WO1997028189A1 - Methods for increasing endogenous levels of corticotropin-releasing factor - Google Patents

Abstract

Levels of free corticotropin-releasing factor (CRF) or CRF-related peptide are increased by administration to a patient of a ligand inhibitor of a CRF/CRF-binding protein complex or CRF-related peptide/CRF-binding protein complex. The ligand inhibitor binds to CRF-binding protein, thereby causing the release of CRF or increasing the level of the CRF-related peptide. The ligand inhibitor may be a peptide derived from CRF or a related protein or a non-peptide. Administration of the ligand inhibitor may provide improvement in learning and memory, in decreasing food intake or in providing treatment for diseases associated with low levels of CRF in the brain, notably Alzheimer's disease. A method is also provided for screening compounds to select particularly effective CRF antagonists for in vivo administration to mammals.

Description

Description

METHODS FOR INCREASING ENDOGENOUS LEVELS OF CORTICOTROPIN-RELEASING FACTOR

Certain aspects of this invention were made with Government support under Grants DK-26741 and HD-13527 awarded by the National Institutes of Health The Government may have certain rights in the invention The University of Reading and the Medical Research Council of Great Britain may have certain rights to this invention

Technical Field

The present invention relates generally to methods of increasing endogenous levels of neuropeptides, and specifically to increasing corticotropin- releasing factor levels in the brain

Background ofthe Invention

Recent clinical data have implicated CRF in neuropsychiatric disorders and in neurodegenerative diseases, such as Alzheimer's disease Alzheimer's disease is a neurodegenerative brain disorder which leads to progressive memory loss and dementia By current estimates, over two million individuals in the United States suffer from this disease In particular, several lines of evidence have implicated CRF in Alzheimer's disease (AD). First, there are dramatic (greater than 50%) decreases in CRF (Bissette et al., JAMA 2543061, 1985, DeSouza et al., Brain Research 397 401 , 1986, Whitehouse et al., Neurology 37:905, 1987, DeSouza, Hospital Practice 23 59, 1988, Nemeroff et al., Regul. Peptides 25: 123, 1989) and reciprocal increases in CRF receptors (DeSouza et al., 1986; DeSouza, 1988) in cerebrocortical areas that are affected in AD, while neither CRF nor CRF receptors are quantitatively changed in non-affected areas of the cortex (DeSouza et al , 1986) Second, chemical affinity cross-linking studies indicate that the increased CRF receptor population in cerebral cortex in AD have normal biochemical properties (Grigoriadis et al , Neυropharmacology 28 761 , 1989) Additionally, observations of decreased concentrations of CRF in the cerebrospinal fluid (Mouradian et al., Neural Peptides 5.393, 1986; May et al , Neurology 37 535, 1987) are significantly correlated with the global neuropsychological impairment ratings, suggesting that greater cognitive impairment is associated with lower CRF concentrations in cerebrospinal fluid (Pomara et al , Biological Psychiatry 26 500, Available therapies for the treatment of dementia are severely limited Tacrine™, a recently approved drug, leads to only marginal memory improvement in Alzheimer's patients, and has the undesirable side effect of elevating liver enzymes

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. In cases of Parkinson's disease, CRF content is decreased and shows a staining pattern similar to cases of AD (Whitehouse et al , 1987, DeSouza, 1988) In progressive supranuclear palsy, 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. 73.1224, 1991 ) Although some depressions have a high improvement rate and many are eventually self-limiting, there are major differences in the rate at which patients recover A major goal of therapy is to decrease the intensity of symptoms and hasten the rate of recovery for this type of depression, as well as preventing relapse and recurrence Anti-depressants are typically administered, but severe side effects may result (e.g., suicidality with fluoxetine, convulsions with bupropion) (See Klerman et al in Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, R F Prien and D S Robinson (eds.), Raven Press, Ltd N Y , 1994, p 281 )

Hypoactivation ofthe stress system as manifested by low CRF levels may play a role in other disorders as well For examples, 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. 774.145, 1986), and patients undergoing withdrawal from smoking have decreased excretion of adrenaline and noradrenaline, as well as decreased amounts of cortisol in blood (West et al., Psychopharmacology 84 141, 1984, Puddy et al , Clm. Exp. Pharmacol. Physiol. 11 423, 1984) These manifestations all point to a central role for CRF in these disorders because CRF is the major regulator of the hypothalamic- pituitary-adrenal axis

Treatments for these disorders have poor efficacy For example, the most effective approach to treatment of obesity is a behavior-change program However, few participants reach goal weight and the relapse rate is high (see Hal i et al. in Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, R F Prien and D.S. Robinson (eds ), Raven Press, Ltd. N.Y , 1994, p. 547)

In view of the deficiencies in treatments for such disorders and diseases, more effective treatments are needed 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.

Summary ofthe Invention The present invention provides methods for increasing the level of free

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 Within one embodiment of the invention, 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 Within a related aspect, therapeutic compositions are provided comprising the ligand inhibitor in combination with a physiologically acceptable carrier or diluent.

Within other aspects of the invention, 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.

Within another aspect of the invention, 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 In one embodiment, the CRF-related peptide is urocortin

It has been found that certain 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 In other words 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 More specifically, 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 As a result, such peptides can be administered to prevent the clearance of endogenous CRF from paπicular regions and thereby stimulate the biological effect of CRF m vivo, and in certain instances, it may be advantageous to administer such peptides along with CRF or a CRF agonist The very nature of these agents is such that potentially undesirable side effects are minimized or totally obviated They might also be administered along with CRF antagonists to prevent the clearance of some CRF antagonists from a target region particularly if the CRF antagonist had a fairly high binding affinity to hCRF-BP, however, the effect is counteracted to some extent by the release of endogenous CRF that would otherwise be bound to CRF-BP These agents are useful for therapeutic treatment to promote parturition in pregnancy, to stimulate the respiratory system, to combat obesity, and to counteract the effects of Alzheimer's disease, and of chronic fatigue syndrome; however, for some of these indications, the agents must be administered in a manner so that they are delivered to the brain

In addition, 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.

Methods are also provided for screening for neuropeptide-binding proteins. In a related aspect, methods are provided for screening for ligand inhibitors of neuropeptide/neuropeptide-binding protein complex, and in particular the CRF/CRF-BP complex Within one embodiment, 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 In certain embodiments, the mixing is performed at a temperature above the cloud point of the nonionic detergent and octylphenoxypolyethoxyethanol is a preferred nonionic detergent

These and other aspects will become evident upon reference to the following detailed description and attached drawings

Brief Description ofthe Drawings

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)

(SEQ ID NO ), sauvagine (SEQ ID NO ), and rat urocortin (r UROCORTIN) (SEQ ID NO )

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, α-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 μg 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 The mean ± SEM number of avoidance responses were determined in 3 and 24 mo old male (Brown-Norway F344)Fι rats treated prior to acquisition training with 25 μg ICV of h/rCRF (6-33) Twenty four hr later, the groups (n = 6 - 7 per group) were tested for retention (8 massed trials) The * indicates p < 0 5 and the + indicates p < 0 5 by the student's t-test

Figure 9 is a graph depicting the effect of administration of control vehicle or 25 μg 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 μg 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

Detailed Description ofthe Invention

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter "CRF" refers to a peptide that regulates the release of adrenocorticotropin (ACTH), β-endorphin, and other pro-opiomelanocortin (POMC)- derived peptides from the pituitary In humans, rats, and other species, the amino acid sequence of CRF has been determined and is presented in Figure 1 (SEQ ID NO 1) The amino acid sequences of rat and human CRFs are identical and the protein is referred to as "h/rCRF " "Free CRF" refers to CRF which is not complexed or bound to CRF-binding protein or CRF receptors

"CRF-binding protein" (CRF-BP) 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" (hCRF-BP) 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. 08/485,984, hereby incorporated by reference Labeled r/hCRF is added (e.g., ¹²⁵1- 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 μM 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 peptide that demonstrates 50% maximal cAMP stimulation (as determined by stimulation with h/rCRF) at 1 μM or less is considered to be a CRF-related peptide

Ligand inhibitor of CRF/CRF-binding protein

As noted above, 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. In addition, 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 based on physical characteristics of the CRF/CRF-BP binding complex, or other ligand inhibitors Ligand inhibitors may also be metabolites of administered compounds. The ligand inhibitors must be accessible to the brain, either administered through the CNS or systemically Preferably, the characteristics of the ligand inhibitor are such that it is a low affinity antagonist at the CRF receptor (Kj > 1 μM) 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 somewhat counteracted by the release of endogenous CRF Examples of CRF-related proteins include urotensin and sauvagine Preferred peptides are those derived from h rCRF In this regard, many different peptides may be used within the context of the subject invention to displace endogenously bound CRF Such peptides include α-helical oCRF (ovine CRF) (9-41 ) (numbers in parentheses refer to amino acids), h rCRF (6-33), h/rCRF (9-33), urotensin 1, sauvagine, and h/rCRF Preferred peptides are h/rCRF (6-33), h rCRF (9-33), and h/rCRF (l-41 )OH, as these peptides bind CRF-BP but do not bind CRF receptors with appreciable affinity The free acid (OH) at the C-terminal end is particularly preferred, rather than the amidation which is found in native CRF Deamidation of the C-terminus amino acid of h rCRF does not affect the binding affinity to CRF-BP, but vastly reduces the affinity of h/rCRF for the CRF receptor The N-terminus of these agents can be protected against degradation by acylation, for example with acetyl (Ac), and such modified peptides are considered equivalents The minimal sequence of CRF which has been found to bind to CRF-BP without significant interaction with the CRF receptor are amino acids 9-33 In particular, amino acid residues 22-25 appear to play a critical role in binding to CRF-BP Other peptides may be designed based on homology to the series of peptides described above As noted above, when designing peptides, it is preferred that the C-terminus amino acid contains a free acid group Figure 1 presents a comparison of the amino acid sequence of known CRFs and CRF-related molecules As mentioned above, residues 9-33 contain the minimal sequence needed to bind to CRF-BP, and residues 22, 23, and 25 are believed to play a critical role in binding A preferred guideline in designing peptides is that the amino acid residue corresponding to residue 22 is alanine, to residue 23 is a basic amino acid (arginine or lysine), and to residue 25 is glutamic acid

Examples of suitable peptides include fragments of human CRF, i.e., hCRF( 1-41 ), which has the sequence

Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-LeuLeu-Arg- Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg- Lys-Leu-Met-Glu-Ile-Ile (SEQ ID NO 1 )

One preferred fragment which is employed is 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 [Nle²¹]-hCRF (6-33), [Nle ^l]-hCRF (9-33), [Ile²⁴]- hCRF (6-33), [Asn²⁶] -hCRF (6-33), [Nle^{18 21}]-hCRF (6-33), [Ile²⁷]-hCRF (6-33), [Val²⁸]-hCRF (6-33), [Asn²⁹ ]-hCRF (6-33), [Lys^l6]-hCRF(6-33), [Asp^{l 7}]-hCRF (6- 33), [Leu^, ]-hCRF (6 - 33), [Arg¹³]-hCRF (6-33), [Glu⁹]-hCRF (6-33) , [VaP'J-hCRF (6-33), [Thr³³]-hCRF (6-33), [Arg³²]-hCRF (6-33), [U e^{l 0}]-hCRF (6-33) and [Ile¹⁴]- CRF (6-33).

Other peptides which may be employed for this purpose are defined by the following sequence (SEQ ID NO.2). Xaa ₄-Xaa₅,-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa_I0- Xaa, _rXaa₁₂-Xaa₁ -Xaa₁₄-Xaa|₅-Xaa_l6-Xaa|₇-Xaa|₈-Xaa₁₉-Xaa₂₀-Xaa_2]-Ala-Xaa₂₃- Xaa₂₄-Glu-Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa_{3 |}-Xaa,₂-Xaa₃₃ or a biologically active fragment thereof which is formed by the deletion of from 1 to 8 residues in sequence from the N-terminus, or from 1 to 5 residues in sequence from the C-terminus, or both, wherein Xaa₂₃ is Arg or Lys, Xaa₂ is Ala, Ile, Asn, Met, Nle or Leu, and each remaining Xaa represents the residue present in the respective position in human CRF (1-41) or another naturally occurring amino acid, preferably a conservative substitute for the naturally occurring residue

Another group of preferred peptides are defined by the following sequence (SEQ ID NO.2) Xaa -Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa,₀-Xaa₁ ,-Xaa,₂- Xaa,₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa^] _{] 7}-Xaa₁₈-Xaa_{] 9}-Xaa₂o-Xaa₂i-AJa-Xaa₂₃-Xaa₂₄-Glu- Xaa₂₆-Xaa₂₇-Xaa₂g-Xaa₂₉-Xaa ₀-Xaa,|-Xaa₃₂-Xaa₃₃ and biologically active fragments thereof which are formed by the deletion of from 1 to 8 residues in sequence from the N-terminus, or from 1 to 5 residues in sequence from the C-terminus, or both, wherein Xaa₆ is Ile, Met, Leu or Nle; Xaa₈ is Leu or Ile; Xaa₁ is Leu, Met or Nle, Xaa₁₇ is Glu or Asn; Xaa₁₈ is Val, Met, Leu or Nle, Xaa,₉ is Leu or Ile, Xaa_2ϋ is Glu or His, Xaa₂, is Met, Leu, Nle or Arg, Xaa₂₃ is Arg or Lys, Xaa₂₄ is Ala, Ile, Asn, Met, Nle or Leu, Xaa₂₆ is Gin, Asn or Gly, Xaa₂₇ is Leu, Glu or Gin, Xaa₂₈, is Ala or Arg, Xaa₂₉ is Gin or Glu, Xaa₃₂ is His, Glu or Leu, Xaa₃^ is Ser, Leu or Ile, and each remaining Xaa represents the residue present in the respective position in human CRF ( 1 -41 ) or another natural amino acid which is preferably a conservative substitution therefor From 2 to 5 residues are preferably deleted from the N-terminus Most preferably, Xaa₇ is Ser, Xaa_<) is Asp, Xaa₁₀ is Leu, Xaa_π is Thr, Xaa₁₂ is Phe, Xaa_n is His, Xaa,₅ is Leu, Xaa₁₆ is Arg, Xaa₃₀, is Gin and Xaa_3] is Ala Another group of preferred peptides are defined by the following sequence (SEQ ID NO:3) Pro-Pro-Ile-Ser-Xaa₈-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg- Xaa₁₇-Xaa,₈-Xaa_|9-Glu-Xaa₂,-AJa-Arg-Xaa₂ -Glu-Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉-Gln-Ala- Xaa₃₂-Xaa₃₃ or a biologically active fragment thereof which is formed by the deletion of from 1 to 8 residues in sequence from the N-terminus, or from 1 to 5 residues in sequence from the C-terminus, or both, wherein Xaa₈ is Leu or Ile, Xaa,₇ is Glu or Asn, Xaa_Ig is Val, Met, Leu or Nle, Xaa_I9 is Leu or Ile, Xaa₂, is Met, Leu or Nle, Xaa₂₄ is Ala, Ile or Asn; Xaa₂₆ is Gin or Asn, Xaa₂₇ is Leu, Glu or Gi n, Xaa₂₈ is Ala or Arg, Xaa₂₉ is Gin or Glu; Xaa₃₂ is His or Glu, and Xaa₃₃ is Ser or Leu

Of the latter group, particularly preferred peptides, other than the aforementioned fragments of hCRF, include the following analogues [He⁸- ¹⁹ -²⁴, Asn¹⁷- ²⁶, Met¹⁸,, Glu²⁷- ²⁹, Arg²⁸, Gly³², Leu³ ]-hCRF(6-33) [Asn¹⁷- ²⁶, Nle¹⁸, He¹⁹- ²⁴, Glu²⁷- ²⁹, Arg²⁸, Gly³², Leu³³]-hCRF(9-33) [He⁸- ^l9- ²⁴, Asn¹⁷- ²⁶, Met¹⁸, Glu²⁷, Arg²⁸]-hCRF(4-28) [He¹⁹- ²⁴, Asn¹⁷- ²⁶, Nle¹⁸, Glu²⁷, Arg²⁸]-hCRF (7-31 ) [He⁸- ¹⁹, Asn¹⁷- ²⁴- ²⁶, Met¹⁸, GLN²⁷, Arg²⁸, Glu²⁹, Gly³², Leu³³]-hCRF (6-33) [Asn'⁷- ²⁴- ²⁶, Met¹⁸, He¹⁹, Gin²⁷, Arg²⁸, Glu²⁹, Gly³², Leu³ ]-hCRF (9-33) [He⁸- ¹⁹, Asn¹⁷- ²⁴- ²⁶, Met¹⁸, Gin²⁷, Arg²⁸]-hCRF (8-28) [He⁸- ¹⁹, Asn¹⁷- ²⁴- ²⁶, Nle¹⁸, Gin²⁷, Arg²⁸, Glu²⁹, Gly³²]-hCRF (8-32) [He⁸- ¹⁹, Asn¹⁷- ²⁴- ²⁶, Nle¹⁸, Gin²⁷, Arg²⁸, Glu²⁹, Gly³²]-HCRF (8-32) [Mef⁵- ¹⁴- ¹⁸-²⁴, lie⁸- ¹⁹-³³, Asn¹⁷, His²⁰, Arg²¹-²⁸, Lys²³, Gly²⁶, Glu²⁷-²⁹, Leu^,2]-hCRF (6-33) [De⁸- ¹⁹-³³, Met¹⁴- ¹⁸-²⁴ Asn¹⁷, His²⁰, Arg²¹-²⁸, Lys²³, Gly²⁶, Glu²⁷-²⁹, Leu³²]-hCRF (9-33) rNigό. 14. i8,2 _{5 De}8. i9.3 ₅ A^Π HJ^ Arg²¹-²⁸ _, Lys²³, Gly²⁶, Glu²⁷-²⁹, Leu ²]-hCRF (6-33) [He⁸- ¹⁹, Nle¹⁴- ¹⁸- ²⁴, Asn¹⁷, His²⁰, Arg²¹- ⁸, Lys²³, Gly²⁶, Glu²⁷- ²⁹]-hCRF (8-30)

Peptide nomenclature may be found in Schroder and Lubke, "The Peptides," Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right The standard 3-letter abbreviations are used to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g., Ser = L-serine, Orn = L- ornithine, Nle = L-norleucine, Nva = L-norvaline, Har = L-homoarginine and CML = L- C^αMeLeu The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution addition Chemical syntheses of peptides employ protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups to prevent a chemical reaction from occurring at that site until the group is ultimately removed Most methods protect an alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at that location. Examples of such syntheses of representative peptides are provided in U.S. Patent No. 5,235,036, issued August 10, 1993, the disclosure of which is incorporated herein by reference.

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. Briefly, tertbutoxycarbonyl (Boc) is used for α-amino protection, and TFA- CH₂C1₂(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) The protected peptide resin is cleaved using anhydrous hydrofluoric acid (HF) in the presence of 3% methyl sulfide, with the HF subsequently being removed in vacuo 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 substituted with an amino acid residue in the positions mentioned hereinbefore. 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. General examples of 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 The phrase "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 more of the residues Xaa₆-Xaa₂, and Xaa₂₆-Xaa₃₃, examples of preferred conservative substitutions are set forth in Table 1.

TABLE 1

"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 For example, 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, and ornithine may be substituted for lysine

In addition, 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 First, 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. 55 984, 1989) In such methods, 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. An alternative system, which presents peptides on the surface of bacteriophage, is a preferred method 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. USA 87 6378, 1990, Markland et al., Gene 109 13, 1991 , U S Patent No 5,223,409) With both phage display and bacterial display methods, there is a physical link between the peptide and the DNA encoding peptide which allows for easy isolation and propagation of the DNA sequence Third, peptides may be attached to plasmids (Cull et al , Proc. Natl. Acad. Sci. USA 59: 1865, 1992) by fusing the peptide to the DNA binding protein Lad at its C-terminus The fusion protein then binds to a Lac operator on the plasmid, such that the peptides become specifically and stably associated with the DNA sequence encoding them Fourth, 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. 1233, 1994), the genetic material encoding the peptide may be recovered In all these methods, libraries of variants based on CRF as a lead peptide may be synthesized by controlling the proportional level of "incorrect" bases which are inserted In addition to biological methods, approaches based on combinatorial chemical technologies may be utilized to generate random peptides A variety of methods have been developed to synthesize multiple, homogeneous peptides which are available for assay. These methods include the multi-pin synthesis (Geysen et al , Proc. Natl. Acad. Sci. USA 57:3998, 1984; Valerio, Anal. Biochem. 797: 168, 1991 , Bray et al., Tetrahedron Lett. 32:6163, 1991 ) and the "tea bag" method (Houghten et al , Proc. Natl. Acad. Sci. USA 52 5131 , 1985, Houghten et a!., Int. J. Pept. Protein Res. 27:673, 1986) Multiple, degenerate peptides may also be synthesized for use. Simultaneous coupling of mixtures of amino acids to a single resin support is a common strategy for synthesizing multiple peptides More recently, combinatorial libraries of soluble peptides (Houghten et al , Nature 354 84, 1991 , Houghten et al., Biotechmqiies 73.412, 1992) and peptides tethered to solid supports (Lam et al., Nature 354 82, 1991) have been synthesized by a "split synthesis" method Many variations on these syntheses have been developed (see Gallop et al., supra, for review), including synthesis of peptides on beads that contain identifiers for the peptide structure (PCT WO 93/06121 ). One such identifier is an oligonucleotide sequence

(Needels et al., Proc. Natl. Acad. Sci. USA 90: 10700, 1993) Other syntheses of random peptides are well known and may be readily performed by one skilled in the art

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. As previously mentioned, key contact residues in CRF for binding to CRF-BP are residues 22, 23, and 25 Thus, 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 μg/L, cortisol, 1 μg/L insulin, 0 1 μg/L EGF₂, 0 4 μg/L T-,, 0 7 μg/L PTH, 10 μg/L glucagon, and 2% fetal bovine serum), centrifuged again, and the resultant pellet is finally reconstituted in BBM-P The cells are then plated at a density of 50,000- 100,000/well in a 48 well plate and incubated for 2 days On the day of assay the cells are washed once with BBM-T in preparation for stimulation with the peptide candidates or CRF Cells are stimulated with a maximally stimulating dose of h/rCRF (1 nM) and ACTH release is measured by RIA or immunoradiometric assay When a blocking concentration of CRF-BP is added, the amount of ACTH that is released is reduced and expressed as a fraction of the maximal release The ligand inhibitor is added at various doses. The potency ofthe peptide in displacing CRF from CRF-BP is measured by the amount of ACTH release expressed as a fraction of the maximal release caused by CRF given alone

A preferred mode of screening candidate ligand inhibitors is by an vitro ligand immunoradiometric assay (LIRMA) For LIRMA, 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. Louis, MO) selection and maintained in Dulbecco's Modified Eagle Medium supplemented with 2 mM L-glutamine and 3% fetal bovine serum In order to scale up production of 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). 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

Active fractions from the secondary purification are then subjected to reversed-phase HPLC 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, and buffer B is 0.1% TF A/80% acetonitrile. Sequential 1 ml injections of affinity-pure, sized hCRF-BP are applied to a semipreparative C4 HPLC column (Vydac, Hesperia, CA) under isocratic conditions with a flow rate of 2.5 ml/minute in 25% B buffer After the passage of the final salt peak, a single gradient elution is performed starting at 25% B buffer and increasing to 95% B buffer over 30 min. The predominant absorbance peak is then quantitated by hCRF-BP IRMA and by amino acid analysis

In 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) ¹²⁵I-h/rCRF (New England Nuclear) and the candidate ligand inhibitor at 10 μM 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. Sci USA 89 4192-4196, 1992) is added to each tube, and after further incubation, bound complexes are precipitated by the further addition of a goat anti-rabbit antibody The precipitate containing ¹²⁵I-CRF is collected by centrifugation and the amount of radioactivity in the pellet is determined by a gamma counter 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 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 μM of the CRF-BP peptide ligand h/rCRF (6-33) Thus, the binding potency of the candidate ligand inhibitor will be measured relative to the potency of the standard h/rCRF (6-33) Preferably, there is at least 50% inhibition when ligand inhibitor is present The inhibitory binding affinity constant (K,) is important, it is viewed in proper perspective as per its value relative to the K, for human CRF which, from this assay, is found to be 0 17 + 0 01 nanomolar (nM) Thus, a ligand having a K, of less than that of hCRF will bind more strongly to hCRF-BP than will hCRF itself, and a ligand having a higher value will have a relatively lower binding affinity Therefore, because the desire is to compete reasonably effectively with hCRF for binding with hCRF-BP, the lower K, value the agent or peptide has, the more valuable it will be for this purpose Preferably, the agent will have a K, value of about 20 nM or less, more preferably a K, value of about 10 nM or less, and most preferably, a K, value of less than about 5 nM hCRF (6-33) has been assayed and found to have a K, value of 3 5 + 0 44 nM Because this agent also has a low binding affinity for the human CRF receptor, / e , an inhibitory binding constant of greater than 1000 nM, and exhibits a CRF agonism of less than about 0 1% of oCRF, it is considered an excellent choice for employment in the method of the present invention Human CRF (9-33) has a K, of 1 1 + 0 36 nM, and it also has a receptor K, of greater than 1000 nM and is an even weaker CRF agonist, rendering it also very useful in the method of the present invention Other similar agents and peptides, particularly those which are analogues of hCRF having between 19 and 28 residues may be synthesized and tested in this straightforward manner to determine their usefulness in these valuable methods for increasing the effective concentration of endogenous hCRF vivo The peptides specifically enumerated herein are felt to be particularly valuable For example (He^{8 1924}, Asn^{17 (}\ Met¹⁸, Glu²⁷, Arg²⁸]-hCRF(4- 28) has a K, of 1 7 + 1 2 and relatively low binding affinity for the hCRF receptor Thus, a high through-put screening using the LIRMA assay as described, or other methods including ACTH release and 2-site ELISA, may be used to identify small molecules that displace CRF from the CRF/CRF-BP complex In a first round of screening, all potential candidates are assayed at a single dose of 10 μM Any compound which gives greater than 50% inhibition at 10 μM is then selected for further screening. The activity of all candidates meeting this criteria is confirmed by a second round of screening using a 6 point-dose response curve IC-50 values are calculated and those candidates with a value in the range of 10-100 μM are further examined to ensure that the candidate compound is displacing CRF from the CRF/CRF-BP complex and not interfering with antibody binding to the CRF-BP Specific displacement of CRF is verified in an assay performed as described for LIRMA, except that 0 2 nM ¹²⁵l-hCRF-BP is added in place of unlabeled CRF-BP

Ligand inhibitors may also be screened by an /// vitro assay in which bound and free CRF are separated by detergent phase separation Briefly, within one embodiment, CRF-BP isolated as described above is incubated with 1-h/rCRF and the candidate ligand inhibitor at lOμM in a binding buffer (0 02% NP-40 in 50 mM phosphate-buffered saline) Following incubation of 1 -2 hr at room temperature, a detergent, such as octylphenoxypolyethoxyethanol, sold as Triton X- l 14™, is added and mixed by vortexing Triton X-l 14™ 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 14™ is 20°C. As such, Triton X-l 14™ is preferred CRF, which has amphiphilic alpha helices, is more soluble in Triton X-l 14™ and thus partitions to the detergent phase In contrast, CRF bound to CRF-BP is more soluble in an aqueous solution Thus, a phase separation of Triton X-l 14™ and the aqueous solution will segregate bound and free CRF. 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. Maximum inhibition (i.e., 100%) of the binding of ¹²⁵I-h/rCRF to the CRF-BP is defined by the amount of radioactivity in the aqueous phase after incubation with 10 μM of the CRF-BP peptide ligand h/rCRF (6-33) Thus, the binding potency of the candidate ligand inhibitor will be measured relative to the potency of the standard h/rCRF (6-33) Preferably, there is at least 50% inhibition when ligand inhibitor is present In addition, this assay has broad application in screening for neuropeptide binding proteins in general Some neuropeptides, such as NPY, have similar physical characteristics to CRF in that they are both very hydrophobic and have alpha helices As such, NPY should be more soluble in a nonionic detergent, such as Triton X-l 14™, than in aqueous solutions. Given that NPY or other neuropeptides of interest will generally partition into the Triton X-l 14™ 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 μl 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 14™ separation is decreased Using this method, an IC-50 value can be established for each neuropeptide and tissue extract

In addition, 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 μM 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 HCI, 95% ethanol. The lysate is lyophilized and subsequently solubilized in a sodium acetate buffer. The levels of cAMP are measured using a single antibody kit, such as the one from Biomedical Technologies (Stoughton, MA).

As an alternative to carrying out the foregoing competitive //; vitro evaluation assays, 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. USA 90 8967-8971, 1993, the disclosure of which is incorporated herein by reference The agent may be evaluated with radioactively labeled [Nle²¹, Tyr³²] oCRF to compute an inhibitory binding affinity constant (K,) Preferably 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-Phe¹², Nl e²¹ ]-rCRF( 12-41 ) or a fragment of alphahelical CRF( AHC), such as AHC (9-41 )

The above-identified /// vitro assays to measure CRF agonist and antagonist activity from the standpoint of stimulation of ACTH secretion may be performed using hCRF (6-33) and hCRF (9-33). As a result of such assays, 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. The desire is that the agent which is employed will not bind strongly to CRF receptors. It is generally believed that 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 [DPhe¹², Nle²¹- ³⁸]-hCRF( 12-41 ) However, it should be understood that 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. In addition to providing methods of therapeutic treatment, the invention also provides methods for screening peptides or other agents to select more effective CRF antagonists for in vivo administration to mammals To carry out this screening procedure, 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 Based upon the evaluation of the results of these two assays, 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„ indicating that it exhibits a relatively low affinity for binding to hCRF-BP The present laboratory standard, CRF antagonist [D-Phe¹², Nle²¹ ]-hCRF( 12-41) has very good antagonist properties as measured by ACTH secretion from cultured pituitary cells, with a K_j value of about 60 ± 10 nM Its (CRF-BP)K, is 300 ± 20 nM Another good CRF antagonist, namely AHC(9-41), which is not as effective as the present standard, has an extremely low K, of 0 10 ± 0 036 nM [D-Phe¹² Nle²¹-³⁸, CML³⁷]- hCRF( 12-41) has a (CRF-BP) K, of greater than 1000 nM and an IC₅₀ of 45 ± 1 1 nM in an ACTH secretion assay performed in pituitary cell cultures, so it ranks even higher than the present laboratory standard Therefore, based upon these screening assays, the latter peptide or the laboratory standard would be the peptides of choice, compared to AHC(9-41), which would have a far greater propensity /// vivo to be complexed and cleared by hCRF-BP This screening assay thus provides a valuable tool for screening newly synthesized peptides to evaluate their overall relative worth as potential CRF antagonists for vivo treatment

Increasing the level of CRF

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 In any of these assays, 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% Within the methods described herein, 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-CRF. The plates are washed, and unbound sites on the plate are blocked with an irrelevant protein, such as casein, bovine serum albumin, or the like Prepared tissue samples and standards containing known amounts of CRF are added to wells and allowed to bind at room temperature Following binding, plates are again washed, and a different anti-CRF antibody, such as RC-70, a rabbit anti-human CRF antibody, is added RC-70 is not only from a different species, but also detects different epitopes than the sheep anti-CRF used to coat the plates After washing, 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 CRF present in the tissue sample One skilled in the art will recognize that monoclonal antibodies or antibody fragments may be used in place of the polyclonal antibodies in this assay

In a similar manner, 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 α- 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. Furthermore, 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, α-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 α-helical ovine CRF resulted in release of all bound CRF (see Figure 2 and Example 5) In addition to the described /// vitro assays that measure the amount of total, bound, and free CRF in tissues or in cerebrospinal fluid, 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., ^πC, ¹⁸F) of single photon-emitting ligands (e.g., ^,2 I-labeled ligand to CRF-BP or to CRF receptors) Free CRF levels are correlated to the amount of binding of the radiolabeled ligand An increase in free CRF levels is manifested by a decreased binding of the radiolabeled ligand to the CRF-BP and CRF receptors Within this imaging technique, an increase in free CRF levels of about 10%-30% or more would be sufficient in the context ofthe present invention. Within the context of the present invention, administration of effective amounts of ligand inhibitor of the CRF/CRF-BP complex 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. , cAMP production, ACTH release, or two-site ELISA) 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-Related Peptides

Other peptide molecules, which are distinct from CRF, bind CRF-BP For example, the human neuropeptide urocortin (Vaughan et al., Nature 378 287, 1995) has a high affinity for human CRF-BP Some non-mammalian peptides such as sauvagine and urotensin, also bind CRF-BP with high affinity Thus, CRF-BP may be a modulator of a family of CRF-related peptides For example, in vitro experiments show that rat urocortin can disrupt the CRF/CRF-BP complexes normally found in human brain tissue. Such disruption has the effect of increasing free CRF levels, thus making more CRF available for binding to its receptors The 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) Moreover, 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 Given the effects of urocortin on lowering blood pressure, such ligand inhibitors may be useful in treating hypertension. In addition, urocortin appears to significantly suppress feeding behavior in animals, and therefore, such ligand inhibitors may be useful in modulating food intake

Improving Learning and Memory

As noted above, 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 Such patients 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 These deficits in language, motor performance, object recognition and abstract thinking, respectively, must be sufficiently severe in conjunction with the memory deficit to cause impairment in occupational or social functioning and must represent a decline from a previously higher level of functioning In addition, a number of biochemical tests that correlate levels of CRF with impaired learning and memory may be utilized. For instance, the level of free CRF in the cerebrospinal fluid may be measured by ELISA or RIA Additionally, or in place ofthe assays, 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 Finally, 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 The patient's minimental status is recorded by the 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). This test involves a number of simple tasks and written questions. For instance, "paired- associate" learning ability is impaired in amnesiac patients of several types including those suffering from head trauma, Korsakoffs disease or stroke (Squire, 1987). Ten pairs of unrelated words (e.g., army-table) are read to the subject Subjects are then asked to recall the second word when given the first word of each pair The measure of memory impairment is a reduced number of paired-associate words recalled relative to a matched control group. This serves as an index of short-term, working memory of the kind that deteriorates rapidly in the early stages of dementing or amnesiac disorders

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 Thus, 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. For example, 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). In animals, several established models of learning and memory are available to examine the beneficial cognitive enhancing effects and potential anxiety- related side effects of activation of CRF-sensitive neurons. The cognitive enhancing effects are measured by the Morris maze (Stewart and Morris, in Behavioral Neuroscience, R. Saghal, Ed. (IRL Press, 1993) p. 107) the Y-maze (Brits et al., Brain Res. Bull. 6, 71 (1981)), one-way active avoidance test, and passive avoidance test; anxiety-related effects are evaluated in the elevated plus-maze. (Pellow et al., J. Neurosci. Meth. 7-7: 149, 1985.) 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. Moreover, improvement in Morris water maze performance is predictive of clinical efficacy of a compound as a cognitive enhancer For example, 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) In addition, 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 As discussed in more detail below (see Example 6), 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 μg of the ligand inhibitor peptide h/rCRF (6-33) or h rCRF, which is additionally an agonist at the CRF receptor When 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. Daily pre-test administration of h/rCRF and h/rCRF (6-33) significantly enhanced learning in the Morris water maze test (Figure 4, upper panel) Somewhat higher doses of CRF (6-33) were necessary to produce increases in learning and memory.

The Y-maze test based on visual discrimination is another assay of learning and memory in animals. In this maze, 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 In the first trial, all animals are allowed to explore the maze for five min, and food pellets are available in each arm On the second day, each animal is placed in the start box with the door closed When the door is opened, the animal is allowed to move down the arms and eat the pellets which are located in both arms On the third day, 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 On 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 effectiveness of a ligand inhibitor to improve learning and memory in the Y-maze is tested as follows Fifteen min pπor to each ofthe blocks of training trials on days 4-10, groups of animals receive ICV injections of control solutions or doses of 1, 5, or 25 μg of peptide ligand inhibitor Again, if a non-peptide ligand inhibitor is used, dosages are adjusted accordingly Control animals are expected to make 50% correct choices The measure of efficacy of treatment on memory is an increase in correct responses Daily pre-test administration of CRF (6-33) ligand inhibitor was shown to significantly increase correct responses (Figure 5)

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. Within this test, an animal is placed in a footshock compartment, an opening door to a safe compartment serves as a signal for avoidance Briefly, in this test 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 At intervals, 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 effectiveness of a ligand inhibitor to improve memory and learning in the one-way avoidance or control solution is tested as follows Animals are given the ligand inhibitor ICV 15 minutes prior to training Twenty-four hrs later, the groups are tested for retention, without further administration of ligand inhibitor The measure of efficacy is a shortened latency time to leaving the footshock compartment Pre-test administration of CRF (6-33) ligand inhibitor significantly improved performance of aged rats relative to young controls

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 effectiveness of a ligand inhibitor to improve learning and memory is tested as follows Immediately prior to training, groups of animals receive control solutions or doses of ligand inhibitor ICV. Dosages are adjusted accordingly for peptide and non-peptide ligand inhibitors Latency time for entering the footshock compartment is significantly increased in animals receiving the ligand inhibitor, CRF (6-33)

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 Thus, 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 In marked contrast, the ligand inhibitor, CRF (6-33), which has low affinity for the CRF receptor, did not alter performance in the elevated plus maze test of anxiety (Figure 4, lower panel). Moreover, there were no overt behavioral alterations such as those seen with administration of h/rCRF These data demonstrate that cognitive enhancement and anxiety effects may be separately controlled These data also demonstrate the therapeutic value of administering a ligand inhibitor of the CRF/CRF-BP complex

In humans, methods for improving learning and memory may be measured by such tests as the Wechsler Memory Scale or a pair-associate memory task 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). During the clinical interview, 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) Apart from the specific diagnosis of dementia or amnestic disorders, 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) As noted above, "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

Decreasing Food Intake

As noted above, 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 For example, administration of CRF agonists or conditions that elevate endogenous CRF levels (e.g., stress) diminish food intake (Appel et al , Endoc. 128 3237, 1991 , Krahn and Gosnell, Psychiat. Med. 7 235, 1989, McCarthy et al , Am. J. Physiol. 264 E638, 1993) Thus, administration of CRF causes significant decrease in nocturnal food intake (Gosnell et al , Peptides 7:807, 1983), lowered body weight in rats (Hotta et al , Life Sci. 48 1483, 1991 ) and increased temperature response in brown adipose tissue (LeFeuvre et al , Neuropharmacol. 26 1217, 1987) Furthermore, neuropeptide Y (NPY), which is the strongest known stimulus of food intake, can be potentiated in its effect upon co-administration of a peptide antagonist ofthe CRF receptor

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 meters²) 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. Government Printing Office, Washington, D.C, 1983) In addition, evidence that CRF is involved for a particular individual may be obtained by demonstrating decreased CRF levels in the cerebrospinal fluid or by brain imaging as described above Because the hypothalamus is a common brain area mediating the effects of CRF on food intake and endocrine parameters, alterations in pituitary hormone concentration may also reflect altered levels in hypothalamic CRF A decrease in food intake may be measured both in the delayed initiation of a meal and the reduction in the overall duration or quantity of food consumption Smith, "Satiety and the Problem of Motivation," in D W Pfaff (ed ), The Physiological 97/28189 PO7US97/01572

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Mechanisms of Motivation, Springer- Verlag, New York, pp 133-143, 1982. In addition, the selection of particular nutrients in a food choice situation serves as a supplemental measure of specific hunger (Rozin, Adv. Study Behav. 6.21 , 1976).

There are two established animal models of appetite regulation. One is a simple measurement of food intake, and the second is a measurement of diet self- selection in a cafeteria environment. In the first method, food intake is limited for 24 hr followed by two hr of access to a preweighed portion of laboratory chow in the animal's home cage. Food intake is measured at 60 and 120 min by weighing the remaining pellets. These tests may also be performed on animals that are obese due to genetic mutations and which effectively reproduce symptoms of overeating and deranged nutrient selection (Argiles, Prog. Lipid Res. 25:53, 1989; Wilding et al., Endocrinol. 732: 1939, 1993).

In the cafeteria environment, 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 μg of a peptide ligand inhibitor, or appropriate doses for a non-peptide ligand inhibitor. 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 In addition, 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. Thus, separate groups treated for 14 days with nicotine or saline (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). in the nicotine withdrawal model, animals are administered nicotine in a chronic fashion These animals show inhibition of normal weight gain and reduction of food and water intake. 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 μg dose of CRF (6-33) ICV diminished food intake in nicotine withdrawal subjects without suppressing feeding in untreated controls A genetic basis for overeating has been discovered in both mice (e.g., ob/ob) and rats (Zucker strain; fa/fa) These animals offer other models of overeating to assess the efficacy of ligand inhibitors. In particular, 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.

In humans, 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. (Drewnowski et al., Am. J. Clin. Nu/r. 46:442, 1987.) Thus, clinical manifestations of appetite regulation are readily detected using controlled experimental diets or cafeteria self-selection protocols which record intake patterns in terms of quantity, meal duration, and choice (Kissileff, Neurosci. Biobehav. Rev. 5: 129, 1984) In these tests, following a baseline determination for each individual, measurement of food intake or self-selection in the cafeteria environment are measured Improvement in the context of the treatment of obesity constitutes a weight loss or reduction in food intake exhibited by treated patients as compared to members of a placebo group. Moreover, this strategy has been successful in identifying serotonergic agonists for obesity.

Diseases Associated with Low Levels of CRF

As noted above, 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. In addition, other conditions associated with decreased CRF levels, such as atypical depression, seasonal depression, chronic fatigue syndrome, obesity, vulnerability to inflammation disease, post-traumatic stress disorder, and psychostimulant withdrawal often present a profile of hypothyroidism and decreased stress system activity which is identified characteristically by a decrease in urinary free cortisol and plasma ACTH Thus, these diseases and conditions would likely be resolved in part by restoration or potentiation of brain CRF levels (Chrousos and Gold, JAMA 267: 1244, 1992)

The hallmark of this diverse set of human disease states is dysregulation of the pituitary-adrenal axis with a presumed derangement of brain CRF. Hence, the fact that experimental alternation of CRF/pituitary-adrenal systems in laboratory animals reproduces essential features of the above syndromes, namely behavioral despair (Pepin et al., 1992), exercise fatigue (Rivest and Richard, 1990), obesity (Rothwell, 1989) and hyperarousal associated with psychostimulant withdrawal (Koob et al , 1993, Swerdlow et al., 1991 ) suggests the broad utility of pharmacotherapies designed to normalize endogenous levels of CRF

The essential feature of seasonal depression (major depressive disorder with seasonal pattern) 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

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 (nicotine-induced disorder) 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. (See Klerman et al , Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, Prien and Robinson (eds ), Raven Press, Ltd , New York, 1994) One test, the Hamilton Rating Scale for Depression, is widely used to evaluate depression and is also used to assess symptom changes in response to treatment Other tests and ratings can be found in the DSM-IV manual. For nicotine withdrawal, as well as the other disorders, tests for evaluation ofthe severity ofthe disorder can be found in the DSM-IV manual.

Alzheimer's Disease

As noted above, 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 In addition, patients are also identified through diagnosis of brain atrophy as determined by magnetic resonance imaging.

Decreased levels of CRF are shown to be implicated in Alzheimer's disease. Brains obtained post-mortem from ten individuals with AD and ten neurologically normal controls were chosen for study. Standard areas of frontal pole, parietal pole, temporal pole, and occipital pole were dissected from fresh brain, frozen in dry ice, and stored at -70°C until they were processed for CRF radioimmunoassay and 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.

Levels of CRF and CRF-BP in the cerebral cortices of Alzheimer's patients and controls were determined. 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. In brains of Alzheimer's patients, the levels of CRF in the frontal, parietal, and temporal cerebral cortex were dramatically reduced compared to normal brains, but the levels in the occipital cortex were only slightly decreased (Figure 3A) In contrast, CRF-BP levels were similar in brains of AD patients and normal controls (Figure 3B) These data provide direct evidence for the presence of CRF-BP in normal and AD brain tissue and suggest that deficits in CRF levels seen in AD may be due to decreased synthesis or increased degradation of CRF rather than neuronal loss If there is neuronal loss in AD, then CRF-BP may be preferentially localized to non-CRF neurons

The nature of the interaction of CRF with CRF-BP in human brain tissue was determined by measuring the proportions of CRF complexed to CRF-BP and in the free pool. Using assays specific for total CRF and bound CRF, approximately 40% and 60% of the total CRF were found complexed with CRF-BP in normal and Alzheimer's cerebrocortical extracts, respectively. Furthermore, CRF was bound to CRF-BP in a reversible manner because treatment of the tissue with human CRF (6-33) or α-helical oCRF (9-41 ) displaced CRF from CRF/CRF-BP complex (Figure 2) These data directly demonstrate that CRF-BP ligand inhibitors increase the free concentration of CRF. Moreover, treatment with a CRF-BP ligand inhibitor raised free CRF levels in AD brains to the level found in normal brains

Several established animal models of Alzheimer's disease which focus on cholinergic deficits are available. The primary role of cholinergic deficits in AD is well established. In AD, there are significant positive correlations between reduced choline acetyltransferase activity and reduced CRF levels in the frontal, occipital, and temporal lobes (DeSouza et al., 1986) Similarly, there are negative correlations between decreased choline acetyltransferase activity and an increased number of CRF receptors in these three cortices (Id.) In two other neurodegenerative diseases, there are highly significant correlations between CRF and choline acetyltransferase activity in Parkinson's disease, but only a slight correlation in progressive supranuclear palsy (Whitehouse et al., 1987).

In rats, anatomic and behavioral studies evidence interactions between CRF and cholinergic systems First, in some brain stem nuclei, CRF and acetylcholinesterase are co-localized, and some cholinergic neurons also contain CRF Second, CRF inhibits carbachol-induced behaviors (carbachol is a muscarinic cholinergic receptor antagonist), suggesting that CRF has effects on cholinergic systems (Crawley et al., Peptides 6:891, 1985). Treatment with another muscarinic cholinergic receptor antagonist, atropine, results in an increase in CRF receptors (DeSouza and Battaglia, Brain Res. 397:401 , 1986). Taken together, these data show that CRF and cholinergic systems interact similarly in humans and animals

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. In these animals, 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. Thus, 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. In the Morris maze, 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 μg 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 μg 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.

Several tests measuring cognitive behavior in AD have been designed. (See Gershon et al., Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, Prien and Robinson (eds ), Raven Press, Ltd., New York, 1994, p. 467.) One of these tests, BCRS, measures concentration, recent memory, past memory, orientation, and functioning and self-care. The BCRS is designed to measure only cognitive functions. This test, as well as the Weschler Memory Scale and the Alzheimer's Disease- Associated Scale, may be used to determine improvement following therapeutic treatment with ligand inhibition As noted above, "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. In addition, scopolamine-induced amnesia in humans can be used as a model system to test the efficacy of the ligand inhibitors

Administration of Ligand Inhibitor

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably. Preferably, 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. It will be apparent to one skilled in the art that 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. In general, dosages for peptide ligand inhibitors are given to achieve an end concentration approximately 50 to 125 μg per 1.5 g of brain tissue or 15 to 38 n oles per 1.5 g of tissue. Dependent, however, on the size of the protein or polypeptide, a relatively larger or smaller amount is employed These treatments are conducted two or three times a week. 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 In addition, 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. These 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) Illustrative of such 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 form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin, a disintegrating agent, such as alginic acid, and a lubricant, such as magnesium stearate If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected

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 μg 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 However, for many of these indications including suppression of appetite, it is necessary for the peptide to be delivered to the brain and preferably it is coupled with an agent capable of penetrating the blood-brain barrier Administration by iv, im, or sc injection effects an increase in cortisol level and can effect a lessening of fatigue Treatment of subjects with these ligand inhibitors can also be carried out to boost the effective biological concentration of free CRF in order to stimulate the human respiratory system by administration to reach the brain For treatment of conditions in medical emergencies, such as cardiovascular arrest and shock due to substances or pathological agents, an increase in cortisol level can be achieved by administration by iv, im or sc injection To promote parturition in pregnancy, the ligand inhibitor, optionally with additional hCRF (or an analogue thereof), is administered to cause the concentration of free hCRF in plasma to rise to at least about 250 pmole/liter or preferably to at least about 0 1 ng/ml It may also be similarly administered to 97/28189 PC17US97/01572

39

patients afflicted with AIDS who frequently have low levels of cortisol so that such a CRF-BP blocker could elevate ACTH and cortisol

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 Examples of 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 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. Suitable CRF antagonists are disclosed in U S Patents Nos 4,605,642, 5, 109,11 1 and 5,245,009, the disclosures of which are incorporated herein by reference Preferred CRF antagonists include [D-Phe¹², Nle^{21 ,8}]-hCRF( 12-41 ) and [D-Phe¹², Nle²¹- ³⁸, CML³⁷]-hCRF( 12-41 ) Preferred CRF agonists include [His²⁰, Nle²¹, Leu³⁸]- hCRF, [D-Phe¹², Nle²¹- ⁸, Leu ⁶]-hCRF, [D-Pro⁴, D-Phe¹², Asp²⁵, Nle²¹ «]-hCRF and [D-Pro⁴, D-Phe¹², Nle²¹- ³⁸, CML³⁷]-hCRF

The following examples are offered by way of illustration and not limitation

Example 1

Ligand - immunoradiometric assay (LIRMA) to assay for ability of ligand inhibitor displacement of CRF from CRF-BP

The assay is performed in 600 μl polypropylene microfuge tubes or a 96-well plate. First, 50 μl of a 250 ng/ml of purified recombinant CRF-BP is added to 150 μl of PBS binding buffer (50 mM sodium phosphate, 0.15 M NaCl, and 0.02% NP-40). Next, ¹²⁵I-h/r CRF at a final concentration of 200 pM and 50 μl of a 10-100 μ M concentration of the ligand inhibitor are added and incubated for 1 hr at room temperature. To the reaction, 50 μl of anti-CRF-BP antibody, 5144, diluted 1.1000 with assay buffer is added to each tube and allowed to bind for a further 1 hr at room temperature. The total volume in all tubes is adjusted to 300 μl with assay buffer Bound complexes are precipitated by the addition of 200 μl of preprecipitated goat anti- rabbit (GAR) second antibody at 1 :20 in 1% normal rabbit serum, 4% PEG, 50 mM sodium phosphate, 0.1% sodium azide, followed by incubation for 1 hr at room temperature. The antibody-bound ^I25I-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 μl 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

Inhibitory binding affinity constant (K,) values are determined using parameters calculated by the LIGAND computer program, Munson et al , Anal Biochem. 107 220, 1980, and a Vax/VMS computer system Errors are shown which are the standard error ofthe mean from 3 replicate binding assays

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 4¹ 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 14™, 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 to CRF-BP, the CRF/CRF-BP complex remains in the aqueous phase Thus, a 50 μl aliquot of the aqueous phase is transferred to a 12 x 74 mm plastic tube and counted The amount of radioactivity left in the supernatant is determined

Example 3 ACTH release assay for free CRF

Four rats are killed by decapitation and the anterior pituitary glands are removed 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 μg/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 BBM-T in preparation for stimulation with the various relevant analogues.

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 potency of the CRF-BP ligand is expressed as a fraction of ACTH release obtained by stimulation with 1 nM CRF alone

Example 4 cAMP production assay to measure free CRF

The assay for detection of CRF-stimulated adenylate cyclase activity is carried out as previously described (Battaglia et al , 1987) with minor modifications

The standard assay mixture contains 2 mM L-glutamine, 20 mM HEPES, 1 mM IBMX

(isobutylmethyl xanthine) in DMEM buffer In stimulation studies, cells that have been transfected with a clone encoding CRF receptor are plated in 24 well plates and incubated for 1 hr at 37°C with various concentrations of CRF-related and unrelated peptides. Following the incubation, the media is aspirated, the wells rinsed once gently with fresh media and aspirated. The amount of intracellular cAMP is determined after lysing the cells in 300 μl of a solution of 95% ethanol and 20 mM HCI at 20°C for 16-18 hr. The lysate is transferred into 1 5 ml Eppendorf tubes, the wells are washed with an additional 200 μl of EtOH/HCl, and the wash is pooled with the lysate The lysates are lyophylized and resuspended in 500 μl of sodium acetate buffer, pH 6 2 cAMP is measured with a single antibody kit from Biomedical Technologies Inc (Stoughton,

MA). For the functional assessment of CRF receptor antagonists, a single concentration of CRF or related peptides causing 80% stimulation of cAMP production is incubated along with various concentrations of competing compounds (10"¹² to 10"⁶ M) The incubation and measurement conditions for cAMP are performed as described

Example 5

Two-site ELISA to measure CRF levels

A Preparation of brain tissue samples

Autopsy samples were weighed and homogenized in 5 ml of 10% sucrose One ml of each sample was centrifuged at 10,000 x g for 10 min at room temperature and the resultant membrane pellets were reconstituted in SPEA (50 mM sodium phosphate pH 7 4, 0 I M NaCl, 25 mM EDTA, 0 1 % sodium azide, containing 0.25% bovine serum albumin (BSA) and 1% NP-40) Eight hundred microliters of cerebral cortex homogenate (in 10% sucrose) was further extracted by the addition of 200 μl of TTBS (Tris-buffered saline with 0 5% Tween -20, 1% NP-40, 1% BSA) followed by vortexing for 1 min The sample was centrifuged at 10,000 x g for 10 min at room temperature The resultant supernatant was kept for analysis of "total CRF," "bound CRF" (i.e., CRF bound to CRF-BP) and "free CRF" using a two-site CRF ELISA

B Measurement of total CRF

Briefly, ELISA plates were coated for 2 hr at 37°C with protein G-purified sheep anti-CRF antibody (20 μg/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 μl of TMB microwell peroxidase substrate solution (Kikegaard and Perry Laboratories, Inc ) Absorbance at 450 nM was determined

C Measurement of bound CRF

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 μg/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

D Measurement of free CRF.

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

G-purified sheep anti-CRF antibody The assay is then performed as described for determining total CRF levels

Example 6 Screening for ligand inhibitors

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

In the LIRMA assay, generally the procedure from Example 1 is followed. The ligand inhibitor at a 10 μM concentration is added to the reaction along with the ¹²⁵h/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 The ligand inhibitors human CRF, α-helical ovine CRF (9-41 ), human

CRF (6-33), and 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)

Results of these assays is shown in the following Table Moreover, these ligand inhibitors were also tested on CRF-BP in cerebral cortices of individuals with Alzheimer's disease and controls Table I Ligand Inhibitors of CRF/CRF-BP

IC50 Values (nM)

Peptide CRF-BP in Cerebral Cortex Recombinant Human Controls Alzheimer's CRF-BP CRF-R

Disease human CRF 0.30 0.18 0 19 1 0 α-helical ovine CRF (9-41 ) 0.16 0.04 0 20 9 9 human CRF (6-33) 1 6 1.1 1 6 >1000 ovine CRF 667 595 471 2 4

These results show that human CRF, α-helical ovine CRF (9-41) and human CRF (6-33) were nearly equally effective at displacing CRF from CRF/CRF-BP complex. In contrast, ovine CRF was not as effective in displacing CRF The CRF-BP in cerebral cortices from diseased or normal individuals was equivalent to the recombinant form. Of the three best ligand inhibitors, human CRF and α-helical ovine CRF (9-41 ) bound human CRF-R approximately equivalently In marked contrast, human CRF (6-33) binds 2-3 logs less efficiently.

Example 7 Treatment with ligand inhibitor

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

The effect of a high affinity CRF-BP ligand to displace bound CRF was assessed after monoclonal capture of the CRF/CRF-BP complex in the presence of 50 nM α-helical ovine CRF (9-41 ) Displaced free CRF was measured in the supematants remaining after CRF-BP monoclonal antibody capture by CRF ELISA As seen in Figure 2, treatment of brain tissues with the ligand inhibitor caused release of all bound CRF in both Alzheimer's and control tissues Moreover, treatment of the Alzheimer's disease cerebral cortex with the ligand inhibitor replenished the free CRF levels to the level seen in age-matched controls

Tissue was also incubated with 50 nM of the ligand inhibitor α-helical ovine CRF (9-41 ) The supernatant was then assayed for displaced CRF by two-site ELISA assay as described in Example 4.

Example 8 Morris water maze test

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 μg; doses of h/rCRF were 0, 0, 1 , 1 or 2 5 μg 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 On the first day of training, 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 On the second day, each rat was allowed one trip down each of the goal arms which were baited with pellets On the third day, 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 On days 4-10, both goal arms were open and the light at the end of one goal arm was illuminated Ten trials were run daily, again in squads of three so that the intertrial interval was about 90 sec Subjects were fed a 15 g portion of laboratory chow in the home cage daily at the conclusion of training

On days 4-10, ICV injections of ligand inhibitor were given immediately prior to testing Groups of 7-9 rats received either 0, 1 , 5, or 25 μg of the ligand inhibitor h/rCRF (6-33) Percent correct responses were recorded Rats receiving 5 and 25 μg CRF (6-33) had statistical significant better performance than rats receiving 0 or 1 μg of ligand inhibitor

Example 10

Elevated plus-maze test

The Elevated plus-maze test predicts how animals respond to an approach-avoidance situation involving an exposed, lighted space versus a dark, enclosed area. In the maze, 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 μg of h/rCRF ( 1 -41 ) Doses of 1 and 25 μg 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 μg) did not alter performance or produce overt behavioral alterations comparable to h/rCRF (1-41 ) h/rCRF (1 -41) is known to be a CRF-receptor agonist Therefore, these data demonstrate a clear-cut functional dissociation of the efficacious cognitive enhancing and anxiogenic side effects for a ligand inhibitor

Example 1 1

Automated One- Way Avoidance Learning

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.

To begin a training session, 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 As soon as the animal enters the safe compartment with all four paws, 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 At the end of the inter-trial interval, the subject is placed back in the footshock compartment facing away from the door On all trials after the first, 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 As soon as the animal enters the safe compartment with all four paws, the door is closed and the inter-trial interval begins If an animal does not escape the shock before it is turned off, it is coaxed the inter-trial interval (return to step 3). This rarely occurs after the first two or three trials This sequence of steps is repeated for the desired number of trials. Typically, for rats, 8-10 trials are run on the training day and an equal number on the testing day, and for mice, 2-4 trials are run on the training day and 10-14 trials on the testing day

When employing this strategy, one must take into account the number of avoidances made on the first day Therefore, 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 relative capacity of young adult (3 mo old) and aged adult (24 mo old) Brown-Norway rats to acquire and retain a one-way active avoidance response was assessed. Aged rats exhibit fewer avoidance responses during initial acquisition training than young rats Moreover, as shown in Figure 1 1 , the deficit in avoidance learning is sensitive to treatment with the ligand inhibitor h/rCRF (6-33) Performance was significantly improved in aged rats relative to young rats Moreover, retention was significant at both 1 and 7 days post-acquisition training (Figure 12) 97/28189 POYUS97/01572

49

Example 12 Automated Passive Avoidance Learning

The apparatus for both rats and mice is the same as used in active avoidance testing (see example 1 1 )

For the training trial, 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 When the animal enters with all four paws, the door is closed, and a 0 5 second AC footshock (Coulburn precision shocker) is delivered After five seconds, the subject is removed and placed in its home cage At the time of testing (usually 1 -7 days later), 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 Hence, 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 intensity ranges from 0 2-1 0 mA For most strains of mice with weights between 24 and 35 grams, an effective footshock intensity ranges from 0 15-0 6 mA The latency time of rats to enter the dark compartment was measured As seen in Figure 13, the median latency was dramatically increased in rats receiving h/rCRF (6-33) ICV 15 min prior to training

Example 13 Nicotine Withdrawal - Induced Overeating

The effect of ligand inhibitors on overeating is assessed in a model of excessive appetite upon withdrawal of nicotine Nicotine is administered chronically by subcutaneous implantation of osmotic mini-pumps infusing a solution of nicotine tartrate salt (9 mg/kg/day, 3.15 mg/kg/day nicotine free base) dissolved in saline or saline alone (vehicle) Nicotine withdrawal is achieved by surgical removal of the pump after 14 days.

Concomitant with nicotine pump removal or sham treatment saline infusion pump removal in controls, subjects were implanted with new osmotic mini- pumps filled with either vehicle(O) or h r CRF (6-33), which was infused ICV continuously over the subsequent seven days (i.e., days 1-7 of nicotine withdrawal) After seven days of vehicle/CRF (6-33) chronic infusion, the pumps were withdrawn so that both nicotine withdrawn (days 8-14 of withdrawal) and nicotine naive control subjects could be monitored in the absence of treatments (over a seven day recovery period). Body weight was recorded daily over the fourteen days of nicotine dependence and the fourteen days of nicotine withdrawal Weight gain constitutes an increase relative to the absolute body weight on the day immediately preceding the measurement period.

Example 14 Effect of Ligand Inhibition of Zucker Rats

Zucker lean (Fa/ ) and obese (fa/fa) rats are treated with vehicle, oCRF or h/rCRF (6-33) and food intake or body weight change is measured

For seven days, baseline measurements are made of the daily food intake and body weight of lean and obese rats Following these measurements, an osmotic pump-delivering vehicle, oCRF, or h/rCRF (6-33) is implanted oCRF is delivered at 5 μg/day and the h/rCRF (6-33) is delivered at 125 μg/day The pumps are surgically removed and for the next seven days, daily food intake and body weight measurements are recorded. As seen in Figures 7 and 8, rats receiving oCRF or h/rCRF (6-33) had reduced body weight gain or exhibited a body weight loss and had decreased food intake relative to rats receiving vehicle alone

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Behan, Dominic P.

Heinrichs, Stephen C. Sutton, Steven W. Lowry, Phillip J. Rivier, Jean E.F. DeSouza, Errol B. Vale Jr. , Wylie W.

(ll) TITLE OF INVENTION: METHODS FOR INCREASING ENDOGENOUS LEVELS OF CORTICOTROPIN-RELEASING FACTOR

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(A) ADDRESSEE: SEED and BERRY

(B) STREET: 6300 Columbia Center, 701 Fifth Avenue

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(D) SOFTWARE: Patentin Release #1.0, Version #1.30

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(A) TELEPHONE: (206) 622-4900

(B) TELEFAX: (206) 682-6031

(2) INFORMATION FOR SEQ ID NO: 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(n) MOLECULE TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 1 : Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg 1 5 10 15

Glu Val Leu Glu Met Ala Arg Ala Glu Gin Leu Ala Gin Gin Ala His 20 25 30

Ser Asn Arg Lys Leu Met Glu Ile Ile 35 40

(2) INFORMATION FOR SEQ ID NO:2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

|iι) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15

Xaa Xaa Ala Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Pro Pro Ile Ser Xaa Asp Leu Thr Phe His Leu Leu Arg Xaa Xaa Xaa

1 5 10 15

Glu Xaa Ala Arg Xaa Glu Xaa Xaa Xaa Xaa Gin Ala Xaa Xaa 20 25 30

(2) INFORMATION FOR SEQ ID NO: :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(il) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ser Gin Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg 1 5 10 15

Glu Val Leu Glu Met Thr Lys Ala Asp Gin Leu Ala Gin Gin Ala His 20 25 30

Ser Asn Arg Lys Leu Leu Asp Ile Ala 35 40

Claims

Claims

1. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for increasing the level of a free CRF- related peptide in the brain, said ligand inhibitor being capable of causing the release of CRF- related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

The ligand inhibitor of claim 1 wherein the CRF-related peptide is urocortin.

3. The ligand inhibitor of claim 1 wherein the ligand inhibitor is a peptide.

The ligand inhibitor of claim 1 wherein the ligand inhibitor is a non- peptide.

5. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for improving learning and memory, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

6. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for modulating food intake, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

7. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating diseases associated with low levels of CRF-related peptide in the brain, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

8. The ligand inhibitor of claim 7 wherein the disease is Alzheimer's disease.

9. The ligand inhibitor of claim 7 wherein the disease is selected from the group consisting of chronic fatigue syndrome, atypical depression, obesity, post-partum depression, and age-related memory loss.

10. The ligand inhibitor according to any one of claims 5-9 wherein the ligand inhibitor is a peptide.

1 1. The ligand inhibitor according to any one of claims 5-9 wherein the ligand inhibitor is a non-peptide.

12. The ligand inhibitor according to any one of claims 5-9 wherein the ligand inhibitor is administered in a pharmaceutically acceptable carrier.

13. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating the symptoms associated with Alzheimer's disease, said ligand inhibitor being capable of causing the release of CRF- related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

14. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating obesity, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

15. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating atypical depression, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF- binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

16. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating substance abuse withdrawal, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF- binding protein.

17. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating post-partum depression, said ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

18. The ligand inhibitor according to any one of claims 13-17 wherein the ligand inhibitor is a peptide.

19. The ligand inhibitor according to any one of claims 13-17 wherein the ligand inhibitor is a non-peptide.

20. A ligand inhibitor capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein, for use as an active therapeutic substance.

21. A method of screening for ligand inhibitors, comprising: incubating a candidate ligand inhibitor with a CRF-related peptide/CRF- binding protein complex; and measuring free CRF-related peptide by an assay.

22. The method according to claim 21 wherein free CRF-related peptide is measured by an in vitro ligand immunoradiometric assay.

23. The method according to claim 21 wherein free CRF-related peptide is measured by a biological assay.

24. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for treating hypertension, the ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

25. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for modulating food intake, the ligand inhibitor being capable of causing the release of CRF-related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

26. A ligand inhibitor of a CRF-related peptide/CRF-binding protein complex, for use in the manufacture of a medicament for inhibiting weight gain associated with nicotine withdrawal, the ligand inhibitor being capable of causing the release of CRF- related peptide from the CRF-binding protein or preventing the binding of free CRF-related peptide to CRF-binding protein.

27. A composition comprising urocortin, for use in the manufacture of a medicament for increasing the level of free CRF in the brain.