JP2001348400A - Corticotropin releasing factor binding protein inhibitor and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective treatment of a disorder and disease caused by various kinds of neurophysiology by increasing the free CRF level by utilizing a relationship between the decreased CRF level and such diseases. SOLUTION: This ligand inhibitor of a CRF/CRF-binding protein complex usable for production of a medicine for increasing the free CRF level in the brain can bring out the release of the CRF from the CRF-binding protein complex, or can inhibit the binding of the free CRF to the CRF-binding protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates generally to methods for increasing endogenous levels of neuropeptides and, more particularly, to methods for increasing corticotropin releasing factor levels in the brain.

[0002]

BACKGROUND OF THE INVENTION Recent clinical data indicate that CRF is associated with neuropsychiatric disorders and neurodegenerative disorders such as Alzheimer's disease. Alzheimer's disease
A neurodegenerative brain disease that results in progressive memory loss and dementia. According to recent estimates, 200 in the United States
More than ten thousand individuals are affected by the disease. In particular, several lines of evidence indicate that CRF may be associated with Alzheimer's disease (A
D). First, CRF
Decreased dramatically (over 50%) and (Bissette
Et al., JAMA 254: 3067, 1985; DeSo.
Uza et al., Brain Research 397: 4.
01, 1986; Whitehouse et al., Neuro.
logic 37: 905, 1987; DeSouza,
Hospital Practice 23:59, 1
988; Nemeroff et al., Regul. Pept
ids 25: 123, 1989), and conversely increases CRF receptors in cerebral cortex areas affected by AD (DeSouza et al., 1986; DeSouza,
1988), but the amount does not change in areas where neither the CRF nor the CRF receptor is affected by the cortex (DeSo).
usa et al., 1986). Second, chemical affinity crosslinking studies indicate that the increased CRF receptor population in the cerebral cortex in AD has normal biochemical properties (Gri
goriadis et al., Neuropharmacolo.
gy 28: 761, 1989). Furthermore, a decrease in CRF concentration in cerebrospinal fluid is observed (Moura).
Dian et al., Neural Peptides 8: 3.
93, 1986; May et al., Neurology 3.
7: 535, 1987), has a significant correlation across grades of neuropsychiatric injury, suggesting that greater cognitive impairment is associated with lower CSF levels in cerebrospinal fluid (Pomara). Et al., Biological P
sychiatry 26: 500, 1989).

[0003] Therapies available for the treatment of dementia are very limited. Tacr, a recently approved drug
Ine ^™ only marginally improves memory in Alzheimer's patients and has undesirable side effects that raise liver enzymes.

[0004] Alterations in brain CRF content have also been found in Parkinson's disease and progressive supranuclear palsy, which are neurological diseases that share certain clinical and pathological features with AD. In the case of Parkinson's disease, CRF content is reduced and shows a similar staining pattern to AD cases (Whitehouse et al., 198).
7; DeSouza, 1988). In the case of progressive supranuclear palsy, CRF is reduced to approximately 50% of control values in the frontal, temporal, and occipital lobes (Whiteh
ouse et al., 1987; DeSouza, 1988).

[0005] Some depression disorders are also associated with reduced levels of CRF. Patients with seasonal depression and chronic fatigue syndrome show lower levels of CRF in cerebrospinal fluid (Vanderpool et al., J. Cl.).
in. Endocrinol. Metab. 7
3: 1224, 1991).

[0006] In some cases, depression has a high rate of improvement and is often self-limiting, but there are major differences in the rate of patient recovery. The primary goal of treatment is to reduce the intensity of symptoms to promote the rate of recovery of this type of depression, and to prevent recurrence and relapse. Although antidepressants are typically administered, severe side effects can occur, such as suicide by fluoxetine, convulsions by bupropion. (Klerman et al., Cl.
initial Evaluation of Psych
otropic Drugs: Principles
and Guidelines, R.A. F. Prie
n and D.I. S. Robinson (ed.), Rave
n Press, Ltd. N. Y. , 1994, 2
See page 81).

[0007] Hypofunction of the stress system, as indicated by low CRF levels, may play a role in other diseases as well. For example, some forms of obesity are characterized by hypothalamic-pituitary-adrenal axis (Kopelman et al., Clin. Endocr.).
inol (Oxford) 28:15, 1988;
Bernini et al., Horm. Res. 31:13
3, 1989), patients with post-traumatic stress syndrome have low cortisol excretion (Mason et al., J. Neu.
Men. Dis. 174: 145, 1986), and patients who quit smoking have reduced excretion of adrenaline and noradrenaline, and decreased cortisol levels in the blood (West et al., Psychopharmacol).
oggy 84: 141, 1984; Puddy et al., Cli.
n. Exp. Pharmacol. Physio
l. 11: 423, 1984). CRF is hypothalamus-
Being a major regulator of the pituitary-adrenal axis, their expression all indicate a central role for CRF in these diseases.

[0008] Treatment of these diseases is not very effective. For example, the most effective approach to treating obesity is a behavior modification program. However, few participants reach the target weight and the recurrence rate is high (Halmi et al., Clinical Evaluati).
on of Psychotropic Drugs:
Principles and Guideline
s, R.S. F. Prien and D.M. S. Robin
son (eds.), Raven Press, Ltd.
N. Y. , 1994, p. 547).

[0009]

In view of the shortcomings in treating such diseases and conditions, more effective treatments are needed. The present invention takes advantage of the correlation between reduced CRF levels and various neurophysiological diseases and conditions to effectively treat such conditions by increasing the level of free CRF, Provide related advantages.

[0010]

SUMMARY OF THE INVENTION The present invention provides an effective amount of CR.
A method is provided for increasing free CRF levels in the brain by administering to a patient a ligand inhibitor of the F / CRF binding protein (CRF / CRF-BP) complex. Administration of the ligand inhibitor causes release of CRF from the CRF binding protein. The ligand inhibitor can be a peptide derived from, homologous to, or unrelated to CRF, as long as it can cause "release" of CRF. A ligand inhibitor can also be a non-peptidic compound isolated from a natural or synthetic chemical library. Within one embodiment of the invention, the ligand inhibitor is h / rCRF (amino acids 6-33), h / rCRF.
rCRF (amino acids 9 to 33) and a peptide selected from the group consisting of h / rCRF. Within a related aspect, there is provided a therapeutic composition, which comprises a ligand inhibitor in combination with a physiologically acceptable carrier or diluent.

[0011] Within other aspects of the invention, a method for improving learning and memory, a method for reducing food intake,
Methods of activating the RF neurocircuitry, treating diseases associated with low levels of CRF in the brain, treating symptoms associated with Alzheimer's disease, treating obesity, treating atypical depression Methods of treating, treating postpartum depression, treating age-related memory loss, and treating withdrawal symptoms of substance abuse are provided. Within such a method,
A therapeutically effective amount of a CRF / CRF binding protein ligand inhibitor is administered to a patient for treatment of these conditions. The criterion for selecting a candidate substance for treatment and a method for evaluating the effectiveness of treatment are shown.

In order to achieve a particular therapeutic goal, human C
It has been found that certain agents with high affinity for RF-BP can be administered. This drug is CRF
-An effective in vivo concentration of endogenous hCRF in a mammal, and / or a CRF agonist optionally administered with such an agent, which effectively competes with human CRF in complexing with BP Or C
Increase the effective concentration of the RF antagonist. That is, these agents are used to block the effects of CRF-BP, and thus increase the level of endogenous CRF in areas of the body where CRF-BP is present.
More specifically, it has been discovered that peptides between about 19-28 residues in length have a high affinity for hCRF-BP, but exhibit a relatively low tendency to themselves bind to the CRF receptor. Have been. As a result, such peptides can be administered to suppress the clearance of endogenous CRF from specific regions, thereby stimulating the biological effects of CRF in vivo and, in some cases, Or it may be advantageous to administer such a peptide together with a CRF agonist. The true nature of these drugs is that potential unwanted side effects are minimized or eliminated entirely. These agents can also be administered with a CRF antagonist to suppress the clearance of some CRF antagonists from the target region, especially if the CRF antagonist has a fairly high binding affinity for hCRF-BP. However, its effect is counteracted to some extent by the release of endogenous CRF that is otherwise bound to CRF-BP. These drugs are used to promote labor in pregnancy, to stimulate the respiratory system,
Useful for therapeutic treatment to combat obesity and to counteract the effects of Alzheimer's disease and chronic fatigue syndrome; however, some of these indications are such that the drug is delivered in the brain Must be administered at

Further, to determine the ability of CRF to block the ability to bind to the native CRF receptor,
And such candidate CRF antagonists and hCRF
Performing such a dual screening assay for potential antagonists also to determine binding affinity to -BP provides a method of screening for particularly effective CRF antagonists.

[0014] Methods for screening for neuropeptide binding proteins are also provided. In a related aspect, there is provided a method of screening for a ligand inhibitor of a neuropeptide / neuropeptide binding protein complex, particularly a CRF / CRF-BP complex. Within one embodiment, the method comprises reacting CRF in the presence of a ligand inhibitor in an aqueous solution.
Contacting with a CRF-BP, further mixing in a nonionic surfactant, separating the nonionic surfactant and the aqueous solution, and detecting the amount of CRF in the aqueous solution, thereby detecting the ligand inhibitor Is CRF / C
Determining whether to disrupt the RF-binding protein complex. In some embodiments, the mixing is performed at a temperature above the cloud point of the nonionic surfactant, and octylphenoxypolyethoxyethanol is the preferred nonionic surfactant.

[0015] These and other aspects will become apparent with reference to the following detailed description and accompanying drawings.

The present invention provides a CRF / CRF for use in the manufacture of a medicament for increasing the level of free CRF in the brain.
Provided is a ligand inhibitor of a binding protein complex, which is capable of causing the release of CRF from the CRF binding protein or inhibiting binding of free CRF to the CRF binding protein.

In one embodiment of the invention, the ligand inhibitor is h / rCRF (amino acids 6 to 3).
3), h / rCRF (amino acids 9-33), and h / rCRF
It is a peptide selected from the group consisting of rCRF.

[0018] In one embodiment of the present invention, the ligand inhibitor is a peptide.

In another embodiment of the invention, the ligand inhibitor is a non-peptide.

The present invention relates to a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for improving learning and memory, wherein the ligand inhibitor can cause the release of CRF from the CRF binding protein, or Provided is a ligand inhibitor capable of suppressing the binding of CRF to a CRF-binding protein.

The present invention also provides a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for reducing food intake, wherein the ligand inhibitor may cause the release of CRF from the CRF binding protein, or Provided is a ligand inhibitor capable of suppressing the binding of CRF to a CRF-binding protein.

The present invention provides a method for producing a medicament for treating a disease associated with low levels of CRF in the brain.
Provided is a ligand inhibitor of an RF / CRF binding protein complex, which is capable of causing the release of CRF from the CRF binding protein or suppressing binding of free CRF to the CRF binding protein.

In one embodiment, the disease is Alzheimer's disease.

In another embodiment, the disease is selected from the group consisting of chronic fatigue syndrome, atypical depression, obesity, postpartum depression, and age-related memory loss or free C
It suppresses the binding of RF to CRF binding protein.

[0025] In one embodiment, the ligand inhibitor is a peptide.

In another embodiment, the ligand inhibitor is a non-peptide.

[0027] In yet another embodiment, the ligand inhibitor is administered in a pharmaceutically acceptable carrier.

The present invention relates to a method for preparing a medicament for treating a symptom associated with Alzheimer's disease, comprising the steps of:
Provided is a ligand inhibitor of a CRF binding protein complex, which is capable of causing the release of CRF from the CRF binding protein or suppressing binding of free CRF to the CRF binding protein.

The present invention relates to a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for treating obesity, wherein the ligand inhibitor can cause the release of CRF from the CRF binding protein or free CRF. Can inhibit binding to CRF binding proteins,
Provide a ligand inhibitor.

The present invention provides a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for treating atypical depression, which may cause the release of CRF from the CRF binding protein or free CRF. Provided is a ligand inhibitor capable of suppressing the binding of CRF to a CRF-binding protein.

The present invention provides a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for the treatment of withdrawal symptoms of substance abuse, comprising a CR
Ligand inhibitors are provided that can cause the release of CRF from an F-binding protein or can inhibit the binding of free CRF to a CRF-binding protein.

The present invention is a ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for treating postpartum depression, which is capable of causing the release of CRF from a CRF binding protein or of freeing it. Provided is a ligand inhibitor capable of suppressing the binding of CRF to a CRF-binding protein.

[0033] In one embodiment, the ligand inhibitor is a peptide.

[0034] In another embodiment, the ligand inhibitor is a non-peptide.

The present invention provides ligand inhibitors that can cause the release of CRF from a CRF binding protein or inhibit the binding of free CRF to the CRF binding protein for use as an active therapeutic substance.

In another aspect, the invention provides a method for screening for a ligand inhibitor, comprising the steps of incubating a candidate ligand inhibitor with a CRF / CRF binding protein complex; and measuring free CRF by an assay. Is provided.

[0037] In one embodiment, free CRF is measured by an in vitro ligand immunoradiometric assay.

[0038] In another embodiment, free CRF is measured by a biological assay.

The present invention also has a high affinity for human CRF binding protein and a low affinity for human CRF receptor for use in the manufacture of a medicament for enhancing the in vivo activity of CRF or CRF analog. An agent is provided that is effective to form a complex with CRF-BP sufficient to increase the concentration of endogenous hCRF available for binding to the CRF receptor. In one embodiment, the agent of the invention further comprises CRF or an analog of CRF that is an agonist or antagonist.

[0040] In one embodiment, the agent is about 19-
The peptide is between about 28 residues in length. In a further embodiment, the peptide is hCRF (9-33). In yet another embodiment, the peptide is hCRF
(6-33).

In a still further embodiment, the above peptide
Is the sequence (SEQ ID NO: 2): Xaa_Four-Xaa_Five-Xaa₆
-Xaa₇-Xaa₈-Xaa₉-Xaa_Ten-Xaa₁₁−
Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa_Fifteen-Xaa₁₆−
Xaa₁₇-Xaa₁₈-Xaa ₁₉-Xaa₂₀-Xaa_{twenty one}−
Ala-Xaa_{twenty three}-Xaa_{twenty four}-Glu-Xaa₂₆-Xa
a₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xa
a₃₂-Xaa₃₃Or 1 to 8 from the N-terminus in the sequence
Residues, or 1 to 5 residues from the C-terminus in the sequence, or both
Its biological activity formed by deletion of
Where Xaa_{twenty three}Is Arg
Is Lys; Xaa_{twenty four}Are Ala, Ile, Asn,
Met, Nle or Leu, and each other
Each Xaa is independently a residue of a natural amino acid. Sa
In another embodiment, each Xaa is independently human CRF.
The residue present at each position of (1-41) or
Shows conservative substitutions for

In yet another embodiment, the pep
Tide has the sequence (SEQ ID NO: 3): Pro-Pro-Ile
-Ser-Xaa₈-Asp-Leu-Thr-Phe
-His-Leu-Leu-Arg-Xaa₁₇-Xaa
₁₈-Xaa₁₉-Glu-Xaa_{twenty one}-Ala-Arg-X
aa_{twenty four}-Glu-Xaa₂₆-Xaa₂₇-Xaa₂₈-Xa
a₂₉-Gln-Ala-Xaa₃₂-Xaa₃₃Or
Represents 1 to 8 residues from the N-terminus in the sequence, or C
By deleting 1-5 residues, or both, from the terminus
Having a biologically active fragment thereof formed,
Where Xaa₈Is Leu or Ile; Xaa
₁₇Is Glu or Asn; Xaa₁₈Is Val, M
et, Leu, or Nle; Xaa₁₉Is Leu
Or Ile; Xaa_{twenty one}Is Met, Leu,
Is Nle; Xaa_{twenty four}Is Ala, Ile, or A
sn; Xaa₂₆Is Gln or Asn; X
aa ₂₇Is Leu, Glu, or Gln; Xaa
₂₈Is Ala or Arg; Xaa₂₉Is Gln again
Is Glu; Xaa₃₂Is His or Glu
And Xaa₃₃Is Ser or Leu.

[0043] In yet another embodiment, the peptide has the ^{formula: (Ile 8,19,24, Asn 17,26,} Met 18, Glu
^27, Arg ^28] having -hCRF (4-28).

[0044] In yet another embodiment, the peptide has the ^{formula: (Ile 8,19,24, Asn 17,26,} Met 18, Glu
^{27,29, Arg 28, Gly 32,} Leu 33] -hCRF
(6-33).

In yet another embodiment, the peptide has the formula: (Asn ^17,24,26 , Met ¹⁸ , Ile ¹⁹ , Gl
^{n 27, Arg 28, Glu 29} , Gly 32, Leu 33] -h
It has a CRF (9-33).

The present invention relates to a medicament having a high affinity for human CRF-binding protein and a low affinity for human CRF receptor for use in the manufacture of a medicament for promoting labor in pregnant females. An agent is provided that is present in an amount sufficient to raise the concentration of free hCRF therein to a level of at least about 250 picomoles / liter.

In one embodiment, the agent is a peptide.

The present invention relates to a medicament for the treatment of Alzheimer's disease, wherein the medicament is a peptide and an increase in free endogenous CRF in the brain results in an improvement in short to medium term memory. The agent is present in an amount effective for the drug.

In another aspect, the sequence (SEQ ID NO: 3): Pr
o-Pro-Ile-Ser-Xaa ₈ -Asp-Le
u-Thr-Phe-His-Leu-Leu-Arg
_{-Xaa 17 -Xaa 18 -Xaa 19 -Glu-} Xaa 21 -
_{Ala-Arg-Xaa 24 -Glu-} Xaa 26 -Xaa
₂₇ -Xaa ₂₈ -Xaa ₂₉ -Gln-Ala-Xaa _32-
Xaa ₃₃ , or 1 to 8 residues from the N-terminus in the sequence,
Or a peptide having a biologically active fragment thereof formed by deleting 1 to 5 residues, or both, from the C-terminus in the sequence, wherein Xaa ₈
Xaa ₁₇ is Glu or Asn; Xaa ₁₈ is Val, Met, Leu, or Nle; Xaa ₁₉ is Leu or Ile; Xaa ₂₁ is Met, Leu, or Nle. ;
Xaa ₂₄ is Ala, Ile, or Asn;
a ₂₆ is located at the Gln or Asn; Xaa ₂₇ is Leu,
Xaa ₂₈ is Ala or Arg; Xaa ₂₉ is Gln or Glu;
Xaa ₃₂ is His or Glu; and Xaa ₃₃
A peptide is provided wherein is Ser or Leu.

In one embodiment, the peptide is h
CRF (6-33); hCRF (9-33); [Ile
^{8,19,24, Asn 17,26, Met 18,} Glu 27,29, Ar
g ²⁸ , Gly ³² , Leu ³³ ] -hCRF (6-33);
[Asn ^17,24,26 , Met ¹⁸ , Ile ¹⁹ , Gln ²⁷ , A
^{rg 28, Glu 29, Gly 32} , Leu 33] -hCRF
(9-33); [Met ^6,14,18,24 , Ile ^8,19,33 ,
Asn ¹⁷ , His ²⁰ , Arg ^21,28 , Lys ²³ , Gly
^{26, Glu 27,29, Leu 32]} -hCRF (6-3
3); and C-terminal and / or N-terminal truncated fragments thereof that bind with high affinity to hCRF-BP.

In yet another aspect, the present invention is a screening method for selecting a CRF antagonist effective for in vivo administration to a mammal, comprising the steps of: (a) significantly activating human CRF receptor (hCRF-R); (B) the affinity of each candidate peptide that binds to the receptor without binding to the receptor;
-Screening a group of CRF antagonist candidate peptides to determine affinity for each candidate peptide that binds to -BP); comparing both the affinity determinations (a) and (b) for each candidate; And exhibits a relatively high affinity for binding to hCRF-R without significantly activating the receptor, and
Selecting a candidate peptide that also exhibits a relatively low affinity for binding to P.

[0052] In one embodiment, the determination (a) according to the first above uses cultured pituitary cells and uses CR
Achieved by testing each candidate for the effect of blocking the secretion of ACTH by a challenge dose of F.

In another embodiment, said second decision (b)
Indicates that each candidate peptide is composed of hCRF-BP and hCRF-B
This is accomplished by performing an inhibitory binding affinity assay that is placed in solution with an appropriately labeled amount of ligand having a known binding affinity for P.

The present invention also provides a method for screening for a ligand inhibitor of a CRF / CRF binding protein complex, comprising: (a) contacting CRF with CRF-BP in the presence of a candidate ligand inhibitor in an aqueous solution; b) adding a non-ionic surfactant to the solution of step (a); (c) separating the non-ionic surfactant from the aqueous solution; and (d) the amount of bound CRF in the aqueous solution or the non-ionic surfactant. Detecting any amount of free CRF in the ionic surfactant so that the candidate ligand inhibitor is CRF / CRF
Determining whether to disrupt the binding protein complex.

In one embodiment, step (b) is performed at a temperature above the cloud point of the nonionic surfactant.

In another embodiment, the non-ionic surfactant is octylphenoxy polyethoxyethanol.

[0057]

DETAILED DESCRIPTION OF THE INVENTION Some aspects of the invention are directed to grant number DK-267, awarded by the National Institutes of Health.
Government support under 41 and HD-13527. The government has certain rights in the invention. the U
diversity of reading and th
e Medical Research Counci
l of Great Britain has certain rights in this application.

No. 08 / 276,276 filed Jul. 15, 1994.
No. 240 is a continuation-in-part application.

DETAILED DESCRIPTION OF THE INVENTION The level of free corticotropin releasing factor (CRF) in the brain is determined by the CRF / CR
It is increased by administration of a ligand inhibitor of the F-binding protein complex to a patient. The ligand inhibitor is C
Binds to RF binding proteins and causes release of CRF. The ligand inhibitor can be a peptide from CRF or a related protein, or a non-peptide. Administration of a ligand inhibitor may provide improved learning and memory, may result in reduced food intake, or may provide treatment for diseases associated with low levels of CRF in the brain, particularly Alzheimer's disease. Also provided are methods of screening compounds to select CRF antagonists that are particularly effective for in vivo administration to mammals.

Before describing the present invention, it is useful to describe the definitions of terms used hereinafter to understand the present invention.

"CRF" refers to a peptide that regulates the release of adrenocorticotropin (ACTH), β-endorphin, and other peptides derived from proopiomelanocortin (POMC) from the pituitary gland. In humans, rats, and other species, the amino acid sequence of CRF has been determined and is shown in FIG. 1 (SEQ ID NO: 1). The amino acid sequences of rat CRF and human CRF are identical, and this protein is referred to as "h / rCRF". "Free CRF" refers to CRF binding protein or CRF.
CRF not complexed or bound to RF receptor
That is.

"CRF binding protein" (CRF-B
P) refers to one or more proteins present as soluble factors in human plasma or associated with cell membranes. It has the ability to inhibit the function of CRF as measured in one of two ways: (1) CRF-induced ACTH release from pituitary cell cultures or perfused rat anterior pituitary system, or (2) C
CRF-induced cAMP formation from cells with RF receptor or cells transfected with cloned CRF receptor. 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” means that CRF and CRF
It is a complex with RF-BP. CRF and CRF-
The binding to BP is caused by hydrophobic interaction, ionic interaction,
Alternatively, it can be effected by covalent interactions.

"Human CRF binding protein" (hCRF
-BP) has been tested for hC in vitro and in vivo.
A 37 kDa serum protein that inactivates hCRF as an actin secretion promoting substance by specifically binding RF. Human CRF-BP is hCRF
Has a high affinity for and a low affinity for oCRF. This suggests that hCRF-BP promotes peripheral plasma hCRF removal.
hCRF loses the ability to stimulate ACTH in vitro and in vivo when bound to hCRF-BP. Although the C-terminus is primarily responsible for receptor affinity, the first eight amino acids of CRF are thought to be involved in receptor activation. hCRF-BP appears to prevent hCRF from stimulating adrenocorticotropic hormone-producing cells by binding to the central domain, so that the ligand cannot interact with the receptor,
ACTH release does not occur.

(Ligand Inhibitor of CRF / CRF Binding Protein) As described above, the present invention provides an effective amount of CRF / CRF-binding protein such that CRF is released from CRF-BP.
Figure 4 shows a method of increasing free CRF levels in the brain by administering a ligand inhibitor of the CRF-BP complex.

A “ligand inhibitor of the CRF / CRF-BP complex” displaces CRF in either a reversible or irreversible manner. Substitution can occur by causing the bound CRF molecule to become free CRF. In addition, binding of the ligand inhibitor was determined by human hCRF-
CRF-BP of free CRF due to high affinity for BP
And can compete with endogenous CRF for binding to hCRF-BP. Reversible or irreversible substitution of CRF can be mediated by a ligand inhibitor that binds directly to the CRF binding site, or by a ligand inhibitor that binds to a site that is not the CRF binding site and causes an allosteric displacement of the bound CRF. The ligand inhibitor can be a peptide derived from CRF or a CRF-related sequence, or a random peptide designed to cause displacement of the bound CRF. In addition, ligand inhibitors can be non-peptide molecules from natural libraries of small molecules, synthetic analogs of natural molecules, small molecules specifically designed based on the physical properties of the CRF / CRF-BP binding complex, or It can be another ligand inhibitor. The ligand inhibitor may also be a metabolite of the administered compound. The ligand inhibitor must be able to be administered by the CNS or systemically to reach the brain. Preferably, the properties of the ligand inhibitor are such that the inhibitor is a low affinity antagonist at the CRF receptor (K _i ≧ 1 μM) or has 100-fold selectivity for CRF binding proteins (K _i ≦ 10 n
M). CRF-BP ligand inhibitors may also exhibit some moderate agonist activity at the CRF receptor (K _i ≧ 50 nM).

CRF-BP binds to endogenous CRF
Is a CRF peptide sequence, CR
It can be derived from an F-related peptide sequence or an unrelated peptide sequence. Relatively short peptides between about 19 and 28 residues in length have been found to be effective in competitively binding to hCRF-BP, while exhibiting very low affinity for the CRF receptor. . As a result, this particular group of peptides binds to hCRF-BP, but
Does not substantially bind to and / or interacts with the receptor. When given alone, these peptides have the effect of increasing the amount of endogenous free CRF. This maintains the ability to interact with the CRF receptor. Thus, these peptides can be used to ensure or increase the effects of endogenous CRF in specific areas or body organs. These peptides, when given with an agonist or antagonist of the CRF receptor in a cocktail, can likewise be used to increase the efficiency of these substances. However, administration with CRF antagonists is somewhat neutralized by the release of endogenous CRF. CR
Examples of F-related proteins include urotensin (urotensin).
sin) and sauvagine. Preferred peptides are those derived from h / rCRF. In this regard, many different peptides can be used within the context of the present invention to replace endogenous binding CRF. Such peptides include α-helix oCRF (sheep CRF) (9-41) (numbers in parentheses refer to amino acids), h / rCRF (6-33), h / rCRF
rCRF (9-33), urotensin I, sovagin,
And h / rCRF. Preferred peptides are h /
rCRF (6-33), h / rCRF (9-33), and h / rCRF (1-41) OH. These peptides bind to CRF-BP with appropriate affinity
This is because it does not bind to the RF receptor. C-terminal free acid (OH) is particularly preferred over amidation found in natural CRF. Deamidation of the C-terminal amino acid of h / rCRF does not affect the binding affinity for CRF-BP but h / rC
Significantly reduces the affinity of RF. The N-terminus of these factors is degraded by acylation (eg, acetyl (Ac)
), And such modified peptides are considered to be equivalent. The minimal sequence of CRF found to bind to CRF-BP without significantly interacting with the CRF receptor is amino acids 9-33. In particular, amino acid residues 22-25 appear to play a critical role in binding to CRF-BP.

Other peptides can be designed based on homology to the peptide series. As described above, when designing a peptide, the C-terminal amino acid preferably contains a free acid group. FIG. 1 shows the known CR
3 shows a comparison of the amino acid sequences of F and CRF related molecules.
As described above, residues 9-33 contain the minimal sequence required for binding to CRF-BP and
3, and 25 are thought to play a critical role in binding. Preferred guidelines in peptide design are that the amino acid residue corresponding to residue 22 is alanine, the amino acid residue corresponding to residue 23 is a basic amino acid (arginine or lysine), and corresponds to residue 25. The amino acid residue is glutamic acid.

An example of a suitable peptide is a fragment of human CRF having the sequence: hCRF (1-
41) including: Ser-Glu-Glu-Pro-Pr
o-Ile-Ser-Leu-Asp-Leu-Thr
-Phe-His-Leu-Leu-Arg-Glu-
Val-Leu-Glu-Met-Ala-Arg-A
la-Glu-Gln-Leu-Ala-Gln-Gl
n-Ala-His-Ser-Asn-Arg-Lys
-Leu-Met-Glu-Ile-Ile (SEQ ID NO: 1).

One preferred fragment used is hCRF (6-33) having the following sequence:
Ile-Ser-Leu-Asp-Leu-Thr-P
he-His-Leu-Leu-Arg-Glu-Va
l-Leu-Glu-Met-Ala-Arg-Ala
-Glu-Gln-Leu-Ala-Gln-Gln-
Ala-His-Ser (SEQ ID NO: 1). This fragment can be shortened to 4 residues at the N-terminus and / or to 5 residues at the C-terminus by removing residues in the sequence. This fragment is, for example, hCRF (9
-33), hCRF (6-30), and hCRF (8
-29). Examples of other peptides that can be used instead include, for example, hCRF (6-33) analogs such as: [Nle ²¹ ] -hCRF (6-33),
[Nle ²¹ ] -hCRF (9-33), [Ile ²⁴ ]-
hCRF (6-33), [Asn ²⁶ ] -hCRF (6-
^{33), [Nle 18,21] -hCRF} (6-33),
[Ile ²⁷ ] -hCRF (6-33), [Va ²⁸ ] -h
CRF (6-33), [Asn ²⁹ , ³⁰ ] -hCRF (6
-33), [Lys ¹⁶ ] -hCRF (6-33), [A
sp ¹⁷ ] -hCRF (6-33), [Leu ¹² ] -hC
RF (6-33), [Arg ¹³ ] -hCRF (6-3
3), [Glu ⁹ ] -hCRF (6-33), [V
a ³¹ ] -hCRF (6-33), [Thr ³³ ] -hCR
F (6-33), [Arg ³² ] -hCRF (6-3
^{3), [Ile 10] -hCRF} (6-33), and ^{[Ile 14] -CRF (6-33)} .

Other pepti that can be used for this purpose
Is defined by the following sequence (SEQ ID NO: 2): X
aa_Four-Xaa_Five, -Xaa₆-Xaa₇-Xaa₈-Xa
a₉-Xaa_Ten-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xa
a₁₄-Xaa_Fifteen-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xa
a₁₉-Xaa₂₀-Xaa_{twenty one}-Ala-Xaa_{twenty three}-Xaa
_{twenty four}-Glu-Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉
-Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃Or creature
Biologically active fragments thereof. This fragment
Represents 1 to 8 residues from the N-terminus in the sequence, or C
Formed by deletion of 1 to 5 residues or both from the end
Is done. Where Xaa_{twenty three}Is Arg or Lys
Xaa_{twenty four}Are Ala, Ile, Asn, Met, N
le, or Leu, and each remaining Xaa is
Present at each position in human CRF (1-41)
Residue or another naturally occurring amino acid, preferably
Or conservative substitution of a naturally occurring residue.

Another preferred group of peptides has the following sequence:
Defined by (SEQ ID NO: 2): Xaa_Four-Xaa_Five−
Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa_Ten-Xa
a₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa_Fifteen-Xa
a₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂₀-Xa
a_{twenty one}-Ala-Xaa_{twenty three}-Xaa_{twenty four}-Glu-Xaa₂₆
-Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁
-Xaa₃₂-Xaa₃₃And those that are biologically active
Fragment. This fragment is the N-terminal
From 1 to 8 residues, or 1 to 5 residues from the C-terminus in the sequence
Formed by deletion of one or both groups. here,
Xaa₆Is Ile, Met, Leu, or Nle
Xaa₈Is Leu or Ile; Xaa₁₄
Is Leu, Met, or Nle; Xaa
₁₇Is Glu or Asn; Xaa ₁₈Is Va
1, Met, Leu, or Nle; Xaa
₁₉Is Leu or Ile; Xaa₂₀Is Glu
Or His; Xaa_{twenty one}Means Met, Leu, N
le or Arg; Xaa_{twenty three}Is Arg or
Lys; Xaa_{twenty four}Are Ala, Ile, Asn,
Met, Nle, or Leu; Xaa₂₆Is G
ln, Asn, or Gly; Xaa₂₇Is Le
u, Glu, or Gln; Xaa₂₈Is Ala
Or Arg; Xaa ₂₉Is Gln or Glu
And Xaa₃₂Is His, Glu, or Leu
Yes; Xaa₃₃Is Ser, Leu, or Ile
And each of the remaining Xaas is a human CRF (1-41)
Residue at each position in
Naturally occurring amino acids, preferably naturally occurring residues
Represents a conservative substitution of Preferably, up to 2-5 residues are N
It is deleted from the end. Most preferably, Xaa₇Is S
er and Xaa₉Is Asp and Xaa_TenIs
Leu, Xaa₁₁Is Thr and Xaa
₁₂Is Phe and Xaa₁₃Is His and Xa
a_FifteenIs Leu and Xaa₁₆Is Arg and Xa
a₃₀Is Gln and Xaa₃₁Is Ala
You.

Another preferred group of peptides has the following sequence:
(SEQ ID NO: 3): Pro-Pro-I
le-Ser-Xaa₈-Asp-Leu-Thr-P
he-His-Leu-Leu-Arg-Xaa₁₇-X
aa₁₈-Xaa₁₉-Glu-Xaa_{twenty one}-Ala-Arg
-Xaa_{twenty four}-Glu-Xaa₂₆-Xaa₂₇-Xaa₂₈−
Xaa₂₉-Gln-Ala-Xaa₃₂-Xaa₃₃,Also
Are biologically active fragments thereof. This flag
Is 1 to 8 residues from the N-terminus in the sequence, or
Deletion of 1 to 5 residues or both from the C-terminus
Formed. Where Xaa₈Is Leu or Il
e; Xaa₁₇Is Glu or Asn; X
aa₁₈Is Val, Met, Leu, or Nle.
Xaa ₁₉Is Leu or Ile; Xaa_{twenty one}
Is Met, Leu, or Nle; Xaa
_{twenty four}Is Ala, Ile, or Asn; Xaa₂₆
Is Gln or Asn; Xaa₂₇Is Leu,
Glu, or Gln; Xaa₂₈Is Ala
Is Arg; Xaa₂₉Is Gln or Glu
Xaa₃₂Is His or Glu; and X
aa₃₃Is Ser or Leu.

Of the latter group, the above-mentioned flag of hCRF
Particularly preferred peptides other than the following are the following analogs
Including: [Ile^8,19,24, Asn^17,26, Met¹⁸, Glu
^27,29, Arg²⁸, Gly³², Leu³³] -HCRF
(6-33) [Asn^17,26, Nle¹⁸, Ile^19,24, Gl
u^27,29, Arg²⁸, Gly³², Leu³³] -HCRF
(9-33) [Ile^8,19,24, Asn^17,26, Met¹⁸, Glu²⁷,
Arg²⁸] -HCRF (4-28) [Ile^19,24, Asn^17,26, Nle¹⁸, Glu²⁷, A
rg²⁸] -HCRF (7-31) [Ile^8,19, Asn^17,24,26, Met¹⁸, GLN²⁷,
Arg²⁸, Glu²⁹, Gly³², Leu³³] -HCRF
(6-33) [Asn^17,24,26, Met¹⁸, Ile¹⁹, Gln²⁷, A
rg²⁸, Glu²⁹, Gly³², Leu³³] -HCRF
(9-33) [Ile^8,19, Asn^17,24,26, Met¹⁸, Gln²⁷,
Arg²⁸] -HCRF (8-28) [Ile^8,19, Asn^17,24,26, Nle¹⁸, Gln²⁷,
Arg²⁸, Glu²⁹, Gly³²] -HCRF (8-3
2) [Ile^8,19, Asn^17,24,26, Nle¹⁸, Gln²⁷,
Arg²⁸, Glu²⁹, Gly³²] -HCRF (8-3
2) [Met^6,14,18,24, Ile^8,19,33, Asn¹⁷, Hi
s²⁰, Arg^21,28, Lys^{twenty three}, Gly²⁶, Gl
u^27,29, Leu³²] -HCRF (6-33) [Ile^8,19,33, Met^14,18,24, Asn¹⁷, His
²⁰, Arg^21,28, Lys ^{twenty three}, Gly²⁶, Glu^27,29,
Leu³²] -HCRF (9-33). [Nle^6,14,18,24, Ile^8,19,33, Asn¹⁷, Hi
s²⁰, Arg^21,28, Lys^{twenty three}, Gly²⁶, Gl
u^27,29, Leu³²] -HCRF (6-33). [Ile^8,19, Nle^14,18,24, Asn¹⁷, His²⁰,
Arg^21,28, Lys^{twenty three}, Gly²⁶, Glu^27,29] -H
CRF (8-30).

The peptide nomenclature is described by Schroder and Lubke, “The Peptides”, Ac
ademic Press (1965), where amino groups are listed on the left and carboxyl groups on the right according to conventional notation. The alpha amino acid residue is identified using the standard three letter code, and if the amino acid has an isomer, the indicated amino acid is in the L form unless otherwise indicated. For example, Ser
= L-serine, Orn = L-ornithine, Nle = L-
Norleucine, Nva = L-norvaline, Har = L-
Homoarginine, and CML = L-CαMeLeu.

The peptide is synthesized by a suitable method. The method may be exclusively by solid phase synthesis, by partial solid phase synthesis, by fragment condensation, or by classical dissociation addition (solution addit).
ion). Chemical synthesis of peptides uses protection of labile side groups of various amino acid moieties with appropriate protecting groups,
Prevent chemical reactions occurring at that site, and finally remove this group. Most methods protect the α-amino group on an amino acid or fragment, while the protected reacts with a carboxyl group and then selectively removes the α-amino protecting group to allow the next reaction at that position. Cause. An example of such a representative peptide synthesis is described in US Pat. No. 5,235,036 (issued Aug. 10, 1993,
This disclosure is incorporated herein by reference).

HCRF (6-33) and hCRF (9
Peptides such as -33), as well as other analogs specifically mentioned above, are disclosed in U.S. Patent No. 4,489,163, the disclosure of which is incorporated herein by reference. Synthesized manually using solid phase methods or automatically on a Beckman Model 990 peptide synthesizer. Briefly, tertiary butoxycarbonyl (Boc) is used for α-amino protection and TFA-CH ₂ Cl ₂ (3: 2) is used for deprotection. The standard coupling is mediated by 1,3-diisopropylcarbodiimide (DIC), while the difficult coupling is 2- (1H-benzotriazole-1-
Yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). The protected peptide resin is cleaved using anhydrous hydrofluoric acid (HF) in the presence of 3% methyl sulfide, and the HF is subsequently removed under vacuum. Crude peptide was obtained by multi-step reverse phase HPL
Purified using C.

[0078] Polypeptide analogs include any polypeptide having substantially the same amino acid residue sequence as the sequences specifically set forth herein. In this polypeptide, one or more residues have been replaced with another amino acid residue at the position described above. Conservative substitutions are made using residues with functionally similar side chains if the polypeptide exhibits the ability to cause an increase in free CFR (eg, by binding strongly to CRF-BP). Can be. A common example of a conservative substitution is replacing one non-polar (hydrophobic) residue (eg, isoleucine, valine, alanine, glycine, leucine, or methionine) with another residue; ) Substituting one residue for another (eg, lysine for arginine, asparagine for glutamine, serine for threonine); one basic residue (eg, lysine, arginine, or Substituting another residue for histidine); and substituting another residue for one acidic residue (eg, aspartic acid or glutamic acid). The expression "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue if such a polypeptide exhibits the desired binding activity. As discussed above, such conservative substitutions may result in residues X
It may be made for one or more residues from aa _{6 to} Xaa ₂₁ and Xaa _{26 to} Xaa ₃₃ ; examples of preferred conservative substitutions are shown in Table 1 below.

[0079]

[Table 1] "Chemical derivative" refers to a subject polypeptide having one or more residues that are chemically derivatized by reaction of a functional side chain. Such derivatized molecules include, for example, derivatized free amino groups, amine hydrochloride, p-
Examples include a molecule that forms a toluenesulfonyl group, a carbobenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group, or a formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups are derivatized and O-
Acyl or O-alkyl derivatives may be formed.
The imidazole nitrogen of histidine is derivatized and Ni
m-Benzyl histidine may be formed. Chemical derivatives also include peptides containing one or more naturally occurring amino acid derivatives of a standard amino acid. For example, proline can be substituted with 4-hydroxyproline, lysine can be substituted with 5-hydroxylysine, histidine can be substituted with 3-methylhistidine, serine can be substituted with homoserine, and lysine can be substituted with homoserine. , Ornithine.

In addition, random peptides can be synthesized and subsequently screened to identify peptides that satisfy functional criteria for substitution of CRF from the CRF / CRF-BP complex, as discussed in more detail below. Random peptides can be generated by biological methods or by combinatorial chemistry techniques (Gallopp et al., JM).
ed. Chem. 37: 1234, 1994).

[0081] Biological methods for producing random peptides include at least four different methods.
First, random nucleotides can be inserted into the host gene to display the peptide on the surface of the microorganism (C
Harbit et al., Embo. Journal 5:30
29, 1986; Agterberg et al., Gene8.
8:37, 1990; Fuchs et al., Bio / Tech.
9: 1369, 1991; Thery et al., Appl.
Environ. Microbiol. 55:98
4, 1989). In such a method, various cell surface proteins of the bacterium are used as fusion partners into which oligonucleotides are inserted to produce a peptide fused to one of the extracellular loops of the protein. Lam
B, OmpA, PhoE, PAL, and pilin proteins all function as vehicles for peptide presentation on bacteria. Another system for providing the peptide on the surface of the bacteriophage is a preferred method. The vector used is a filamentous phage (eg, M13, f1,
And fd). Typically, a coat protein (eg, pill or pVIII) functions as a peptide expression vehicle. (Smith, Scie
228: 1315, 1985; Parmley
And Smith, Gene 73: 305, 198.
8; Cwirla et al., Proc. Natl. Aca
d. Sci. USA 87: 6378, 1990;
Markland et al., Gene 109: 13,199.
1, U.S. Pat. No. 5,223,409). With both phage and bacterial display methods, there is a physical link between the peptide and the DNA encoding the peptide,
This allows for easy isolation and propagation of the DNA sequence. Third, the peptide can be bound to a plasmid by fusing the peptide at its C-terminus to the DNA binding protein LacI (Cull et al., Proc. Na.
tl. Acad. Sci. USA 89: 186
5, 1992). The fusion protein is then ligated to the Lac operator on the plasmid such that the peptide specifically and stably binds to the DNA sequence encoding it. Fourth, the peptide is required to be stalled.
After translation, they can be displayed on polysomes. By causing the accumulation of RNA and polysomes, including nascent peptides still linked to the RNA encoding them (Gallop et al., J. Me.
diginal Chem. 37: 1233,199
4), the genetic material encoding the peptide can be recovered. In all of these methods, CRF was used as the lead peptide.
Can be synthesized by controlling the proportional level of inserted "incorrect" bases.

[0082] In addition to biological methods, approaches based on combinatorial chemistry techniques can be used to generate random peptides. Various methods have been developed to synthesize multiple homogeneous peptides that can be used in assays. These methods include multi-pin (multi-pin).
pin) synthesis (Geysen et al., Proc. Nat.
l. Acad. Sci. USA 81: 399
8, 1984; Valerio, Anal. Bioc
hem. 197: 168, 1991; Ray et al., T.
etrahedron Lett. 32: 6163,
1991) and "tea bag"
(Houghten et al., Proc. Natl. A.
cad. Sci. USA 82: 5131, 198
5; Houghten et al., Int. J. Pept.
Protein Res. 27: 673, 198
6). Multiple degenerate peptides can also be synthesized for use. Simultaneous attachment of a mixture of amino acids to a single resin support is a common strategy for synthesizing multiple peptides. Recently, soluble peptides (Hought
en et al., Nature 354: 84, 1991; Ho.
Ughten et al., Biotechniques 13:
412, 1992) and peptides attached to a solid support (Lam et al., Nature 354: 82, 199).
The combination library of 1) is “split synthesis (spli
t synthesis) method.
Peptide synthesis on beads containing an identifier for the peptide structure (PCTWO 93/06121).
Numerous modifications to these syntheses have been developed (for review, see Gallop et al., Supra).
One such identifier is an oligonucleotide sequence (Needels et al., Proc. Natl. A
cad. Sci. USA 90: 10700, 19
93). Other syntheses of random peptides are well known and can be readily performed by those skilled in the art.

[0083] Small peptides, natural or synthetic, may also be used as ligand inhibitors. CRF
Libraries of small molecules screened for ligand inhibitors of the / CRF-BP complex include soil samples, plant extracts, marine microorganisms, fermentation broths, fungal broths, pharmaceutical chemical libraries, combinatorial libraries (chemical and Biological)). Such libraries can be obtained from a variety of sources, both commercial and proprietary.

Molecules with similar but not identical structures to candidate compounds can be synthesized and tested for ligand inhibition as described below. The small peptide sequence is
It can be designed for the solution NMR structure of the CRF. As noted above, key contact residues in the CRF for binding to CRF-BP are residues 22, 23, and 25. Thus, the peptide sequence from residues 20-27 can be designed from the reference solution structure of human CRF, which structure is
1 H NMR and limited molecular dynamics (restra
Distance geometries with ined molecular dynamics
(Protein En)
ginering 6: 149, 1993). Intact CRF molecules are used as the basis for the design. The reason for this is that when the deletion is large, the α-helical structure of CRF may be lost. The 3-D structure of the small peptide spanning the key contact residues can then be used to search a computer bank for non-peptide molecules that mimic the peptide structure. Molecules with lower IC-50 values than the starting molecule are selected. Additional molecules are synthesized based on the starting compounds and the physical and biochemical properties of those molecules. Particularly preferred candidates have IC-50 values of 10 nM or less.

The determination of whether any of the inhibitors has the requisite properties depends on the CRF / CR of the inhibitor.
This can be done by assaying the ability to displace CRF from the F-BP binding complex and then assessing the ability of the inhibitor to bind the CRF receptor.

[0086] Candidate ligand inhibitors can be identified by CRF / CR by biological or in vitro assays.
One can screen for their ability to displace CRF from the F-BP complex. One suitable biological assay is the measurement of ACTH release from cultured pituitary cells. This assay is performed in the following manner.
Rat anterior pituitary glands were treated with sterile HEPES buffer for 6 hours.
Washed once and transferred to a solution containing collagenase. After subsequent transfer to a 25 ml Bellco dispersion flask, the pituitary is agitated at 37 ° C. for 30 minutes, ground, incubated for a further 30 minutes, and ground again. The partially dispersed cells are then collected by centrifugation. The cell pellet is resuspended in 10 ml neuraminidase and collected again by centrifugation. This pellet was mixed with 25 ml of BBM-P (BBM (I
rvine Scientific) +100 μg / L
Cortisol, 1 μg / L insulin, 0.1 μg / L
LEGF ₂ , 0.4 μg / L T ₃ , 0.7 μg / L
Reconstitute in PTH, 10 μg / L glucagon, and 2% fetal calf serum), centrifuge again, and finally reconstitute the resulting pellet in BBM-P. The cells are then plated at a density of 50,000-100,000 / well in 48-well plates and incubated for 2 days. On the day of the assay, cells are washed once with BBM-T in preparation for stimulation with peptide candidates or CRF. Cells are stimulated with a maximal stimulating dose of h / rCRF (1 nM) and ACTH release is measured by RIA or immunoradiometric assay. Inhibitory concentration CR
When F-BP is added, the amount of ACTH released decreases and is expressed as a fraction of maximum release. The ligand inhibitor is added at various doses. CRF
-The ability of the peptide to displace CRF from BP is measured by the amount of ACTH release expressed as a fraction of the maximum release caused by a given CRF alone.

The preferred mode of screening for candidate ligand inhibitors is by the in vitro ligand immunoradiometric assay (LIRMA). For LIRMA, CRF-BP can be isolated from brain tissue, serum or cells expressing a recombinant form. Recombinant hCRF-BP
Can be produced in Chinese hamster ovary (CHO) cells with the pSG5-hA3 and RSV-neo plasmids. A stable CHO transfectant is G418 (Sigma Chemical, S.D.).
t. (Louis, MO) by dilution under selection and maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine and 3% fetal calf serum. To scale up production of hCRF-BP, transfected CH
O cells were obtained from a 10,000 MWCO bioreactor (Cell Pharm Micro Mouse, U.S.A.).
nisyn Technologies, Tusti
n, CA). The enriched media is harvested daily from the bioreactor and stored at −20 ° C. until purification. Alternatively, a closed roller bottle containing recombinant cells and tissue culture medium
(rotate slowly in a 37 ° C. environment) may be used.

HCRF-BP can be purified by a three-step process in which the fraction from each step is evaluated using the following assay. First, the enrichment medium is Bio P
ilot chromatography equipment (Pharmacia)
LKB Biotechnology, Uppsal
a, Sweden). Human CRF was prepared using N-hydroxysuccinimide via an amino group via Affi-Prep 10
(Bio Rad Laboratories, Ric
hmond, CA). After binding, the affinity gel was run on XK16 or equivalent column (Pharm
acia LKB Biotechnology, Up
psala, Sweden). Affinity purification involves filtering the enriched media through a column at 2 ml / min, 10 bed volumes of 100 mM at 5 ml / min.
Washing with HEPES HCl (pH 7.5),
And 8% containing 20% acetonitrile at 5 ml / min.
0 mM triethylammonium formate (pH 3.
0) to elute fractions of one bed volume. Alternatively, under mild basic conditions (for example, a pH of about 1
The elution under 0.5) can be used.

The second purification utilizes gel chromatography. Affinity pure hCRF-BP is
Lyophilized and 0.1 M ammonium acetate (pH
4.75) reconstituted in 6M guanidine-HCl buffered. The FPLC apparatus consists of two Superose 12 HR connected in series for this purification step.
10/30 column (Pharmacia LKB B
iotechnology, Uppsala, Swed
en). H pure affinity
CRF-BP is filled in 1 ml, followed by 0.4 m
Elution is carried out at 1 / min with 6M guanidine HCl / 0.1M ammonium acetate (pH 4.75) and fractions are collected every minute.

The active fraction from the second purification is then subjected to a reverse phase HPLC. The HPLC instrument has two mods
el 100A pump (Beckman, Palo A
lto, CA), Axxiom HPLC controller (Cole Scientific, Calabas)
as, CA), Spectroflow 773 absorbance detector set (for 214 nm) (Kratos
Analytical, Ramsey, NJ), and Pharmacia. model 482 chart recorder (Pharmacia LKB Biotec)
hnology, Uppsala, Sweden). Buffer A is 0.1% trifluoroacetic acid (TF
A) / 5% acetonitrile, buffer B was 0.1%
% TFA / 80% acetonitrile. Subsequently, a 1 ml infusion of affinity-pure, same-size hCRF-BP was added to a semi-preparative C4 HPL under isocratic conditions at a flow rate of 2.5 ml / min in 25% B buffer.
Apply to column C (Vydac, Hesperia, CA). After passage of the final salt peak, a single gradient elution
Perform starting from 5% B buffer and increasing to 95% B buffer over 30 minutes. The dominant absorbance peak is then quantified by hCRF-BP IRMA and amino acid analysis.

In LIRMA, CRF-B isolated from brain tissue, serum, or cells expressing a recombinant form
P is added in binding buffer (0.02% NP-40 in 50 mM phosphate buffered saline) to the wells of a 96-well plate, small polypropylene microcentrifuge tubes, or borosilicate glass tubes. ¹²⁵ I-
h / rCRF (New England Nuclea
r) and 10 μM of candidate ligand inhibitor are added and the reaction is incubated for 1 hour at room temperature. Properly diluted anti-CRF-BP antibodies (eg, rabbit anti-hCRF-BP (Potter et al., Proc.
Natl. Acad. Sci. USA 89: 4
192-4196, 1992) to each tube, and after further incubation, the bound conjugate is precipitated by further adding goat anti-rabbit antibody. ^The precipitate containing ¹²⁵ I-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 pellet will contain less radioactivity as compared to the control without the addition of the candidate peptide. ¹²⁵ Ih / rCRF
Maximal inhibition of binding to CRF-BP (ie, 100
%) Is 10 μM CRF-BP peptide ligand h /
Defined by the amount of radioactivity remaining in the pellet after incubation with rCRF (6-33). Thus, the binding capacity of the candidate ligand inhibitor is the standard h /
Measured for rCRF (6-33) capability. Preferably, when a ligand inhibitor is present, there is at least 50% inhibition.

The binding affinity constant (K _i ) of the inhibition is important; the assay examines with appropriate expectation according to its value for K _i for human CRF found to be 0.17 + 0.01 nanomolar (nM). You. Thus, ligands with a K _i lower than the _h CRF K _i bind more strongly to hCRF-BP than hCRF itself, and ligands with higher values have a relatively lower binding affinity. Therefore, hC
Since it is desired to reasonably and effectively compete with hCRF for binding to RF-BP, the lower the reagent or peptide has a lower K _i value, the more beneficial for this purpose. Preferably, the reagent has a K of about 20 nM or less.
_{It has an i} value, more preferably a K _i value of about 10 nM or less, most preferably a K _i value of less than about 5 nM. hCRF (6-
33) was assayed and found to have a K _i value of 3.5 + 0.44 nM. This reagent also contains human CR
It has a low binding affinity for the F receptor (ie, a binding constant of inhibition greater than 1000 nM) and a CRF agonism (a
gonism) is considered to be an excellent choice for use in the method of the present invention. Human CRF
(9-33) also has a K _i value of 11 + 0.36 nM and is also very useful in the method of the invention since it also has a receptor K _i of more than 1000 nM and is a weaker CRF agonist It is. Other similar reagents and peptides (especially analogs of hCRF having between 19 and 28 residues) have been synthesized and tested in this simple manner, and the endogenous hCR in vivo
Their effectiveness in these beneficial methods for increasing the effective concentration of F can be measured. The peptides specifically recited herein appear to be particularly beneficial.
For ^{example, (Ile 8,19,24, Asn 17,26,} Met 18, G
lu ²⁷ , Arg ²⁸ ] -hCRF (4-28) is 1.7
Has a relatively low binding affinity for a K _{i of} +1.2 and the hCRF receptor.

Thus, high-throughput screening using the LIRMA assay as described or other methods involving ACTH release and a two-site ELISA has been used to replace small molecules that displace CRF from the CRF / CRF-BP complex. Can be identified. In the first round of screening, all potential candidates are assayed at a single dose of 10 μM. Any compound that gives more than 50% inhibition at 10 μM is then selected for further screening. The activity of all candidates meeting this criterion is confirmed by a second round of screening using a 6-point dose response curve. IC-50 values are calculated and candidates with values ranging from 10 μM to 100 μM are further tested, and the candidate compound is identified as CRF / CRF
Displacing CRF from the BP complex, and CRF-BP
Ensure that it does not interfere with antibody binding to The specific substitution of CRF is 0.2n instead of unlabeled CRF-BP.
LI except that M ¹²⁵ I-hCRF-BP was added.
Demonstrated in assays performed as described for RMA.

The ligand inhibitor may also bind to CRF
And free CRF are separated by surfactant phase separation
It can be screened by an in vitro assay. Simple
In brief, within one embodiment, isolated as described above
The CRF-BP thus obtained was added to a binding buffer (50 mM phosphate buffer).
In 0.02% NP-40) in opiated saline^{12 Five}
Ih / rCRF and 10 μM candidate ligand inhibitor
Incubate with bitter. 1 hour to 2 hours at room temperature
After incubation with the detergent (eg, Octi
Ruphenoxypolyethoxyethanol, Triton
X-114^TMIs commercially available as)
And mix by vortexing. Triton X-
114^TMAnd other nonionic surfactants
At temperatures above the cloud point it is insoluble in water. At this temperature,
Visual phase separation occurs. Below this temperature, the surfactant
Form a clear micellar solution, above which temperature clear
Two phases (one phase is depleted of surfactant and the other phase
Is concentrated in the surfactant). Tri
ton X-114^TMHas a cloud point temperature of 20 ° C. this
So, Triton X-114^TMIs preferred. parents
The CRF with the amphipathic α helix is Triton X-1
14^TMAre more soluble in the
Be placed. In contrast, CRF bound to CRF-BP
Are more soluble in aqueous solutions. Therefore, Triton
 X-114^TMPhase separation between water and aqueous solution
And free CRF. Phase separation is facilitated by centrifugation.
Well achieved. The aqueous phase (top) is removed¹²⁵I
The amount of -h / rCRF can be measured. Ligand inhibitor
Of radioactivity relative to radioactivity obtained in the absence of
At least, the ligand inhibitor is CRF-BP to CRF
Is replaced.¹²⁵C of Ih / rCRF
Maximum inhibition of binding to RF-BP (ie, 100%)
Is 10 μM CRF-BP peptide ligand h / rC
In the aqueous phase after incubation with RF (6-33)
It is defined by the amount of radioactivity. Therefore, the candidate ligand
Inhibitor binding capacity was determined by standard h / rCRF (6-3
It is measured for the ability of 3). Preferably, the ligand
At least 50% inhibition when inhibitor is present
Exists.

Furthermore, this assay has widespread use in screening for neuropeptide binding proteins in general. Some neuropeptides (eg, NPY)
Has similar physical properties to CRF. Because both are very hydrophobic and have an alpha helix. Thus, NPY is more nonionic than aqueous solution (eg, Triton X-11).
4 ^™ ) should be more soluble in The NPY or other neuropeptide of interest is generally Triton X-1
Assuming partitioning into the 14 ^TM surfactant phase, the above method can be commonly used to screen for neuropeptide binding proteins. Briefly, by way of example, tissues from various organs are 1% NP-40 / PB
Homogenized in S solubilization buffer. The particulate matter is 30
Removed by centrifugation at 00xg for 10 minutes. A 50 μl aliquot from the supernatant was
Incubated with ¹²⁵ I-labeled neuropeptide and the assay is performed as described above. Serum or plasma can also be used as a potential source of neuropeptide binding proteins. A predetermined concentration range (0.1 nM to 10
(00 nM) unlabeled neuropeptide is co-incubated with the radiolabeled neuropeptide to assess whether the putative binding protein specifically binds the radiolabeled neuropeptide. If the binding is specific, Triton X
-114 radioactivity remaining in the aqueous phase after ^TM separation decreases. Using this method, IC-50 values can be established for each of the neuropeptides and tissue extracts.

In addition, this method can be used to screen for neuropeptide ligand inhibitors of the neuropeptide binding protein. Briefly, a radiolabeled neuropeptide is incubated with a neuropeptide binding protein or a soluble receptor, and the reaction is performed as described above. These assays can use either recombinant neuropeptide binding proteins or receptors, or crude neuropeptide binding proteins isolated from tissue samples.

A preferred ligand inhibitor is CRF
It has either a low affinity antagonist effect on the receptor or has a 100-fold selectivity for CRF binding proteins. Thus, compounds with IC-50 values ranging from 10 μM to 100 μM and specific inhibition of the CRF / CRF-BP complex are further tested for binding to the CRF receptor. The ability of the ligand inhibitor to antagonize the CRF receptor is evaluated in a cAMP production assay. This assay increases the level of free CRF binding to the CRF receptor and compares the ability of the ligand inhibitor to stimulate cAMP production. The test cell line is CRF as a stable transfectant.
Express the receptor. This assay is based on the technique of Battaglia et al. (Synapse 1:
572, 1987). The test cells are
1 at various CRF and ligand inhibitor concentrations
Incubate for hours. The cells are washed and intracellular cAMP is released upon incubation of the cells for 16-18 hours, followed by 20 mM HCl, 9
Extracted in 5% ethanol. The lysate is lyophilized and subsequently solubilized in sodium acetate buffer. c
AMP levels can be determined using a single antibody kit (eg, Bio
medicalTechnologies (Stoug
hton, MA).

As an alternative to performing the competitive in vitro evaluation assays described above, ligand inhibitors can be evaluated in binding assays using the human CRF receptor. The human CRF receptor and the binding assay for such receptor and human CRF are
hen et al., Proc. Natl. Acad. Sc
i. USA 90: 8967-8971, 1993.
(This disclosure is incorporated herein by reference). The reagents were radiolabeled [Nle ²¹ ,
Tyr ³² ] oCRF can be used to calculate the binding affinity constant (K _i ) for inhibition. Preferably, the reagent is at least about 100 nM, more preferably 100 nM.
With a receptor K _i of more than 0nM. Alternatively, the use of the rat CRF receptor has been satisfactory.
This is because human CRF and rat CRF have the same amino acid sequence.

Further assays using rat anterior pituitary cells to measure ACTH secretion can be performed to determine whether a ligand inhibitor functions as a CRF agonist of the hCRF receptor. The technique used is generally as described above, except that only the ligand inhibitor is added to the cells. Antagonism can be determined by performing the assay in the presence of an inducing challenge dose of CRF. The performance of the ligand inhibitors may be a standard antagonist for this purpose (eg, CD-Phe ¹² , Nle ^21,38 ]-
rCRF (12-41) or α helix CRF (AH
C) compared to the performance of fragments (eg, AHC (9-41)).

The in vitro assay identified above for measuring CRF agonist and antagonist activity in terms of stimulating ACTH secretion is based on hCRF (6-
33) and hCRF (9-33). As a result of such an assay, hCRF (6-
33) is the standard oCR which is considered to be 1.0
It is shown to have a significantly lower CRF agonist biological activity than F. This peptide does not show any substantial CRF antagonist activity. This peptide is acceptable from this point of view as it has less than about 0.1% CRF agonist activity of the standard peptide. Peptide hCRF (9-33) accounts for 0.01% of the activity of oCRF
It is also a weak CRF agonist with substantially less. It is desirable that the reagents used do not bind strongly to the CRF receptor. It is generally believed that the reagent should have less than about 25% of the CRF agonist activity of oCRF and should not exhibit substantial CRF competitive antagonist activity. Preferably, the reagent is a standard peptide of the invention [DPhe ¹² , Nle ^21,38 ] -h
It should have less than 5% of the antagonist activity of CRF (12-41). However, since the potential blocking effect is minimized as a result of binding to the CRF receptor, if its value decreases in such an assay,
It should be understood that the reagents work better in this way.

In addition to providing a method of therapeutic treatment,
Thus, the present invention also provides for in vivo administration to mammals.
To select more effective CRF antagonists
To screen for peptides or other drugs
provide. Candidates for this screening procedure
The peptide is first evaluated in the well-known assay described above. This
Assay was performed with rat test pituitary gland at test dose of CRF
ACTH content from a culture of leaf (interior) cells
Measuring the biological effects of candidate peptides that inhibit stimulation of ligation
It is done by setting. Then, the candidate peptide
Is the K_iHCRF-BP to determine
Assessed in a competitive binding assay. K_iIs h as described above.
Indicator of binding affinity to CRF-BP, which is
Injection peptide from the target cell site to which the selected peptide is directed
Shows a tendency for removal of tide. The conclusion of these two assays
Particularly effective CRF antagonists based on fruit evaluation
May be selected. This antagonist is a CRF anta
Gonism assay has a high value,
F-BP) K_iAlso has a relatively low hCR
It shows that it shows the binding affinity to F-BP. Current
CRF antagonist [D-Phe
¹², Nle^{twenty one},³⁸] -HCRF (12-41)
Measured by ACTH secretion from cultured pituitary cells
Have very good antagonist properties when
, K_iThe value is about 60 ± 10 nM. The (CRF
-BP) K_iIs 300 ± 20 nM. Another good
A CRF antagonist, namely AHC (9-41),
This is not as effective as the current standard, but very low
K of 0.10 ± 0.036 nM_iHas a value. [D
-Phe^{1 Two}, Nle^{twenty one},³⁸, CML³⁷] -HCRF
(12-41) shows that ACTH secretion is up-regulated in pituitary cell culture.
When Say was performed, it was greater than 1000 nM (CRF
-BP) K_iAnd 45 ± 11 nM IC₅₀To
Had. Therefore, more than the current experimental standard
Positioned high. Therefore, these screening
Based on the assay, the latter peptide or this experimental standard
The quasi product is a better choice of pep than AHC (9-41)
And complexed by hCRF-BP in vivo
And have a greater tendency to be removed. This
Thus, this screening assay is newly synthesized
Screened peptides for in vivo treatment
These pairs as potential CRF antagonists
Valuable for assessing the relative value of a peptide overall
Provide tools.

(Elevation of CRF level)
Methods are provided for increasing the level of free CRF in the brain via administration of a ligand inhibitor of the F / CRF-BP complex. Elevated levels of free CRF can be measured by in vitro assays (eg, ELISA, stimulation of ACTH release,
Or stimulation of cAMP production). In any of these assays, the increase in free CRF upon administration of the ligand inhibitor is measured relative to a reference ligand inhibitor, in this case h / rCRF (6-33). The minimum acceptable increase is h / rC
10% of the value for RF (6-33). Moderate values are 50%, preferred values are 80%, and particularly preferred values are 100%.

Within the scope of the method described herein,
The level of free CRF is higher than that of brain samples, cerebrospinal fluid or other body tissues and homogenates of body fluids.
It can be measured by site ELISA. Total CRF is first quantified as follows. The wells of the ELISA plate are coated with an anti-CRF antibody (eg, protein G purification-sheep anti-
CRF). Wash the plate and block unbound sites on the plate with extraneous proteins (eg, casein, bovine serum albumin, etc.). A prepared tissue sample and a standard containing a known amount of CRF are added to the wells and allowed to bind at room temperature. After binding, the plates are washed again and a different anti-CRF antibody (eg, RC-70, rabbit anti-human CRF antibody) is added. RC-70 not only is obtained from different species, but also detects different epitopes other than the sheep anti-CRF used to coat the plate. After washing, RC-70
Is added.
Alternatively, an RC-70 antibody can be conjugated to the enzyme. Suitable conjugates are horseradish peroxidase and alkaline phosphatase, but those skilled in the art will recognize that many different and acceptable alternatives (including radiolabels instead of enzymes) are available. An enzyme substrate is added and color is developed. After completion of the reaction, the total amount of CRF present in the tissue sample is quantified using absorbance measurement. One skilled in the art will recognize that monoclonal antibodies or antibody fragments can be used in place of polyclonal antibodies in this assay.

In the same manner, the plate was placed on the anti-CRF-BP
Bound CRF can be quantified by ELISA, coated with antibody and then detecting bound CRF with an anti-CRF antibody. Bound CRF is CRF-BP ligand α
It is specifically displaced by the helix oCRF (9-41) and reduces the signal detected. α helix oCRF
(9-41) is used for substitution since it does not cross-react with RC-70 (anti-CRF antibody). Furthermore, the displaced CRF in the supernatant was then analyzed by the two-site ELISA described above.
Can be assayed. The free CRF then becomes the total C
By calculating the difference between RF and combined CRF,
Alternatively, it can be measured by a direct assay. In a direct assay, after capturing the binding complex with an anti-CRF-BP monoclonal antibody, the supernatant is removed and free CRF is measured in a two-site ELISA as described above.

It has been found that the ligand inhibitor α-helical sheep CRF increases the level of free CRF in brain tissue of both normal individuals and patients with Alzheimer's disease.
Shown herein. A two-site ELISA was used to determine the amount of total and bound CRF. Addition of alpha helix sheep CRF released all bound CRF (see FIG. 2 and Example 5).

In addition to the described in vitro assays that measure the amount of total CRF, bound CRF and free CRF in tissue or cerebrospinal fluid, other procedures can be performed in vivo. These include MRI, PETSCAN, spectral scanning or other similar imaging techniques, some of which are
A radiolabeled ligand for the RF-BP or CRF receptor is used. Preferred methods include PET positron-emitting ligands (eg, ¹¹ C, ¹⁸ F) or single photon emitting ligands (eg, CRF-BP or CRF receptor).
Image analysis using ( ¹²³ I-labeled ligand).
Free CRF levels correlate with the amount of radiolabeled ligand bound. Elevated free CRF levels are manifested by reduced binding of radiolabeled ligand to CRF-BP and CRF receptors. Within this imaging technology,
Elevated levels of free CRF of about 10-30% or more are sufficient in the context of the present invention.

In the context of the present invention, CRF / CRF
The administration of an effective amount of a ligand inhibitor of the -BP complex can be used to treat a disease or syndrome in which CRF levels are reduced. CRF levels can be measured directly in the cerebrospinal fluid or in the brain by imaging methods or by other methods (eg, cAMP production, ACTH release or 2-
Site ELISA). Such diseases or syndromes include: dementia or symptoms of learning and memory loss, obesity, chronic fatigue syndrome, atypical depression, postpartum depression, seasonal depression.
pressure, hypothyroidism, post-traumatic stress syndrome, nicotine withdrawal symptoms, susceptibility to inflammatory disease (vulnerability to inflamm)
atry disease). The definition of these syndromes (except for obesity, chronic fatigue syndrome, and susceptibility to inflammatory diseases) is defined as Diagnosis and St.
atomic Manual of Menta
l Disorders (4th edition), American
Psychiatric Association,
Washington, D.C. C. , 1994 (hereinafter DSM-IV).

(Improvement of Learning and Memory) As described above, the present invention provides a method for improving learning and memory by administering to a patient a therapeutically effective amount of a ligand inhibitor of the CRF / CRF-BP complex. . Such patients can be identified through clinical diagnosis based on symptoms of dementia, or learning and memory loss. Individuals with amnesia have an impaired ability to learn new information or cannot remember previously learned information or past events. Memory loss
This is most evident in tasks requiring spontaneous recall, and also when the tester provided the individual with a stimulus to recall at a later point in time. Memory impairment must be sufficiently severe to cause significant impairment of social or occupational function, and exhibit significant changes from previous functional levels.

[0109] Dementia is characterized by a number of clinically important cognitive deficits that represent significant changes from previous functional levels. Memory deficits, including the inability to learn new subjects or forgetting previously learned subjects, are required to make a diagnosis of dementia. Memories can be formally tested by asking individuals to record, retain, recall and recognize information. Diagnosis of dementia also requires at least one cognitive impairment: aphasia, apraxia, agnosia,
Or failure of execution function. Language, motor activity,
These deficits in object recognition and abstract thinking
Must be severe enough to cause an impairment of occupational or social functioning in association with memory deficits, and must indicate a decline from previous higher functional levels.

In addition, a number of biochemical tests that correlate the level of CRF with impaired learning and memory can be utilized. For example, the level of free CRF in cerebrospinal fluid can be measured by ELISA or RIA. Additionally or alternatively to this assay, the CRF-
Free receptor or CRF-BP can be quantified using brain imaging using a labeled ligand specific for the BP or CRF receptor. Therefore, it can be known that the free CRF has decreased. Finally, imaging of the brain with ligands specific for unbound CRF can be used to directly assay the amount of free CRF in the brain.

Minimenta of the patient
l) State is recorded in learning and memory mini-mental tests. This test is a standard test used by clinicians to determine whether a patient has impaired learning and memory (Folstein et al., J. Ps.
ychiatric Res. 12: 185, 19
75). This exam includes a number of simple tasks and written questions. For example, the learning abilities of "pair allies" are impaired in some types of amnesic patients, including those with head trauma, Korsakoff's disease, or stroke (Squ
ire, 1987). 10 sets of unrelated words (for example,
Have the subject read an army-table). The subject is then asked to remember the second word when given the first word of each set. The measure of memory deficits is the reduced number of remembered paired associates' words corresponding to the matched control group. It serves as an indicator of a short-term working memory of the type that rapidly declines in the early stages of a dementia-prone or amnesic disorder.

Improvements in learning and memory were attributed to (a) a statistically significant difference between the work of the ligand-inhibitor treated patients as compared to the work of members of the placebo group; or, (b)
A statistically significant change in work towards normality on a scale appropriate to the disease model. This strategy has been successfully applied to identify therapeutically useful cholinergic substances to improve memory. Animal models or clinical examples of the disease show symptoms that are, by definition, distinguishable from normal controls. Thus, a measure of effective pharmacotherapy is a significant, but not necessarily complete, reversal of symptoms. Clinically effective "cognition-enhancing" agents that act to improve the performance of memory tasks may facilitate improvement in both animal and human models of memory pathology. For example, cognitive enhancers that act as cholinergic agents in replacement therapy in patients suffering from Alzheimer-type dementia and amnesia significantly improve short-term working memory in paradigms such as the anti-allergic task (Davidson and Stern, 1991). ).
Other potential applications for therapeutic intervention for memory deficits include:
With a long-term study of recent memory in aging mice,
Age-related deficits in work that are modeled effectively (Forster and Lal, 199).
2).

In animals, several established models of learning and memory are utilized to examine the beneficial cognitive enhancing and potential anxiety-related side effects of activation of CRF-sensitive neurons. The cognition-enhancing effect can be seen in the Morris maze (Stewart and Morris, Behavi
oral Neuroscience, R.A. Sag
hal (IRL Press, 1993) 107
P.) And the Y maze (Brits et al., Brain Re).
s. Bull. 6, 71 (1981)); anxiety-related effects are elevated plus maze (el
Evaluated plus-maze test (Pellow et al., J. Neurosci. M.)
eth. 14: 149, 1985).

The Morris water maze is one of the most effective models of learning and memory and is sensitive to the cognitive enhancing effects of various pharmacological agents (McNamara and Skelton, Brain Res. Rev.
18:33, 1993). Tasks performed in the maze are particularly sensitive to manipulating the hippocampus of the brain. The hippocampus is an important area of the brain for spatial learning in animals and memory enhancement in humans. Furthermore, improvements in Morris water maze behavior predict the clinical efficacy of compounds as cognitive enhancers. For example, treatment with cholinesterase inhibitors or selective muscarinic cholinergic agonists reverses learning deficits in the Morris maze animal model for learning and memory, as well as in clinical populations with dementia (MacNamara)
And Skelton, 1993; Davidson and Stern, 1991; McEntee and Cr.
book, 1992; Dawson et al., 1992). In addition, this animal paradigm shows the extent to which age-related defects increase (Le
vy et al., 1994) and increased susceptibility of memory traces to delays or interference in preliminary testing (vulnera).
(Billy) (Stewart and Morri)
s, 1993), which is characteristic of amnestic patients.

This test is a simple spatial learning task, where the animal is placed in lukewarm water that is opaque by the addition of powdered milk. The animal learns the position of the platform in relation to the maze and the visual cues located in the test room; this learning is called place learning.

This will be described in more detail below (see Embodiment 6).
As such, 15 minutes prior to each training on days 1-3, groups of animals received control solution or 0.1, 1,.
5 or 25 μg of ligand inhibitor peptide h
/ RCRF (6-33) or h / rCRF (this is
In addition, an ICV injection of (which is an agonist at the CRF receptor) is given. If a non-peptide inhibitor is used, the injection volume will be adjusted accordingly. Control animals are typically 5 to 10 days after 3 days of training.
Arrives at the platform within seconds. A measure of the memory-modulating effect of a ligand inhibitor is this shift in time. Synaptic CR by administration of ligand inhibitor
There is a dose-dependent increase in the effectiveness of F and a dose-dependent increase in behavior in acquisition and memory retention. H / rCRF and h / rCRF (6-33) before daily test
Administration significantly enhanced learning in the Morris water maze test (FIG. 4, upper panels (FIGS. 4A and B)). To produce increased learning and memory, a slightly higher CRF (6-3
The dose of 3) was required.

The Y-maze test based on visual discrimination is another assay for learning and memory in animals. In this maze, the ends of the two arms of the maze are translucent plastic panels, behind which there is a 40 watt light bulb. The start box is separated from the third arm by a manual guillotine door. In the first trial, all animals are allowed to explore the maze for 5 minutes and food pellets are available in each arm. On the second day,
Place each animal in the start box with the door closed. When you open the door, the animal moves along the arm,
Food pellets located on both arms can be consumed. On the third day, six trials are performed in groups of three. At this time, one arm is closed at the selected point, there is no discernible stimulus, and 2
Two diet pellets are available in the open goal box. On days 4-10, the light at the end of the arm with the food pellet is illuminated and again 10 trials are performed in groups of 3 animals. The time required for the animal to reach the diet pellet is recorded.

The effects of ligand inhibitors on improving learning and memory in the Y maze are tested as follows.
15 of the training trial on days 4-10 of each block
Minutes before, animals receive control or 1, 5, or 25 μg of peptide ligand inhibitor ICV
Perform injection. In addition, if a non-peptide ligand inhibitor is used, the dose will be adjusted accordingly. Control animals are expected to make 50% of the correct selections. A measure of the efficacy of a treatment on memory is an increase in the correct response. Administration of the CRF (6-33) ligand inhibitor daily prior to the test showed a significant increase in correct response (FIG. 5).

The elevated plus maze test is an anxiogenic response in approach avoidance situations.
response, this situation includes spaces that radiate open light to dark enclosed spaces. Both spaces are elevated and are assembled as two runways intersecting in the form of a plus sign.
This type of avoidance situation is a classic test of "emotional" and is very sensitive to disinhibition and stress-producing treatments. The animal is placed in the center of the maze and has free access to all four arms during the 5 minute test period. Record the time spent in each arm.

The daily pre-test administration of a dose of h / rCRF by ICV infusion, which increases learning and memory, also
This caused anxiety as indicated by the results of the elevated plus maze test (FIG. 4, lower panels (FIGS. 4C and D)). A dose-dependent suppression of exploration was observed. Thus, loss of memory and learning (This is because the level of CRF is decreased) CRF receptor agonists for the treatment of (i.e., K _i ≦ 1 nM) utility of, for the side effects associated with this, unambiguous Worth it. In marked contrast, the ligand inhibitor CRF (6-3
3), which has low affinity for the CRF receptor, did not alter anxiety behavior in the elevated plus maze test (Figure 4, lower panel). Furthermore, h / r
There were no apparent behavioral changes as shown by administration of CRF. These data indicate that cognitive enhancement and anxiety effects can be controlled separately. These data also
9 shows therapeutic value of administering a ligand inhibitor of the CRF / CRF-BP complex.

[0121] Methods for improving learning and memory in humans can be measured by tests such as the Wexler memory scale or the paired associates memory task. The Wexler memory scale is a widely used pencil and paper test of cognitive function and storage capacity. In a normal population, the standard test gives an average of 100 and a standard deviation of 15.
Therefore, in mild amnesia, this score can be detected by a reduction of 10 to 15 points, and in more severe amnesia, 2 points can be detected.
0-30 points, etc. (Squire, 198
7). During clinical interviews, including mini-mental exams, Wexler memory scale, or paired associate learning,
A series of non-limiting tests apply to the diagnosis of symptomatic memory loss. These tests provide general sensitivity to both general cognitive deficits and specific loss of learning / storage capacity (Squire, 1987). Apart from the specific diagnosis of dementia or amnesia, these clinical instruments also identify age-related cognitive decline. This cognitive decline reflects an objective decline in mental function as a result of the aging process that is within the normal limits imposed on the age of the person (DSM IV, 1994). As described above, an "improvement" in learning and memory is associated with a statistically significant difference between, for example, the behavior of a ligand inhibitor-treated patient and a member of the placebo group, or between successive trials given to the same patient. It is within the scope of the present invention if present in the direction of normality in the alliance test.

(Decreased Food Intake) As described above, the present invention provides a method for reducing food intake through administering to a patient a therapeutically effective amount of a ligand inhibitor of the CRF / CRF-BP complex. provide. Such patients can be identified by being obese. Obese individuals,
A weight index that is heavier than the target weight considered normal for the person's age, gender, and height and is objectively the 85th or higher percentile (B) of the same reference population
MI-calculated as weight (Kg) / height (m) ² )
(National Center
for Health Statistics, “O
bese and Overweight Adult
s inthe United States. "First
Vol. 1, Book BO, U.S. S. Government
Printing Office, Washington
on D. C. , 1983). Further, evidence that CRF is involved in a particular individual can be obtained by showing a decrease in CRF levels in cerebrospinal fluid or by brain imaging as described above. Because the hypothalamus is a common brain area that mediates the effects of CRF on food intake and endocrine parameters, changes in pituitary hormone concentrations may also reflect changes in levels of hypothalamic CRF.

A decrease in food intake can be measured by a delay in starting a meal and an overall decrease in the duration or amount of food consumption. Smith, “Satiety and Pr
Oblem of Motivation, "D.
W. Pfaff (ed.), ThePhysiological
al Mechanisms of Motivati
on, Springer-Verlag, New
York, 133-143, 1982. further,
The selection of specific nutrients under a food selection situation serves as a supplementary measure of specific hunger (Rozin, Adv. S.
study Behav. 6:21, 1976).

There are two established animal models of appetite regulation.
The first is a simple measure of food intake, and the second is
It is a measure of food self-selection in a cafeteria environment.
In the first method, food intake is restricted for 24 hours, and then the animals are allowed to access pre-weighed experimental food in their home cages for 2 hours. Food intake is 60 minutes and 1
At 20 minutes, measured by weighing the remaining pellets. These tests can also be performed on obese animals due to genetic mutations, which effectively replicates the symptoms of overeating and confused nutritional choices (Argiles, P.
log. Lipid Res. 28:53, 19
89; Wilding et al., Endocrinol.
132: 1939, 1993).

In a cafeteria environment, food is specially formulated with different proportions of macronutrients (eg, carbohydrates, proteins, and fats) to create a preference for specific nutrients based on sensory triggers or benefits after food intake. Measurement was made possible. Food choices vary, in part, with a wide variety of neurochemical systems. These tests are
Useful for detecting subtle changes in food intake regulation. Food intake regulation strongly affects phenomena such as aspirations or fuss and is associated with the diagnosis of eating disorders (eg, anorexia nervosa and obesity). After establishing the animal baseline, each animal was given an ICV infusion of a control solution or a peptide ligand inhibitor at a dose of 1, 5, or 25 μg, or a non-peptide ligand inhibitor at an appropriate dose, 15 minutes prior to testing. Receive. Food intake is measured as described in the intake test or self-selection of food in a cafeteria environment, and test results are compared to baseline.

In humans, obesity is not only associated with overeating, but may also be associated with the consumption of nutritionally unbalanced foods such as disproportionately large intakes of sweet or fatty foods. (Drewnowski et al., Am.
Clin. Nutr. 46: 442, 198
7). Thus, the clinical manifestation of dietary control is easily detected by using a controlled experimental food or a cafeteria self-selection protocol that records the intake pattern in terms of quantity, mealtime and choice. (Kiss
ileff, Neurosci. Biobeha
v. Rev .. 8: 129, 1984). In these tests, measures of food intake or self-selection in a cafeteria environment are determined after each individual's baseline is determined. The improvement associated with obesity treatment constitutes a decrease in body weight or food intake as represented by the treated patient as compared to members of the placebo group. In addition, this strategy has successfully identified serotonergic agonists for obesity.

(Disease Related to Low Level CRF) As described above, the present invention provides a method for treating a disease associated with low level CRF by administering a therapeutically effective amount of a ligand inhibitor of the CRF / CRF-BP complex to a patient. Provide a way to Such patients can be identified by the diagnosis of cognitive disorders such as intake disorders, neuroendocrine disorders and Alzheimer's disease. In addition, other conditions associated with decreased CRF levels, such as atypical depression, seasonal depression, chronic fatigue syndrome, obesity, vulnerability to inflammatory disease, post-traumatic stress disorder, psychostimulant withdrawal symptoms, and thyroid dysfunction Frequently, there is a manifestation of the disease profile and reduced stress system activity. These are characteristically identified by reduced urinary free cortisol and plasma ACTH. As such, these diseases and conditions are associated with CRF in the brain.
It is likely that some will be resolved by levels of repair or efficacy (Chrousos and Gold, JAMA
267: 1244, 1992).

[0128] Such various disease states in humans are associated with pituitary-adrenal axis dysregulation (dysre), which is thought to be accompanied by brain CRF derangement.
gulation). Therefore,
By experimentally altering the CRF / pituitary-adrenal system in experimental animals, the syndrome described above, ie, behavioral despair, (Pepin et al., 199).
2) Fatigue to exercise (Rivest and Ric
Hard, 1990), obesity (Rothwell,
1989) and hyperarousals associated with psychostimulant withdrawal symptoms (Koob et al.,
The fact that the main features of Swedlow et al., 1991) are reproduced suggests the widespread utility of drug treatments designed to normalize endogenous CRF levels.

A major feature of seasonal depression (severe depressive disorder with a seasonal pattern) is the onset and remission of severe depression symptoms at characteristic times of the year. In most cases, onset begins in the fall or winter and remits in the spring. Severe depression episodes that occur in a seasonal pattern are often characterized by significant anergy, excessive sleep, overeating, weight gain, and craving for carbohydrates and last for at least two weeks. During this time, almost all activities become depressed or lose interest or joy.

The main features of post-traumatic stress disorder include direct personal experience of events involving actual or moribund conditions or severe injury to the physical integrity of the individual or others. The characteristic symptoms develop after exposure to extreme trauma stressors. The personal response to this event included intense fear, helplessness, or fear. The traumatic event is re-experienced as a memory or nightmare that persists when paid, and triggers a severe psychological distress or physiological response. Complete symptomatic conditions exist for over a month, causing clinically significant distress or impairment of social or occupational functioning.

Nicotine withdrawal symptoms (nicotine-induced disorder)
The main feature of is the use of products containing nicotine for a long time (at least a few weeks) and the presence of a characteristic withdrawal syndrome that develops after suddenly discontinuing or reducing the number of uses is there. To be diagnosed with nicotine withdrawal symptoms are four of the following: mental anxiety or depressed mood, insomnia, tantrum or anger, anxiety, inability to concentrate, restless or impatient, decreased heart rate and increased appetite or weight gain More than one needs to be true. These symptoms cause clinically significant distress or impaired social or occupational functioning.

The improvement is (a) a statistically significant change in the symptoms of the treated individual compared to a baseline or pre-treatment condition based on a scale associated with the disease model, or
(B) Consisting of any of the statistically significant symptom differences between patients treated with the ligand inhibitor and members of the placebo group. Clinical cases of the disease have, by definition, symptoms that are distinct from normal controls. For depression, several depression rating scales are used. (Kl
erman et al., Clinical Evaluatio
no of Psychotropic Drugs:
Principles and Guideline
s, Prien and Robinson (eds.), R
aven Press, Ltd. , New Yor
k, 1994). Depression was evaluated using the Hamilton Rating Scale (Hamilton Rat).
ing Scale for Depression)
Test is widely used. This test has also been used to assess changes in symptoms in treatment response. For other tests and evaluations, see DSM-IV
It is described in the manual. For nicotine withdrawal symptoms and other disorders, tests for assessing the severity of the disorder are described in the DSM-IV manual.

(Alzheimer's Disease) As described above, the present invention provides a method for treating Alzheimer's disease by administering a therapeutically effective amount of a ligand inhibitor of the CRF / CRF-BP complex to a patient. Such patients are identified by clinical diagnosis based on symptoms of dementia or learning and memory loss that are not due to other causes. Further, the patient can be identified as a patient by diagnosing brain atrophy by nuclear magnetic resonance imaging.

[0134] Reduced CRF levels have been shown to be involved in Alzheimer's disease. Postmortem brains from 10 individuals with AD and 10 neurologically normal controls were selected for study. Standard areas of the frontal, parietal, temporal and occipital poles were cut from fresh brain, frozen in dry ice and stored at -70 ° C before CRP radioimmunoassay and CRF-BP. Processed by assay. Formalin-fixed samples of cerebral cortex and hippocampus were embedded in paraffin and subsequently sectioned and stained by hematoxylin / eosin and silver immersion. When a stained section obtained from the brain of an AD patient was examined,
AD showed abundant neuritic plaques and neurofibrillary tangles typical of AD, but none were shown in control patients.

CRF and CRF-BP levels were measured in the cerebral cortex of Alzheimer patients and controls. CRF-BP was previously identified and characterized in rat brain, sheep brain and human plasma. In the cerebral cortex of the brain studied here, most (> 85%) of CRF-BP was associated with the membrane. Solubilized CR from either control or AD human brain membrane
The pharmacological characteristics of F-BP are that soluble plasma CRF-BP
Found no difference in binding properties to CRF and ligand inhibitors as compared to the recombinant form of In the brains of Alzheimer's patients, CRF levels in the frontal, parietal and temporal cerebral cortices were significantly reduced when compared to normal brain, while levels in the occipital cortex were only slightly reduced (FIG. 3A). In contrast, CRF-BP levels were similar in the brains of AD patients and normal control brains (FIG. 3B). These data show that normal and AD brain tissues have CRF-B
Is direct evidence that P exists, and A
D suggests that the decrease in CRF levels observed in D was not due to neuronal loss, but could be due to decreased CRF synthesis or increased degradation. When neurons are lost in AD, CRF-BP becomes non-CR
It can be preferentially localized for F neurons.

Complexed CRF against CRF-BP
Of the ratio of brain tissue and free pool
l), the C in human brain tissue
The nature of the interaction between RF and CRF-BP was determined. Using an assay specific for total CRF and bound CRF, normal cerebral cortex extracts showed about 40% of total CRF,
Approximately 60% of total CRF in Alzheimer's cerebral cortex extract
Was found to be complexed with CRF-BP.
In addition, CRF was reversibly bound to CRF-BP. Treatment of tissue with human CRF (6-33) or α-helical oCRF (9-41) results in CRF / C
This is because CRF was displaced from the RF-BP complex (FIG. 2). These data directly indicate that CRF-BP ligand inhibitors increase the free concentration of CRF. In addition, treatment with CRF-BP inhibitors results in free C
Free CRF levels in AD brains were elevated relative to RF levels.

Several animal models of Alzheimer's disease that have been established with a focus on cholinergic reduction are available. The major role of hypocholinergic in AD is well established. In AD, there is a significant positive correlation between reduced choline acetyltransferase activity in the frontal, occipital and temporal lobes and reduced CRF levels (DeSouza et al., 1986). Similarly, there is a negative correlation between a decrease in choline acetyltransferase activity and an increase in the number of CRF receptors in these three cortices (Id). In the other two neurodegenerative diseases, a very significant correlation between CRF and choline acetyltransferase activity is seen in Parkinson's disease, but only marginally in progressive supranuclear palsy (Whitehouse et al., 1987).

Anatomic and behavioral studies in rats demonstrate the interaction between CRF and the cholinergic system. First, in some brain stem nuclei, CR
F and acetylcholinesterase are co-localized, and some cholinergic neurons contain CRF. Second, CRF inhibits carbachol-induced behavior, which is an antagonist of the muscarinic cholinergic receptor, suggesting that CRF affects the cholinergic system (Crawley et al., Peptides). 6: 891,
1985). Treatment with atropine, another antagonist of muscarinic cholinergic receptors, results in increased CRF receptors (DeSouz
a and Bartaglia, Brain Res.
397-401, 1986). Taken together, these data indicate that CRF and the cholinergic system interact similarly in humans and animals.

Scopolamine is a non-selective antagonist of the postsynaptic muscarinic receptor that blocks the stimulation of the postsynaptic receptor by acetylcholine. By administering scopolamine, an animal model of Alzheimer's disease is obtained that focuses on reduced cholinergic activity. In these animals, passive avoidance or delayed-matching-to-
Loss of memory is readily apparent as measured by the position test. These tests distinguish between the hypomotility or hypoesthesia of the experimental treatment and amnesia or cognitive enhancing effects. Thus, using the Morris maze test and the Y-maze test after scopolamine-induced amnesia, memory loss and memory enhancement after subsequent administration of a ligand inhibitor are tested. Morri
In the s maze, the experiment is designed essentially as described above, but is treated on days 1 to 3 daily with an ip injection of scopolamine hydrobromide 30 minutes before training (0.3 mg).
/ Kg). Scopolamine at amnesic doses impairs the acquisition and retention of the spatial and avoidance learning paradigms in rats. 1 μg, 5 μg or 25 μ of peptide ligand inhibitor
The anti-amnesic effect of g or an appropriate dose of a non-peptide ligand inhibitor is measured relative to a concurrent control group with or without scopolamine. Evaluation of ligand inhibitors for reversibility of scopolamine-induced amnesia using the Y-maze is performed in the same manner as the Y-maze test described above. On days 5-10,
The test is modified to be treated daily (30 mg / kg) with ip injection of scopolamine hydrobromide 30 minutes before training. 1 μg, 5 μg or 25 μg of I
The anti-amnesic effect of a peptide ligand inhibitor administered CV or an equal dose of a non-peptide ligand inhibitor administered centrally or systemically is measured in comparison to a concurrent control and scopolamine-treated control group.

Several test designs have been designed to measure cognitive behavior in AD. (Gershon et al., Cl.
initial Evaluation of Psyc
hotpic Drugs: Principle
s and Guidelines, Prien and Robinson (eds.), Raven Pres.
s, Ltd. , New York, 1994,
p. See 467. 1) BCRS, one of these tests, measures concentration, memorization, past memory, adaptability, functionality and self-care. BC
RS is designed to measure cognitive function only. This test and the Weschler memory scale (Wesch
er Memory Scale) and the Alzheimer's Dis-related scale (Alzheimer's Dis).
ease-Associated-Scale) can be used to measure the improvement after therapeutic treatment due to ligand inhibition. As described above, in the Wechsler memory scale test, a statistical comparison of the normality, for example, when comparing the behavior of a patient treated with a ligand inhibitor with members of a placebo group, or between subsequent tests performed on the same patient. If a significant difference is observed, “improvement” in Alzheimer's disease is considered an intended improvement of the present invention.
In addition, scopolamine-induced amnesia in humans can be used as a model system to test the efficacy of ligand inhibitors.

(Administration of Ligand Inhibitors) As used herein, the terms “pharmaceutically acceptable”, “physiologically acceptable”, and grammatical variants thereof may refer to compositions, carriers, dilutions The terms liquid and drug are used interchangeably. Preferably, these substances can be administered to mammals without producing undesirable physiological effects such as nausea, dizziness, upset stomach and the like.

The patient is administered a therapeutically effective amount of a ligand inhibitor of the CRF / CRF-BP complex. A therapeutically effective amount refers to increasing brain free CRF levels, improving learning and memory, reducing food intake, brain CRF
Activate neural circuits, treat diseases involving low CRF levels in the brain, treat symptoms related to Alzheimer's disease, treat obesity, treat dysmorphic depression, treat substance abuse withdrawal symptoms, postpartum depression Or treating age-related memory loss is the amount calculated to achieve any desired effect. It will be apparent to one of skill in the art that the route of administration may vary depending on the particular treatment, and whether a peptide ligand inhibitor or a non-peptide ligand inhibitor is administered. The route of administration may be non-invasive or invasive. Non-invasive routes of administration include oral, buccal / sublingual, rectal, nasal, topical (including transdermal and ocular), vaginal, intravesical and pulmonary and the like. Invasive routes of administration include ICV, intraarterial, intravenous, intradermal, intramuscular, subcutaneous, intraperitoneal, intrathecal, and intraocular.

In animals, intraventricular (IC)
V) Perform injection. Anesthetize animal with halothane, KOP
Fix to the F localization. With two stainless steel screws and dental cement, a targeted guide cannula over the lateral ventricle is implanted and secured in the skull. 6 for injection
A 30 gauge stainless steel cannula attached to a 0 cm PE 10 tube is inserted through the guide until it exits 1 mm from the tip. Inject 2 microliters of ligand inhibitor over 1 minute by gravity flow by simply raising the tube above the animal's head until flow occurs. Procedures for other routes of administration are well known in the art.

The required dosage may vary with the particular treatment and route of administration. Generally, the dose of the peptide ligand inhibitor is about 50-125 μ / 1.5 g of brain tissue.
g, i.e. an amount such that a final concentration of 15-38 nmol per 1.5 g tissue is obtained. However, larger or smaller doses may be used depending on the size of the protein or polypeptide. These treatments are performed a few times a week. The treatment may need to be continuous to maintain therapeutic efficacy. The patient is monitored by assessing CRF levels in the cerebrospinal fluid or brain by imaging as described above. In addition, patients are monitored by assessing behavior under various tests as described for each treatment.

[0145] Therapeutic administration is performed under the guidance of a physician, and the pharmaceutical composition contains 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 include physiologically buffered saline, buffers such as phosphate buffered saline, glucose, mannose, sucrose, carbohydrates such as mannitol or dextran, amino acids such as glycine, antioxidants. Agents, chelating agents such as EDTA or glutathione, adjuvants and preservatives. Acceptable non-toxic salts include acid addition salts or, for example, zinc, iron, calcium, barium, magnesium,
Metal complexes with aluminum and the like (considered as additive salts for the purposes of the present invention). Examples of such acid addition salts include hydrochloride, hydrobromide, sulfate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, Examples include acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. When the active ingredient is administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrant such as alginic acid; a lubricant such as magnesium stearate. If administration in liquid form is desired, sweetening and / or flavoring agents may be used, and intravenous administration in isotonic saline, phosphate buffer may be effective.

The peptides administered under the guidance of a physician are usually in the form of a pharmaceutical composition containing a ligand inhibitor and a conventional pharmaceutically acceptable carrier. Typically, dosages will be from about 1 to about 1000 micrograms of peptide / kg of host animal body weight per day, often between about 100 μg and about 1 mg, but can vary up to about 10 mg. Treatment of subjects with these peptides can be used to reduce symptoms or correct the onset of adverse symptoms that are characteristic of relatively low peripheral CRF levels occurring in Alzheimer's disease patients and chronic fatigue syndrome patients. It is possible. In the former case, the treatment improves short-term and mid-term memory. However, for many of these indications, including appetite suppression, it is necessary to deliver the peptide to the brain, preferably combining the peptide with an agent that can penetrate the blood-brain barrier. Administration by intravenous, intramuscular or subcutaneous injection can increase cortisol levels and reduce fatigue. Treatment of subjects with these ligand inhibitors can be performed to rapidly increase the biologically effective concentration of free CRF by administration to the brain and stimulate the human respiratory system. For treatment in conditions of medical emergencies such as cardiovascular arrest and shock with substances or pathological agents, cortisol levels may be increased by administration by intravenous, intramuscular or subcutaneous injection. HC as needed to promote delivery during pregnancy
A ligand inhibitor further comprising RF (or an analog thereof) can be administered to increase the concentration of free hCRF in plasma to at least about 250 pmole / l, preferably to at least about 0.1 ng / ml. It can also be administered to AIDS-affected patients, who often have low cortisol levels, so that such CRF-BP blockers can increase ACTH and cortisol.

Drugs with CRF or Agonists of CRF
When administering an agent, such a CRF peptide will
About 1 to about 200 CRF peptides per kg of animal body weight
It may be administered in microgram daily doses. Proper CRF
No. 4,415,558 as examples of agonists.
No. 4,489,163, No. 4,594,329
No. 5,112,809, 5,235,036
And 5,278,146 (these disclosures are hereby incorporated by reference).
As described in the detailed description)
Agonists. With CRF antagonist
When the reagent is administered to the
Is about 0.01 peptide / kg body weight of the host animal.
May be administered in an amount of up to about 10 mg. Appropriate CRF
No. 4,605,642 as an antagonist
No. 5,109,111 and 5,245,009
(These disclosures are incorporated herein by reference.)
And antagonists as disclosed in US Pat.
Preferred CRF antagonists include [D-Ph
e¹², N^21,38] -HCRF (12-41) and
[D-Phe¹², Nle^21,38, CML³⁷] -HC
RF (1241). Preferred CRF jaws
[His²⁰, Nle^{twenty one}, Le
u ³⁸] -HCRF, [D-Phe¹², Nle^21,38,
 Leu³⁶] -HCRF, [D-Pro^Four, D-Ph
e¹², Asp^{twenty five}, Nle^21,38] -HCRF and
[D-Pro^Four, D-Phe¹², Nle^21,38, C
ML³⁷] -HCRF.

The following examples are for illustrative purposes.
It is not limited.

[0149]

EXAMPLES Example 1 Ligand-Immune Radiation Assay (LIRMA) 600 to Assess the Ability of a Ligand Inhibitor to Displace CRF from CRF-BR
The assay is performed in μl polypropylene centrifuge tubes or 96 well plates. First, 50 μl of the purified recombinant CRF-BP (250 ng / ml) was added to 150 μl.
μl of PBS binding buffer (50 mM sodium phosphate,
0.15 M NaCl and 0.02% NP-40). Next, ¹²⁵ Ih / rCRF was added to a final concentration of 20
Add to 0 pM and add 50 μl of ligand inhibitor at a concentration of 10-100 μM and incubate for 1 hour at room temperature. For the reaction, 50 μl of anti-CRF-BP diluted 1: 1000 in assay buffer
Antibody 5144 is added to each tube and allowed to bind for an additional hour at room temperature. Adjust the total volume of all tubes to 300 μl with assay buffer. 1% normal rabbit serum, 4% P
The binding complex was precipitated by the addition of precipitated goat anti-rabbit (GAR) secondary antibody (20: 1) in EG, 50 mM sodium phosphate, 0.1% sodium azide, followed by 1 hour at room temperature. Incubate for hours. next,
Antibody binding by centrifugation (3000 × g) at 4 ° C. for 20 minutes in a Beckman GS-15R centrifuge
Collect the ¹²⁵ I-CRF precipitate. Beckman B
Aspirate the tube and wash once with PBS and 0.02% NP-40 (600 μl) by using an iomer 1000 robotic workstation. Next, the tube containing the pellet is transferred to a 12 × 74 mm counting tube, and counted by a γ counter.

See, Munson et al., Anal. Bioch
em. The inhibitory binding affinity constant (Ki) value is determined using the LIGAND computer program at 107: 220, 1980 and parameters calculated on a Vax / VMS computer system. Errors represent the average standard error of the triplicate binding assay.

Example 2: Detergent phase separation for assaying the displacement of ligand inhibitors from CRF-BR of CRF. 200 ng / ml of human recombinant CRF-BP was radiolabeled with ¹²⁵ I-h / r CRF ( 8
0 pM) along with binding buffer (phosphate buffered saline, pH
Incubate for 2 hours at room temperature in 7.4 / 0.02% NP-40). After incubation, the assay buffer octylphenoxypolyethoxyethanol (oct
ylphenoxypolyethoxyethano
l) Separate bound and free CRF by adding a 1:10 dilution of Triton X-114 ^™ in (SIGMA). Triton X-114 ^TM
Is insoluble in water at room temperature and separates into a detergent phase in aqueous solution. After adding Triton X-114 ^™ , vortex the tube and immediately add 1 tube.
Centrifuge at 2,000 xg for 5 minutes at room temperature. The detergent phase is found at the bottom of the tube and the aqueous phase remains on top. CRF as an amphipathic α helix separates into the detergent phase. However, CRF
Is bound to CRF-BP, CRF / CR
The F-BP complex remains in the aqueous phase. Therefore, 50 μl aliquots of the aqueous phase are transferred to 12 × 74 mm plastic tubes and counted. The amount of radioactivity remaining in the supernatant is measured.

Example 3 ACTH Release Assay of Free CRF Four rats are decapitated and sacrificed, and the anterior pituitary is removed. The anterior pituitary gland was washed with sterile HEPES buffer for 6 hours.
After washing twice, 20 ml of collagenase solution (4 mg / m
Transfer to l). Next, 25 ml of pituitary gland in collagenase
Into a Bellco dispersion flask and stirred at 37 ° C. for 30 minutes. After 30 minutes, the pituitary cell suspension is triturated by aspirating the pituitary with a 10 ml pipette and further triturated after an additional 30 minutes incubation. The cell suspension was incubated for an additional 45 minutes and the partially dispersed cells were transferred to a 50 ml sterile tube and
Centrifuge at rpm for 4 minutes. 10 ml of cell pellet
Reconstituted in neuraminidase (8 μg / ml),
Vortex. The suspension is placed in a water bath for 9 minutes, vortexed again for 4 minutes and centrifuged again. The supernatant is poured off and the cell pellet is reconstituted by vortexing in 25 ml of BBM-P (250 ml of BBM-T plus 5 ml of 2% fetal calf serum). The cells are collected by centrifugation and finally the suspension is washed with BBM-P2
Reconstitute in 2 ml. Next, 48-well plates Cells are plated at a density of 50 to 100,000 / well in, and incubated for 2 days in a humidified CO ₂ chamber. On the day of the assay, cells are washed once with BBM-T for stimulation with various relevant analogs.

The blocking concentration of CRF-BP (5n
Maximum stimulation volume of h / rC in the presence and absence of M)
Stimulate cells with RF (1 nM). This concentration of CRF-
BP reduces the amount of ACTH released from pituitary cells by binding to h / rCRF. This decrease is
Expressed as a fraction of the amount of ACTH released by 1 nM CRF in the absence of CRF-BP. h / rCR
CRF-BP bound to F (1 nM) (5 nM)
With a range of ligand inhibitors (eg, CR
The typical concentration of the F-BP ligand h / rCRF (6-33) is 0.1-1000 nM). The ligand inhibitor binds CRF-BP and displaces the complex to CRF. Thus, h / rCRF induced AC by CRF-BP
The reversibility of the inhibition of TH secretion becomes dose dependent. CRF-B
P-ligand potency is expressed as the fraction of ACTH release obtained by stimulation with 1 nM CRF alone.

(Example 4: Measurement of free CRF)
cAMP production assay) CRF-stimulated adenylate cycler
Assays for the detection of enzyme activity with minor modifications.
(Battaglia et al.,.,
1987). The standard assay mixture is D
2 mM L-glutamine, 20 mM in MEM buffer
HEPES, 1 mM IBMX (isobutyl methyl oxalate)
Cinchin (isobutylmethyl xanthi)
ne)). In stimulation studies, CRF receptors
Transfected with a clone encoding
Cells are plated in 24-well plates and various concentrations
CRF-related peptides and non-CRF-related peptides
Incubate at 37 ° C. for 1 hour. Incubate
After the application, aspirate the medium and gently quench the wells with fresh medium.
Wash once and aspirate. 95% ethanol and 2
16 ° C. at 20 ° C. in 300 μl of 0 mM HCl solution
Measure the amount of intracellular cAMP after lysing the cells for 18 hours
I do. Lysate 1.5 ml Eppendorf
And add another 200 μl of EtOH /
Wash the wells with HCl and wash with lysate
Pool together. Lyophilize the lysate and add
Resuspend in 500 μl of 6.2 sodium acetate buffer,
cAMP was purchased from Biomedical Technology
gies Inc. (Stoughton, MA)
Measure with the following single antibody kit. CRF receptor ann
In order to evaluate the function of the agonist, cAMP generation was
Single concentration of CRF or related peptide stimulates 0%
At various concentrations (10 ^-12-10^-6M) with the competing composition
Incubate together. Incubation on cAMP
Execution conditions and measurement conditions as described above.
You.

(Example 5: Two-site ELISA for measuring CRF concentration) (A. Preparation of brain tissue sample) An autopsy sample was weighed and 10%
Homogenized in 5 ml sucrose. 1 ml of each sample was centrifuged at 10,000 xg for 10 minutes at room temperature, and the resulting membrane pellet was SPEA (50 mM phosphoric acid containing 0.25% bovine serum albumin (BSA) and 1% NP-40). Sodium acid, pH 7.4, 0.1M
NaCl, 25 mM EDTA, 0.1% sodium azide). TTBS (0.5% Tween
T with n-20, 1% NP-40, 1% BSA
ris buffered saline) by adding 200 μl cerebral cortex homogenate (in 10% sucrose) 8
Another 100 μl was extracted and vortexed for 1 minute. Samples were centrifuged at 10,000 xg for 10 minutes at room temperature. The supernatant obtained as described above was used for two-site CRF
“Total CRF”, “Bound CRF” (ie, CRF bound to CRF-BP) and “Free CRF” using ELISA
RF "for analysis.

(B. Measurement of total CRF) Briefly, ELISA plates were incubated at 37 ° C for 2 hours at pH9.
5. Protein G purified sheep anti-CRF antibody (20 μg / ml) diluted in 50 mM sodium bicarbonate buffer
Covered. Wash the plate once with TTBS,
Blocked with 1% casein in for 1 hour at room temperature. 10
Add 0 μl of sample or standard to each well,
Bound at room temperature. Plates were washed 5 times with TTBS. RC-70 rabbit anti-human CRF antibody (TTBS / 1
(Diluted 1: 1000 in% BSA). After incubation for 1 hour at room temperature, the plate was washed with TTBS
Washed 5 times with buffer, then exposed the plate to horseradish peroxidase-conjugated goat anti-rabbit (GAR) secondary antibody for 1 hour at room temperature. Plates are finally washed with TTBS for 5
Washed twice and washed with TMB microwell peroxidase substrate solution (Kikegaard and Perry La
boratories, Inc. ) Color was developed by addition of 100 μl. The absorbance at 450 nM was measured.

(C. Measurement of bound CRF) Anti-human CRF
After measuring bound CRF by capture of CRF / CRF-BP complex in wells pre-coated with -BP monoclonal antibody (5 μg / ml of antibody in 50 mM sodium bicarbonate buffer at pH 9.5), essentially total CRF
Bound CRF was detected with the RC-70 anti-human CRF antibody as described above for the ELISA.

(D. Measurement of Free CRF) Anti-CRF-B
After capturing the bound complex with the P monoclonal antibody, the free CRF is measured in the supernatant. After binding of the sample material, the supernatant is transferred to a new ELISA plate coated with protein G purified sheep anti-CRF antibody. The assay is then performed as described above to determine total CRF levels.

(Example 6: Screening of ligand inhibitor) A candidate ligand inhibitor was identified as CRF / C
Regarding the ability to replace the RF-BP complex with CRF,
Can be screened. ACTH from cultured pituitary cells
Appropriate assays such as release (see Example 2) or two-site LIMRA (see Example 1) are used to measure free CRF and CRF-BP concentrations, respectively.

In the LIMRA assay, the procedure of Example 1 is almost followed. 10 μM concentration of ligand inhibitor
Add to the reaction together with ¹²⁵ h / r CRF. If the candidate ligand inhibitor replaces CRF-BP with CRF, the pellet will contain lower radioactivity as compared to a control without the addition of the candidate peptide. Rescreen candidate peptides using a 6-point dose curve. Calculate the IC-50 value.

The ligand inhibitors human CRF, α-helix sheep CRF (9-41), human CRF (6-3
3) and sheep CRF were screened by this method against the CRF / CRF-BP complex. The affinity of these peptides for the cloned human pituitary CRF receptor was determined using a pre-characterized radioligand binding assay using membrane preparations of stable transfectants of the receptor in LtK ^- mouse fibroblasts. (DeSouza, J. Neuro).
sci. 7:88, 1987).

The results of these assays are shown in the table below. In addition, these ligand inhibitors were tested for CRF-BP in the cerebral cortex of individuals affected with Alzheimer's disease and control individuals.

[0163]

[Table 2] These results indicate that human CRF, α-helical sheep CRF
(9-41), and human CRF (6-33)
9 shows that replacing the F / CRF-BP complex with CRF was almost equally effective. In contrast,
Sheep CRF was not effective in replacing CRF. CRF-BP in the cerebral cortex from affected or normal individuals was equivalent to the recombinant form. These three
Among the best ligand inhibitors, human C
RF and α-helical sheep CRF (9-41) bound human CRF-R in much the same way. In sharp contrast, human CRF (6-33) has 2-3 log
Combine with low efficiency.

Example 7 Treatment with Ligand Inhibitors Five cerebral cortex samples obtained from Alzheimer's patients and five cerebral cortex samples obtained from normal controls of the same age were prepared as in Example 4. Pooled. The concentration of total CRF, bound CRF, and free CRF was measured as described in Example 4. As shown in FIG. 2, the brain extract from a normal individual had a total CRF of 4
0% complexed with CRF-BP and 60% of the total CRF in CR in brain extracts from Alzheimer's patients
It was complexed with F-BP.

Binding CR of High Affinity CRF-BP Ligands
The effect of replacing F was demonstrated by 50 nM α-helix sheep C
CRF / CRF-B in the presence of RF (9-41)
Evaluated after monoclonal capture of P-complex. The displaced free CRF was determined by CRF ELISA
The measurement was performed on the supernatant remaining after capturing the RF-BP monoclonal antibody. As shown in FIG. 2, treatment of brain tissue with a ligand inhibitor released all bound CRF in both Alzheimer's disease and control tissues. In addition, treatment of Alzheimer's disease cerebral cortex with ligand inhibitors returned free CRF levels to levels found in age-matched controls.

The tissues were also incubated with 50 nM of the ligand inhibitor α-helix sheep CRF (9-41). Next, the supernatant was subjected to 2 described in Example 4.
Displaced CRF by site ELISA assay
Assayed.

(Example 8: Morris water maze test) M
The orris water maze test is a simple spatial learning task that requires minimal stress and experience. No motivation, such as shock or reduced food intake, is required. Animals are placed in lukewarm water which has been rendered opaque by the addition of milk powder. Monitor the potential time to find hidden platforms. The animal learns the position of the platform relative to the maze and visible cues in the test room; this learning is called position learning. This test is particularly sensitive to manipulation of the hippocampus, a key brain region involved in spatial learning in animals and memory consolidation in humans.

The equipment used in this test was a pool (46.4 cm diameter, 45.7 cm high) filled with opaque water (22 ° C. to 25 ° C.) to a depth of 23 cm. The top 10 cm diameter section of the weighted target platform is located 1-2 cm below the water surface. Pool 4
The four quadrants are distinguished by the inner design. Animals are placed in a defined quadrant of the tank and the time to approach and climb the hidden platform is measured; the location of the subject and the location of the platform are constant throughout the experiment. After climbing to the top of the platform, the animals are rested for 20 seconds. The subject who did not find the platform within 60 seconds is placed on the platform and
Rest for a second.

Rats were treated with ICV injection of either the ligand inhibitor h / rCRF (6-33) or the CRF receptor agonist h / rCRF 15 minutes prior to testing. The dose of h / rCRF (6-33)
0, 1, 15, 25, 50 or 125 μg.
The dose of h / rCRF is 0, 0, 1, 1, or 2.5 μ
g. Seven to ten rats were treated per group. Statistical analysis showed that h / rCRF (6-3
3) It was confirmed that the behavior after treatment with (p <0.05) or CRF (p <0.05) was significantly improved (FIG. 4). Similar improvements in behavior were seen after time (p <0.0001).

(Example 9: Y-maze visual discrimination test) The Y-maze visual discrimination test is a learning test in which a minimum stress is applied to an animal to utilize positive reinforcement for learning. Subjects are not fed and are fed only after the end of the training session. Animals may choose to not respond, but in most cases respond due to the positive enhancing properties of the diet pellets that rats prefer over normal food.

The Y maze has the same length (61 cm long, 1 width)
4 cm, height 30 cm). One arm is used as a start box and is separated from the other two goal arms by a manual guillotine door. The vertical surfaces at the ends of the two distal arms are equipped with an 8 watt light bulb. On the first day of training, two food pellets (45 mg Noye
s) was placed and rats unfed to 80% of body weight were released into the maze for 5 minutes. On the second day, each rat had one trip under each goal arm with the pellet. On the third day, one goal arm was closed at the selected point and two 45 mg pellets were obtained in the open goal box, but without any visible stimulus to distinguish ( Rats were subjected to six spatial tests in groups of three animals (lights off). The open arm was reciprocated from left to right throughout the six tests and from subject to subject. From day 4 to 10
On day one, both goal arms were opened and the lights at the ends of the goal arms were lit. One in a group of three animals so that the time between tests is approximately 90 seconds.
The test was performed ten times a day. At the end of training, subjects received 15 g of laboratory chow in the cage daily.

On days 4-10, an immediate ICV injection of the ligand inhibitor was given immediately prior to testing. 0, 1, 5, or 25 μg in groups of 7-9 rats
Any of the ligand inhibitors h / rCRF (6-3
3) was given. The percent corrected response was recorded. 5 μg
And rats receiving 25 μg of CRF (6-33) performed statistically significantly better than rats receiving 0 or 1 μg of ligand inhibitor.

Example 10: Elevated Plus Maze Test The elevated plus maze test demonstrates how an animal responds to a proximity avoidance situation involving a dark, closed area against an exposed and illuminated space. Can be predicted. In the maze, both spaces are lifted off the ground, forming two passages that intersect in a positive sign. This type of proximity avoidance situation is a classic test for "emotional" and is extremely sensitive to disinhibition (sedatives or hypnotics) and stress-producing processes. No motivational coercion is required, and the animal can remain in the dark or exit the open arm.

The elevated plus maze device has four arms (50 cm long and 10 cm wide) that are orthogonally lifted from the floor (50 cm). Two of these arms are surrounded by walls (40 cm high), and two arms have no walls (open arms). Subjects were each placed in the center of the maze with free access to all four arms during the 5 minute test period. The time spent in each arm was recorded automatically by the light beam of the photocell and the computer interface.

A group consisting of 7 to 10 rats contains the ligand inhibitor h / rCRF (6-33) or h
An ICV injection of / rCRF (1-41) was given. Rats are given h / rCRF (1-41) at 0, 0.1, 1 or 2
5 μg was given. 1 and 2 of h / rCRF (1-41)
At the 5 μg dose, there was a statistically significantly longer stay in the open arm, indicating increased anxiety (FIG. 5). In sharp contrast, CRF (6-3)
3) memory improvement dose and 2-5 times higher dose (50-
125 μg), there was no change in behavior and h / rCRF
No apparent behavior change comparable to (1-41) was produced. h / rCRF (1-41) is known to be a CRF receptor agonist. Thus, these data indicate efficient cognitive improvement and anxiogenicity for ligand inhibitors
It shows a clear functional dissociation of side effects.

(Example 11: Obesity animal model)
It relates to excess body fat resulting from consuming more energy than energy expenditure. The combined etiology of obesity in the clinical population may be associated with overeating, abnormal lipid metabolism, insulin overload, and lack of exercise. These phenomena and the resulting increase in body size have been demonstrated in genetically obese rats and mice, animals with lesions in the hypothalamus region of the brain, and animals that have been treated with various medications over time. , Can be modeled in animals.

Centrally administered CRF produces anorexic effects that are effective in stopping excessive weight gain in genetically obese Zucker rats. Endogenous C
The appetite-suppressing effect of RF has recently been elucidated using the CRF / CRF-binding protein complex or CRF (6-33). This ligand inhibitor acts as an indirect CRF agonist, increasing synaptic levels of free unbound CRF in the brain. H / rCR immediately before feeding the animals fasted for 24 hours for 2 hours
When F (6-33) was centrally administered, a dose-dependent suppression of appetite was observed. H / r CRF compared to CRF
(6-33) are significantly less effective and less efficient in reducing appetite. In contrast to CRF, it does not change the 2-hour average intake in subjects who have not fasted. Furthermore, in another study, anorexic doses of h / rCRF (6-33) did not induce the fear-like behavior expected after central administration of CRF itself. These results show that h /
Pharmaceutical treatment with rCRF (6-33)
It can be seen that appetite is selectively and moderately suppressed by stimulating physiologically significant increases in levels.

Weight gain and overeating are unfavorable features of nicotine withdrawal, and by pharmacologically targeting deregulated biological and neurochemical substances for energy balance and appetite control. Can be solved. Increased hunger and weight gain last for at least 6 months, often averaging 4 to 6 pounds a year after quitting smoking. Obesity, a situation that occurs in more than three-quarters of smokers who quit smoking, is not efficiently remedied by standard behavioral weight loss strategies. As an example of a clinical phenomenon, investigators reported that abstinent women gained 9 pounds above baseline at 26 weeks after quitting drinking, although their caloric intake was within normal limits. There is. Such long-term withdrawal symptoms may play a major role in replacing smoking.

Appetite and weight disorders after smoking cessation are reproducible components of nicotine withdrawal syndrome modeled in animals. FIG. 6 shows changes in body weight and food intake in laboratory rats obtained by continuous infusion of nicotine at more than two weeks at dependent induction concentrations and withdrawal for two weeks after cessation of nicotine administration. Chronic nicotine administration reduces food intake and body weight gain for the vehicle-treated group, but subsequent nicotine withdrawal induces normalization of body weight for overeating and controls. Since nicotine replacement therapy is detoxifying for appetite and weight disturbances in withdrawal symptoms, the effect on energy balance of smoking cessation appears to be caused by sudden removal of nicotine. This clinical observation is confirmed by an animal model in which disturbances in behavior and signs of withdrawal as measured 24 hours after cessation of nicotine infusion are alleviated by acute systemic administration of nicotine.

In nicotine withdrawal induced binge eating C
In experiments designed to demonstrate the beneficial anorexic properties of RF-BP antagonists, h / rCRF (6- to 10%) were given to independent and nicotine withdrawal subjects immediately prior to a 2 hour diet on day 4 after cessation of nicotine administration. 33) was administered. FIG. 7 shows the ligand inhibitor WrCRF (6-3
3) did not change appetite in nicotine-untreated controls, but nicotine withdrawal subjects had increased food intake levels, which were significantly reduced by administering h / rCRF (6-33). Is shown.
Taken together, these results indicate that doses of ligand inhibitors that did not alter appetite in no-fed or spontaneously hungry animals affected excessive appetite stimulated by food fasting or nicotine withdrawal. It is disclosed that it is attenuated.

While the specific embodiments of the present invention have been described for purposes of illustration, it will be apparent that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0182]

Utilizing the correlation between reduced CRF levels and various neurophysiological diseases and conditions, increasing the level of free CRF provides an effective treatment for such diseases. .

[0183]

[Table 3]

[Brief description of the drawings]

FIG. 1 shows the amino acid sequences of human-derived CRF (hCRF) (SEQ ID NO: 1) and sheep-derived CRF (oCRF) (SEQ ID NO: 4).

FIG. 2 is a graph showing the levels of CRF bound to CRF-BP and free CRF. CRF levels were measured in brain tissue from Alzheimer's disease patients and normal age-appropriate controls. CRF-B
Α-helix sheep C, a P-ligand inhibitor
Levels were established with or without the addition of RF (9-41).

FIG. 3 shows C in four regions of brain tissue collected from normal control or Alzheimer's disease patients.
It is a graph which shows the level of RF (panel A) and CRF-BP (panel B).

FIG. 3 shows C in four regions of brain tissue collected from normal control or Alzheimer's disease patients.
It is a graph which shows the level of RF (panel A) and CRF-BP (panel B).

FIG. 4 shows vehicle or CRF (6-33).
Or Morris water maze and elevated plus-maze after intracerebroventricular (ICV) injection of CRF (1-41).
Shows the test results. Rats received ICV injections in groups of 7-10 animals 15 minutes before testing.
In the Morris underwater maze test, the time to reach the platform was recorded. The elevated maze test recorded the percentage of time spent in open arms. An asterisk indicates that there is a statistically significant difference.

FIG. 4 shows vehicle or CRF (6-33).
Or Morris water maze and elevated plus-maze after intracerebroventricular (ICV) injection of CRF (1-41).
Shows the test results. Rats received ICV injections in groups of 7-10 animals 15 minutes before testing.
In the Morris underwater maze test, the time to reach the platform was recorded. The elevated maze test recorded the percentage of time spent in open arms. An asterisk indicates that there is a statistically significant difference.

FIG. 4C shows vehicle or CRF (6-33)
Or Morris water maze and elevated plus-maze after intracerebroventricular (ICV) injection of CRF (1-41).
Shows the test results. Rats received ICV injections in groups of 7-10 animals 15 minutes before testing.
In the Morris underwater maze test, the time to reach the platform was recorded. The elevated maze test recorded the percentage of time spent in open arms. An asterisk indicates that there is a statistically significant difference.

FIG. 4 shows vehicle or CRF (6-33).
Or Morris water maze and elevated plus-maze after intracerebroventricular (ICV) injection of CRF (1-41).
Shows the test results. Rats received ICV injections in groups of 7-10 animals 15 minutes before testing.
In the Morris underwater maze test, the time to reach the platform was recorded. The elevated maze test recorded the percentage of time spent in open arms. An asterisk indicates that there is a statistically significant difference.

FIG. 5 is a diagram showing test results of rats in a Y maze test. Groups of 7-10 rats were given ICV injections 0, 1, 15 minutes prior to testing.
5 or 25 μg of either CRF (6-33) was given. The exact percent response was measured. The stars are
Indicates that there is a statistically significant difference.

FIG. 6 shows the test results of weight change and food intake in rats during continuous infusion of addiction-inducing levels of nicotine (addiction phase) and during 2 weeks withdrawal (withdrawal phase). Weight is indicated by a hatched bar and expressed in grams;
Food intake is indicated by lines and displayed in grams.

FIG. 6 shows the test results of weight change and food intake of rats during continuous infusion of addiction-inducing levels of nicotine (dependency phase) and during 2 weeks withdrawal (withdrawal phase). Weight is indicated by a hatched bar and expressed in grams;
Food intake is indicated by lines and displayed in grams.

FIG. 7 shows that the ligand inhibitor h / r
FIG. 4 is a diagram showing the effect of withdrawal symptoms from nicotine dependence on food intake after administration of CRF (6-33).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) // A61K 38/00 A61K 37/24 38/22 37/02 (71) Applicant 501111005 The University of Reading United Kingdom Country ARG6 2 AJ Reading, P.K. Oh. Box 228, White Knights (no address) (72) Inventor Dominique P .. Behan United States of America California 92122, San Diego, Avenida Navidad Number 282 79015 (72) Inventor Philip Jay. Raleigh UK RGS 6 2 H. Barks, Reading, Malden Arley Drive 19 (72) Inventor Willie W. Lee. Burr Jr. United States of America California 92037, La Jolla, Valdez 1643 (72) Inventor Stephen Sea. Heinrich USA California 92037, La Jolla, Via Maloka Number Sea 8611 (72) Inventor Stephen W. Sutton United States California 92014, Del Mar, P. Oh. Box 2964 (No address) (72) Inventor Gene E. F. Libya United States California 92037, La Jolla, Black Gold Road 9674 (72) Inventor Erol De Sauza United States California 92014, Del Mar, South Lane 4507 F-term (Reference) 4C084 AA02 BA01 BA19 BA44 DB12 ZA122 ZA162 ZA702 ZC032 4H045 AA30 BA10 BA18 BA19 CA45 EA21 FA71

Claims (1)

[Claims] 1. A ligand inhibitor of a CRF / CRF binding protein complex for use in the manufacture of a medicament for increasing the level of free CRF in the brain, comprising a CRF.
Can cause the release of CRF from the binding protein;
Alternatively, a ligand inhibitor capable of suppressing binding of free CRF to a CRF binding protein.