1 (fr) * 2013-06-13 2019-08-07 Biodesy, Inc. Procédé de criblage d'entités biochimiques candidates ciblant une entité biochimique cible

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WO1996002569A1 (fr) * 1994-07-15 1996-02-01 Neurocrine Biosciences, Inc. Inhibiteurs de la proteine de liaison du facteur de liberation de la corticotropine

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WO1996002569A1 (fr) * 1994-07-15 1996-02-01 Neurocrine Biosciences, Inc. Inhibiteurs de la proteine de liaison du facteur de liberation de la corticotropine

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CHEMICAL ABSTRACTS, vol. 126, no. 5, 3 February 1997, Columbus, Ohio, US; abstract no. 54976, BEHAN, DP ET AL: "Neurobiology of corticotropin releasing factor ( CRF ) receptors and CRF -binding protein: implications for the treatment of CNS disorders" XP002034102 *
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HEINRICHS S C ET AL: "Corticotropin - releasing factor- binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 93 (26). 1996. 15475-15480, XP002034101 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10768174B2 (en) 2014-12-23 2020-09-08 Bluelight Therapeutics, Inc. Attachment of proteins to interfaces for use in nonlinear optical detection

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