CA2223792A1 - Urocortin peptides - Google Patents

Abstract

Urocortin (Ucn) is a native mammalian peptide generally related to Urotensin I
and Corticotropin Releasing Factor (CRF). Human Ucn has the formula: Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 (SEQ ID NO:15). Rat-derived Ucn is identical but for 2 substitutions, Asp2 for Asn2 and Pro4 for Ser4. Ucn or analogs thereof or pharmaceutically acceptable salts can be administered to humans and other mammals to achieve substantial elevation of ACTH, .beta.-endorphin, .beta.-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone. They can also be used to lower blood pressure over an extended period of time, as stimulants to elevate mood and to improve memory and learning performance, as well as diagnostically. Shortened fragments may be administered to release endogenous CRF and/or Ucn in the brain and peripherally. Ucn antagonists can be used to block the action of Ucn and/or CRF, as can antibodies to Ucn. Labelled Ucn agonists and antagonists can be used in drug screening assays along with CRF
receptors; they may also be used diagnostically along with Ucn antibodies.

Description

CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96110240 r~ E8 This invention was made with Government support under Grant Number DK-26741 awarded by the National Institutes of Health. The Government has certain rights in this invention.
This application claims priority from Provisional Application Serial No. 60/002223, filed August 11, 1995 and from Serial No. 08/490,314, filed June 13, 1995, which is being converted to a provisional application.
This invention is directed to peptide hormones, to 10 methods for treatment of mammals, including humans, using such peptides, to antibodies which bind such peptides, to methods for diagnosis and drug screening using such peptides and/or antibodies, and to nucleic acid encoding such peptides. More specifically, the invention relates to 15 a native peptide having certain pharmacological properties in common with urotensin and with CRF, which is termed urocortin (Ucn), to analogs and fragments thereof ~broadly termed Ucn-like peptides), to pharmaceutical compositions containing such Ucn peptides and to methods of treatment of 20 mammals, method of diagnosis and methods of screening using such Ucn peptides and antibodies thereto.

BAC~GRO~ID OF THE lNV~
Experimental and clinical observations have supported 25 the concept that the hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells secretory functions. Although over 40 years ago, Guillemin, Rosenberg and Saffran and Schally independently demonstrated the presence of factors in hypothalamus which 30 would increase the rate of ACTH secretion by the pituitary gland incubated in vitro or maintained in an organ culture, a physiologic corticotropin releasing factor (CRF) was not characterized until ovine CRF (oCRF) was characterized in 1981. It was disclosed in U.S. Patent No. 4,415,558, as 35 having the amino acid sequence (SEQ ID NO:1):
-CA 02223792 1997-12-0~

W O 97/00063 PCT~US96tlO240 H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-Although originally isolated and characterized on the basis of its role in this hypothalamopituitary-adrenal (HPA) axis, CRF has been found to be distributed broadly throughout the central nervous system as well as in extraneural tissues, such as the adrenal glands, placenta 10 and testes, where it may also act as a paracrine regulator or a neurotransmitter. Moreover, thellikely involvement of CRF in affective disorders, such as anxiety, depression, alcoholism and anorexia nervosa, and in modulating reproduction and immune responses suggests that changes in 15 CRF expression may have important physiological and pathophysiological consequences. For example, perturbations in the regulatory loops comprising the HPA
axis often produce chronically elevated levels of circulating glucocorticoids; such patients display the 20 physical hallmarks of C~lching's syndrome, including truncal obesity, muscle-wasting, and reduced fertility.
In addition to its role in mediating activation of the hypothalamic-pituitary-adrenal, CRF has also been shown to modulate autonomic and behavioral changes, some of which 25 occur during the stress response. Many of these behavioral changes have been shown to occur independently of HPA
activation in that they are not duplicated by dexamethasone treatment and are insensitive to hypophysectomy. In addition, direct infusion of CRF into the CNS mimics 30 autonomic and behavioral responses to a variety of stressors. Because peripheral administration of CRF or a CRF antagonist fails to affect certain of these changes, it appears that CRF exhibits a direct brain action with respect to such functions, which include appetite 35 suppression, increased arousal and leaining ability.
However, CRF antagonists given peripherally attenuate stress-mediated increases in ACTH secretion, and when CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 dellvered into the cerebral ventricles can mitigate stress-n~-lce~ changes in autonomic activity and behavior.
As a result of the e~tensive anatomical distribution and multiple biological actions of CRF, this regulatory 5 peptide is believed to be involved in the regulation of numerous biological processes. CRF has also been implicated in the regulation of inflammatory responses.
Although it has been observed that CRF plays a pro-inflammatory role in certain animal models, CRF appears to 10 suppress inflammation in others by reducing injury-induced increases in vascular permeability.
In about 1981, a 40-residue amidated peptide generally similar to CRF was isolated from the skin of the South American frog Phyllomedusa sauvagei; it is referred to as lS sauvagine. It was characterized by Erspamer et al. and was described in Requlatorv Pe~tides, Vol. 2 (1981), pp. 1-13.
Sauvagine has the amino acid sequence (SEQ ID N0:2):
pGlu Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser~Leu-Glu-Leu-Leu-Ar g-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-20 Gln-Ala--Ala--Asn--Asn-Arg-Leu-Leu--Leu-Asp-Thr-Ile NEI2. When given intravenously(iv), sauvagine and oCRF have been reported to cause vasodilation of the mesenteric arterie~
80 as to lower blood pressure in mammals and also in stimulating the secretion of ACTH and ~-endorphin.
25 However, when administered intracerebroventricularly(icv), there is an elevation of heart rate and mean arterial blood pressure, which are secondary to activation of ~he sympathetic nervous system.
Rat CRF(rCRF) was isolated, purified and characterized 30 in about 1982-1983 as a hentetracontapeptide having the amino acid sequence (SEQ ID N0:3):
H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu Gln-Leu-~ Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu Ile-Ile-35 NH2. The formula of human CRF was subsequently determined ~ to be the same as that of rCRF. The compound is often CA 02223792 1997-12-0~

WO ~7/00063 PCTAUS96/10240 referred to as r/hCRF and is covered in U.S. Patent No.
4,489,163.
At about the same time, two homol!ogous polypeptides were isolated from the urophyses of different species of 5 fish. These isolated peptides were generally homologous to CRF, i.e. about 54% homology, and were termed Urotensin I
(UI). Catostomus commersoni(white sucker or suckerfish) UI
is a polypeptide having the amino acid sequence (SEQ ID
NO:4):
10 H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Ile-Glu-Asn-Glu-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2; it is sometimes referred to as suckerfish(sf) urotensin or sfUI. Its purification and characterization 15 are described in an article by Lederis et al., Science Vol.
218, No. 4568, 162-164 (Oct. 8, 1982). The homolog, carp urotensin, was obtained from Cyprinus carpio and has the amino acid sequence (SEQ ID NO:5):
H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-20 Leu-Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala--Gly-Leu-Asn-Arg-Lys-Tyr-Leu--Asp-Glu-Val-NH2.
Another urotensin homolog having the following amino acid se~uence (SEQ ID NO:6):
H-Ser-Glu-Glu-Pro-Pro-Met-Ser-Ile-Asp-Leu-Thr-Phe-His-25 Met-Leu-Arg-Asn-Met-Ile-His-Arg-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln-Ala-Leu-Ile-Asn-Arg-Asn-Leu-Leu-Asp-Glu-Val-NH2 was later isolated from the urophyses of Hippoglossoides elassodon or Flathead (Maggy) Sole; it is sometimes referred to as Maggy urotensin. Synthetic UIs 30 have been found to also stimulate ACTH and ~-endorphin activities in vitro and in vivo and to have many of the same general biological activities of CRFs and sauvagine.
Since the discovery of the original discoveries of CRFs in mammals and urotensins in fish, CRFs have now been 35 shown to exist in other animal species. For example, fish CRF was found to be a 41-residue peptide having high CA 02223792 1997-12-0~

W O 97t00063 PCTrUS96/10240 homology to r~hCRF; it has the amino acid sequence (SEQ ID
NO:7): H-Ser-Glu-Glu-Pro~Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu -Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg Lys-Met-Met-Glu-Il 5 e-Phe-NH2. Synthetic fish CRF (fCRF) stimulates ACTH and ~-endorphin activities in vitro and in vivo and has similar biological activitie to mammalian CRFs. Because of the high homology between fCRF and r/hCRF, it is thought that other mammalian hormones ~ay exist which would be the 10 counterparts of urotensin and/or sauvagine.

8~MNARY OF TB INVENTlON
Another pepti~e 40 residues in length has now been discovered, which is related to urotensin and CRF; it is 15 arbitrarily referred to as urocortin (Ucn). It has less than 50% homology with rat/human CRF. Although it has the same length as sauvagine, it shares less than 4~% homology with sauvagine. It has 62.5~ homology with the closest urotensin sequence, i.e. carp urotensin. Thus Ucn has less 20 than about 80% homology with any other previously known native peptide. Rat Ucn has the following amino acid sequence (SEQ ID NO:8): Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu~Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg Ile-Ile-25 Phe-Asp-Ser-Val-NH2. Human Ucn has the following amino acid sequence (residues 83-122 of SEQ ID NO:15)o Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu Arg-Ala-Glu-~ln--Asn--Arg-Ile-Ile--Phe-Asp-Ser-Val-NH2. Thus, hUcn is 30 the same as rUcn except for Asn2 and Ser4. Ucn has biological properties which are considered to generally resemble those of known CRFs, urotensins and sauvagine but is more biopotent in a number of respects.
~ The present invention provides Ucn-like peptides, 35 including human and rat Ucn and Analogs thereof, which have substantially all the properties of known CRFs. These Ucn-like peptides not only are potent hypotensive agents, but _5_ CA 02223792 1997-12-0~ ~

they have additional pharmacological and physiological properties over and beyond those of heretofore known CRFs.
More specifically, agonists are provided for the stimulation of the known CRF receptors (referred to as CRF-5 Rs), i.e., CRF-Rl and CRF-R2 and their splice varients, as well as the putative novel receptor for Ucn.
Ucn competitive antagonists are also provided which bind the CRF-Rs and the putative Ucn receptor with high affinity but do not significantly stimulate or activate 10 such receptors. Such antagonists are broadly created by deleting a sequence of from 7 to 10 residues beginning at the N-terminus from the amino acid sequence of Ucn or from an analog seguence that is substantially the same.
Preferably 9 or 10 residues are deleted, and most 15 preferably 9 are deleted. It may be preferred that the shortened N-terminus be acylated with a group having 7 or less carbon atoms, e.g. [Ac-Thr1~~-Ucn(10-40), and the inclusion of one, two or three other residues at the N-terminus, e.g., Prol~, does not markedly affect biopotency.
20 Other substitutions may be effectively made as described hereinafter. Particularly, D-Phe11 or D-Tyr11 may be present at the N-terminus and/or a lactam bond created between residues 29 and 32. These antagonists can be administered to achieve at least the same physiological 25 effects as the known CRF antagonists, and such more effective methods of treatment are thus provided.
Fragments of Ucn and Ucn Analogs that are useful to block CRF-binding protein (CRF-BP) are further provided which are effective to elevate levels of endogenous 30 peptides normally cleared by the binding protein. More ~pecifically, these Ucn-like peptides and blocking fragments bind to human CRF-binding protein with high affinity and effectively compete with human CRF and human Ucn (hUcn) in the formation of complexes with hCRF-BP; in 35 this ~nn~r, they increase the effective in vivo concentration of endogenous hCRF and/or hUcn, as well as the effective concentration of any CRF agonist or CRF

j CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 antagonist that may be optionally administered along therewith for the purpose of achieving a particular therapeutic pul~o_e. As a result of blocking the effect of CRF-BP, these fragments effectively increase the 5 concentration of endogenous CRF in those regions of the body where CRF-BP is normally present.
The invention also provides pharmaceutical compositions which include such Ucn-like peptides, or nontoxic salts thereof, dispersed in a pharmaceutically 10 acceptable liquid or solid carrier. The administration of such peptides or pharmaceutically acceptable salts thereof to ma ~l~, particularly humans, in accordance with the invention may be carried out for regulation of the secretion of ACTH, ~-endorphin, ~-lipotropin, other 15 products of the pro-opiomelanocortin (POMC) gene and corticosterone and/or for lowering blood pressure or increasing coronary flow and/or decreasing swelling and inflammation and/or for affecting learning, mood, behavior, appetite, gastrointestinal and intestinal functions and 20 autonomic nervous system activities.
The invention also provides antibodies which recognize Ucn, and assays for practically employing Ucn and analog~
and/or such antibodies for the evaluation of the status of pituitary, cardiovascular, reproductive, hepatic, immune, 25 gastrointestinal or central nervous system functions. For example, such antibodies can be used diagnostically to monitor the level of therapeutically administered Ucn, to facilitate the maintenance of therapeutically e fective amounts thereof, as well as for the diagnosis o~ potential 30 physiological disorders that result from abnormal levels of Ucn. An~; hoA i es of the invention may be therapeutically administered to neutralize endogenous Ucn; alternatively DNA ~ncoA; ng such antibodies might be employed in gene ~ therapy. Anti-Ucn ant; ho~; es can also be used to purify 35 CRF-R protein as well as to therapeutically counteract the biological action of Ucn in vivo.

CA 02223792 1997-12-0~

WO 97/00063 PCT~US96/10240 The invention also provides competitive binding assays which are particularly useful for screening candidates for new drugs, e.g. to identify new Ucn-like peptides or other compounds having even greater or more selective b;n~;n~
5 affinity for CRF receptors and/or for CRF-BP than Ucn, which candidates would therefore be potentially useful as drugs. Such screening assays may be used to screen for potential agonists of Ucn, and other assays employing a labelled Ucn antagonist with high affinity may be used to 10 screen for more potent antagonists of Ucn. In addition, there is provided a method for screening for particularly effective peptides or other compounds which will block the ability of CRF-BP to bind to CRF and Ucn and therefore increase the concentration of CRF andjor Ucn in locations 15 where hCRF-BP is present.
The present invention further provides nucleic acid hybridization probes in the form of isolated nucleic acid encoA; ng native rat Ucn and isolated nucleic acid enco~;ng native human Ucn, which are useful for detecting other Ucn-20 encoding nucleic acids in biological samples or inlibraries of other species in order to identify additional native Ucn or Ucn-like peptides. Such nucleotide sequences also can be used as coding sequences for the recombinant expression of complete Ucn-like peptides or desired 25 biologically active fragments thereof. Fragments of Ucn-encoding nucleic acid can also be employed as primers for PCR amplification of Ucn-encoding DNA. In addition, sequences derived from sequences encoding Ucn or analogs thereof can also be used in gene therapy applications to 30 target the expression of vectors carrying useful genes to specific cell types, and antisense polynucleotides that hybridize with Ucn mRNA may also be used to reduce Ucn levels to counteract certain conditions, e.g. Ucn-secreting tumors. More specifically, the present invention further 35 provides isolated nucleic acids encoding Ucn as well as Ucn analogs cont~;n;ng L-isomers of the 20 natural amino acids.
Such nucleic acids comprise:

- CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 (a) nucleic acids that encode the amino acid sequence of rat Ucn set forth in SEQ ID No:8 and that encode the amino acid sequence of human Ucn set forth in SEQ ID N0:15;
(b) nucleic acids which hybridize to the nucleic acids 5 of (a) wherein said hybridizing nucleic acids encode biologically active Ucn-like peptides; or (c) nucleic acids which encode frag~ents of Ucn or analogs thereof which are CRF-antagonists or CRF-BP
blockers.

D~T~TT-~n DE8~UTPTIO~ OF THE PREFEFURED ~MRODIMEIrT8 Unless defined otherwise, all technical and scientific terms used herein have the same ~~n;ng as is commonly understood by one of skill in the art to which ~his 15 invention belongs. The nomenclature used to define the peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the rightO The 20 stAn~Ard 3-letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g. Ser = L-serine. The nucleotides, which occur in the various 2S nucleic acids, are designated with the st~n~rd single-letter designations used routinely in the art.
The term "homology" is used in its usual and well know~ sense of indicating correspondence between members in a sequence, e.g. either on an amino acid (AA) level or at 30 the nucleotide level. For purposes of this application, the term homologous refers to at least about 70~
correspondence, the term substantially homologous refers to a correspon~nce of at least about 80%, and the term highly ~ homologous refers to a correspondence of at least about 90%
35 or preferably about 95% or higher. The term "homolog" is generally considered to include analogous proteins, peptides and DNA sequences from other mammalian species _g_ CA 02223792 1997-12-0~ ' wherein insignificant changes have evolved but the homolog still performs the same biological function in substantially the same way.
Protein, polypeptide and peptide are used to designate 5 linear seq~l~nc~C of amino acid residues connected one to the other by peptide bonds between the alpha-amino and alpha-carboxy groups of adjacent residues. The term polypeptide may be used interchangeably with peptide and with the term protein; unless otherwise limited, protein is 10 generally used to describe a sequence of about 75 or more residues.
The term "analog" includes any polypeptide having an amino acid residue sequence generally identical to a seguence specifically shown herein, e.g. rUcn or hUcn, 15 wherein one or more residues has been replaced (with at least about 80% and preferably at least about 90% of the residues being the same) and wherein the analog displays the ability to biologically mimic the parent molecule as described herein in some particular function. Preferably, 20 most if not all of such substitutions are replacements of a residue with a functionally similar residue, i.e.
conservative substitutions. Examples of such conservative substitutions include: the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, alanine, 25 glycine, leucine or methionine for another non-polar residue; the substitution of one polar (hydrophilic) residue for another polar residue, such as arginine for lysine, glutamine for asparagine, threonine for serine; the substitution of one basic residue such as lysine, arginine 30 or histidine for another basic residue; and the substitution of one acidic residue, i.e. aspartic acid or glutamic acid, for the other. The phrase "conservative substitution" is also intended to include the use of a chemically derivatized residue in place of a 35 non-derivatized residue provided that the resultant polypeptide displays the requisite biological activity, e.g. binding activity. For purposes of this application, -W O 97/00063 ~C~r~US96/10240 two peptides are considered to be substantially the s ame when they only differ from each other by conservative substitutions. Examples of preferred conservative substitutions are set forth in Table 1.

Preferred Original ConservativeMost Preferred Residue SubstitutionsSubstitution 10Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser 15 Gln (Q) Asn Asn Glu (Ej Asp Asp Gly (G) Pro Pro His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Leu Phe; Nle 20 Leu (L) Nle; Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly Gly 25 Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Nle -CA 02223792 1997-12-0~

"Chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized polypeptides include, for example, those in which free 5 amino yLOu~ have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of 10 esters or hydrazides. Free hydroxyl groups may be derivatized to from 0-acyl or 0-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Chemical derivatives also include those peptides which contain one or more naturally 15 occurring amino acid derivatives of the twenty stAn~rd amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and 20 ornithine may be substituted for lysine. Peptides embraced by the present invention also include peptides having one or more residue additions and/or deletions relative to the specific peptide whose sequence is shown herein, so long as the modified peptide maintains the requisite biological 25 activity.
As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to co ~sitions, carriers, diluents and reagents, are used interchangeably 30 and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The term "biologically active fragment" as used herein 35 refers to (a) a fragment of a peptide of the invention which has been truncated with respect~to either the N- or C-termini, or both; or (b) a fragment of nucleic acid CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 corresponding to a coding region for rUcn or a highly homologous native peptide of another ~ ~lian species which has been truncated at the 5' or 3' end, o~ both, and is useful in antisense applications. The peptide fragment 5 shown performs substantially the same function or a directly related biological function as does the parent.
The phrase "modulating the transactivation of CRF
receptors" as used herein refers to administering a therapeutically effective amount of a physiologically 10 tolerable composition containing a Ucn-like peptide to thereby modulate CRF actions in mammals by means of direct or induced antagonistic(competitive) association with CRF
receptors (CRF-Rs).
CRF-R is used to refer to a family of receptor protein 15 subtypes which participate in the G-protein-coupled response of cells to CRF and Ucn-like ligands. CRF-Rs are coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels, and transporters. The G-proteins associate with the receptor proteins at the 20 intracellular face of the plasma membrane. An agonist binding to a CRF-R catalyzes the exchanges of GTP for GDP
on the ~-subunit (G-protein "activation"), resulting in its dissociation and stimulation of one (or more) of the various signal-transducing enzymes and channels. G-protein 25 preferentially stimulates particular effectors, and the specificity of signal transduction may be determined, therefore, by the specificity of G-protein/receptor interaction. CRF-R proteins mediate signal transduction through the modulation of adenylate cyclase and perhaps 30 through PI turnover. For example, when CRF or Ucn binds to and activates the CRF-R, adenylate cyclase causes an elevation in the level of intracellular cAMP. An effective bioassay for evaluating whether a test compound is capable of elevating intracellular cAMP is carried out by culturing 35 cells cont~; n; ng cDNA which expresses CRF receptor proteins in the presence of a potential agonist or antagonist whose ability to modulate signal transduction activity of CRF

CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 recep~or protein is sought to be determined. Such transformed cells are monitored for either an increase or decrease in the level of intracellular cAMP which provides a determination of the effectiveness of the potential 5 agonist or antagonist. Methods for measuring intracellular levels of cAMP, or measuring cyclase activity, are well known in the art.
A human CRF receptor was the first to be reported, and it was cloned from a human Cushing pituitary tumor as 10 described in Chen R., et al, P.N.A.S.~ 90, 8967-8971 (October 1993). It is referred to as hCRF-Rl or hCRF-RA
and has 415 amino acids; a splice variant thereof includes an insert of 29-amino acids. A rat CRF receptor was isolated, approximately contemporaneously, by hybridization 15 from a rat brain cDNA library. It is referred to as rCRF-Rl; it has the 415 amino acid sequence which is set forth hereinafter as SEQ ID NO:10. It was disclosed in Perrin, M., et al., Endocrinology, 133, 3058-3061 (1993). It was found to be 97% identical at the amino acid level to the 20 human CRF-R1, differing by only 12 amino acids. The receptor has since been reported to be widely distributed throughout the brain and the pituitary and to be likely present in the adrenals and spleen.
A second subclass of CRF receptors has more recently 25 been found, and such receptors are arbitrarily referred to herein as CRF-R2 but are sometimes referred to as CRF-RB.
One such receptor, having the amino acid sequence set forth hereinafter as SEQ ID NO:ll, was obtained by the cloning and characterization of a cDNA from a mouse heart cDNA
30 library. It is 431 amino acid residues in length, and the details of the receptor are set forth in Perrin, M., et al ., P.N.A.S, 92 , 2969-2973 (March 1995). It is hereinafter referred to as CRF-R2~, but has been referred to as CRF-RBL.
Another, slightly shorter receptor of this C~con~
subclass was independently obtained from a rat hypothalamus cDNA library. It is referred to herein as CRF-R2~ and has , CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 the 411 amino acid residue sequence set forth hereinafter as SEQ ID NO:12. The details of its cloning are set forth in Lovenberg, T., et al ., P.N.A.S., 92, 836 - 84 o (January 1995~, wherein a second spliced variant was also identified 5 via PCR as being a putative protein of 431 amino acids that would be the rat homolog of mCRF-R2~ identified above. The 431 amino acid sequence is set forth hereinafter as SEQ ID
N0:13 and can be seen to be homologous with mCRF-R2~. The distribution of Ucn throughout the rat brain is consistent 10 with its being the endogenous ligand for CRF-R2~ as is the fact that it exhibits a much higher binding affinity, than does CRF, for the receptor, particularly the R2s which is believed to be the main CRF-R in the brain.
Ucn-like peptides, including rUcn, hUcn and analogs 15 thereof, can be easily synthesized as described in Example I hereinafter and then individually tested for binding affi~ity. Binding affinity refers to the strength of interaction between ligand and receptor. To demonstrate binding affinity for a CRF receptor, the peptides of the 20 invention are easily evaluated using a tracer ligand of known affinity, such as 125I-radiolabeled oCRF, in binding assay experiments which are well known in this art. The results of such assays indicate the affinity at which each Ucn-like ligand binds to a CRF receptor, expressed in terms 25 of K;, an inhibitory binding affinity constant relative to such a known standard. K; (inhibitory binding a~finity constant) is determined using a "st~n~rd" or "tracer"
radioactive ligand and thus measures the displacement of the tracer from the receptor or binding protein; it is most 30 properly expressed with re~erence to such tracer. So long as these assays are carefully performed under specific conditions with relatively low concentrations of receptor or the like, the calculated Kj will be substantially the ~ same as itc dissociation constant KD~ It is particularly 35 efficient to test for Kj because only a single tracer need be labelled, e.g. radioiodinated. Dissociation constant is representative of the concentration of ligand n~cecs~ry CA 02223792 1997-12-0~

WO 97/00063 PCT~US96/10240 to occupy one-half (50%) of the binding sites of a receptor or the like. A given ligand having a high binding affinity for a CRF receptor will require the presence of very little ligand to bind at least 50% of the available binding sites 5 so that the ~ value for that ligand and receptor will be a small number. On the other hand, a given ligand having a low binding affinity for a particular CRF receptor will require the presence of a relatively high level of the ligand to bind 50% of the sites, so that the ~ value for 10 that ligand and receptor will be a large number.
With respect to a particular receptor protein, a Ucn-like peptide having a ~ of about 10 nM or less means that a concentration of the ligand (i.e., the Ucn-like peptide) of no greater than about lo nM will be required to occupy 15 at least 50% of the active binding sites of the receptor protein. Such values may be fairly determined from the results obtained using a radioiodinated st~n~rd and no more than approximately 0.8 nM of the receptor (approximately 10-20 pmol receptor/mg membrane protein).
20 Preferred Ucn-like peptides have a binding affinity (~) such that a ligand concentration of about 10 nanomolar or less is required in order to occupy (or bind to) at least 50% of the receptor binding sites, and particularly preferred Ucn-like peptides have a binding affinity of 1 nM
25 or less. Generally, a dissociation co,nstant of about 5 nanomolar or lower is considered to bel an indication of fairly strong affinity, and a ~ of about 1 nanomolar or less is an indication of very strong affinity. For example, rUcn binds CRF-Rl with very strong affinity, 30 having a ~ = about 0.18 nanomolar and binds CRF-R2~ with similar strong affinity. It is also considered to be particularly advantageous to provide Ucn-like peptides which have a substantially higher affinity for CRF-R2, compared to CRF-R1, and which will thus be selective in 35 their biological effect. Because CRF-R2 receptors are distributed widely throughout the body, Ucn will have a substantially greater effect than CRF in modulating many , CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 peripheral actions, and because the native peptide or fragments thereof should not be immunogenic, it should be a very good drug physiologically.
These binding assays employing CRF recepto~s are ~ 5 straightforward to perform and can be readily carried out with initially identified or synthesized peptides to determine whether such peptides are effective agonists of CRF, or alternatively to determine whether other shortened candidates are effective antagonists of CRF. Such b;n~;~g 10 assays can be carried out in a variety of ways as well known to one of skill in the art. A detailed e~ample of such an assay is set forth in Perrin, M., et al~, Endocrinology, 118, 1171-1179 (1986). Competitive binding assays employing Ucn are particularly contemplated to 15 evaluate whether candidate peptides are effective agonists with respect to each of the receptors previously described, i.e. CRF-Rl, CRF-R2~ and CRF-R2~ as well as assays with Ucn antagonists to determine whether candidates are effective antagonists. In such assays, Ucn can be appropriately 20 labeled with a substance that is readily detected, such as a radioactive isotope, e.g. 1ZsI, or an enzyme or some other suitable tag. For example, suitably labelled ayonists, such as l25I-Tyr~-Ucn, or suitably labelled antagonists, such as 125I-(cyclo 29-3~)tD-Tyr11, Glu29, Lys32]-Ucn(l1-40), 25 are particularly useful tracers for use in such receptor assays. Such receptor assays can be used as screens for potential drugs which interact with CRF and/or CRF
receptors.
Very generally, the invention provides Ucn-like 30 peptides, including Ucn and analogs of Ucn, having an amino acid sequence which is substantially the same as the following amino acid sequence based upon SEQ ID NO:8 and upon SEQ ID NO:15 (Formula I): Y-R1-Pro-R4-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-35 Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-- Phe-Asp-Ser-Val-NH2, wherein Y is an acyl group llaving 7 or less carbon atoms, preferably acetyl, or hydrogen; R1 is CA 02223792 1997-12-0~

Wos7/00063 PCT~S96/10240 Asp-Asp or Asp-Asn or Asp or Asn or desR1; and R4 is Pro or Ser; as well as nontoxic salts thereof. When the N-terminus is shortened by the deletion of 2 residues, it is preferably acylated, e.g. acetylated. These peptides have 5 pharmacological properties somewhat similar to those of oCRF or r/hCRF and additional properties as described hereinafter. As indicated hereinbefore, analogs of the above having at least about 80% homology with the amino acid sequence of either hUcn or rUcn are preferred for the l0 Ucn-like peptides of the invention, although peptides having at least 66% homology with either hUcn or rUcn, wherein all or all but one of the substitutions are conservative substitutions, are considered to be biologically active and to have advantages over known CRF
15 peptides. Particularly preferred are analogs which are substantially the same as either hUcn or rUcn (as defined hereinhefore) and which have D-isomer amino acid substitutions and/or cyclizing bonds between the side ~-h;~ in~s of specific residues in the sequence which are known 20 to increase ligand binding affinity for CRF receptors.
In addition to the foregoing general group of Ucn-like peptides, two additional groups are disclosed hereinafter based upon related syntheses and testing carried out in this general area with regard to ligands which bind to CRF-25 R.
The following group of analogs of Ucn does not merelyinclude one or more conservative substitutions. Instead, bioactive Ucn analogs are found to be defined according to the following amino acid sequence: Y-Asp-R2-Pro-R4-Leu-30 Ser-Ile-Asp-Leu-Thr-D-Phe-His-heu-Leu-Arg-Thr-Leu-Leu-R19-Leu--Ala--Arg--Thr--Gln--Ser--Gln--Arg--Glu--R29--Ala--Glu--R32--Asn--ArgIle-R36-Phe-R3~-Ser-Val-NH2, wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R2 is Asp or Asn; R4 is Pro or Ser; R19 is Glu or Ala; R29 is Arg, Glu, 35 Lys or Orn; R32 is Gln, Lys, Orn or Glu; R36 is Ile, CQMeIle or CQMeLeu; R38 is Asp or Ala; provided that when R29 is Glu, R3z is either Lys or Orn and the side chains thereof are CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 lin~e~ ~y an amide bond and tha~ when R32 is Glu, R29 is eithQr Lys or Orn and the side chains thereof are linked by an a~ide bond; and provided further that D-Phe11 can be substituted by another D-isomer amino acid, pre~erably a D-5 iSomer of a natural amino acid, such as D-Leu and more preferably one other than D-Cys; that Glu in the 31-position can be substituted by any D-amino acid~ e.g. D-Glu, D-Asp, D-Arg, (imBzl)D-His, ~-(2naphthyl)-D-Ala, etc., again preferably a D-isomer of a natural amino acid other 10 than Cys; and that the N-terminus can be shortened by 1 or

2 residues. One particularly preferred Ucn analog is (cyclo 29-32)tLys29, D-Glu31, Glu32]-Ucn, with Ucn being either hUcn or rUcn; others are described in the Examples hereinafter. When N-terminally shortened by 7 to 10 15 residues, these Ucn analogs are effective antagonists.
Extensive synthesis and testing over the past 10+
years have shown that ligands for the C~F receptors can tolerate a number of changes in amino acid sequence of native r/hCRF which do not result in significant changes in 20 bioactivity, such as would be indicative of the resulting analog being no longer able to bind and/or activate CRF
receptors, particularly CR~-R1. As a result of this extensive earlier work, it has been found that one, two, or three substitutions can be made in the Ucn amino acid 25 sequence, within certain limits as set forth hereinafter, that will result in Ucn analogs which retain the bioac~ivity of Ucn and, in some instances, may have even more desirable pharmacological characteristics.
Using the amino acid sequence of Ucn as a reference, 30 these analogs should differ only by one, two or three substitutions from SEQ ID NO: 8 or from SEQ ID NO:15. The invention thus provides Ucn analogs according to the follo~ing amino acid sequence (SEQ ID NO: 14):
Y-Xaa1-Xaa2-Pro-Xaa4-Xaa5-Ser-Xaa7-Asp-Leu-Xaa10-Xaa11-Xaa12-Leu Arg-Xaa16-Xaa17-xaa1g-xaa1g-xaa2o-xaa21 Xaa22 23 Xaa24-xaazs-xaa26-xaa27-xaa28-xaa29-Ala-xaa31 Xaa32 Asn A g 35 Xaa36 xaa37 Xaa38-Xaa39-Xaa40-NH2, wherein Y is an acyl group CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 having 7 or less carbon atoms or hydrogen; Xaa1 is Asp, Glu or Gln; Xaaz is Asn, Asp, Glu or Gly; Xaa4 is Ser or Pro;
Xaa5 is Leu, Ile or Met; Xaa7 is Ile or Leu; Xaa10 is Thr or Ser; Xaa~ is Phe or Leu; Xaa12 is His or Glu; Xaa13 is ~eu 5 or Met; Xaa~6 is Thr, Asn, Glu, or Lysi Xaa17 is Leu, Met or Val; Xaa18 is Leu or Ile; Xaa19 is Glu or His; Xaa20 is Leu, Met, Ile or Arg; Xaa21 is Ala, Glu or Thr; Xaa22 is Arg or Lys; Xaa~ is any natural amino acid and preferably Thr, Ser, Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu lO or Asp; Xaa24 is Gln, Glu or Asp; Xaa25 is any natural amino Acid and preferably Ser, Thr, Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp; Xaa26 is Gln, Leu or Glu;
Xaa27 is Arg, Ala or Lys; Xaa28 is Glu or Gln; Xaa29 is Arg or Gln; Xaa31 is any natural amino acid and preferably Ala, 15 Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp;
Xaa32 is any natural àmino acid and preferably Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp; Xaa35 is Ile, Lys, Leu or Asn; Xaa36 is Ile, Tyr, Met or Leu;
Xaa37 is Phe, Leu or Met; Xaa38 is Asp or Glu; Xaa39 is Ser, 20 Ile, Glu or Thr; and Xaa40 is Val, Ile, Phe or Ala;
provided that there are no more than 3 residues different from the hUcn or the rUcn amino acid sequence and that the N-terminus may be shortened by l or 2 residues. Moreover, these Ucn analogs when N-terminally shortened by 7 to lO
25 residues constitute effective antagonists.
The Ucn-like peptides of the invention may be chemically synthesized by any suitable method, such as by exclusively solid-phase t~chn; ques, byipartial solid-phase tec-hn;ques, by fragment condensation ~F by classical 30 soluti~n addition.
Ucn may also be synthesized by recombinant DNA
techn;ques as may its analogs which include only natural amino acids. The amino acid sequence for rUcn (SEQ ID
N0:8) was deduced from a partial cDNA clone isolated from a 35 rat brain cDNA library. Set forth in Table 2 hereinafter is the native rat nucleic acid sequence encoding Ucn (SEQ
ID N0:9). The additional codon encoding glycine that is -W 0 97/00063 P~TAUS96/10240 present at the end of the native sequence is expected to account for the C-terminal amidation of rUcn.

TILB~E 2 - A~p A~p Pro Pro Leu Ser Ile Asp Leu Thr Phe Hi~ Leu Leu Arg Thr 1 S lO 15 CTG CTA GAG CTA GCT CGG ACA CAG AGC CAG CGC GAG CGC GCA ~AG CAG
10 Leu L~u Glu Lau Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln AAC CGC ATC ATA TTC GAT TCG GTG GGCAAGTGA
A~n Arg Ile Ile Phe Asp Ser Val ~ 15 35 40 Using the nucleic acid encoding rUcn as a probe, the 20 nucleic acid encoding the mature hUcn was isolated from a human genomic placental library. Set forth in TABLE 2A
hereinafter is the portion of native human nucleic acid sequence encoding the mature Ucn peptide (see SEQ ID
NO:16), with the additional codon for glycine at the end 25 being expected to account for C-terminal amidation.

~ABLE 2A
GAC AAC CCT TCT CTG TCC ATT GAC CTC ACC TTT QC CTG CTG CGG ACC
Al-p AE~n Pro Ser Leu Ser I le Anp Leu Thr Phe Hi~ Leu Leu Arg Thr

3 01 5 10 15 CTG CTG GAG CTG GCG CGG ACG CAG AGC CAG CGG GAG CGC GCC GAG CAG
L~u Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln AAC CGC ATC ATA TTC GAC TCG GTG GGCAAGTGA
Ann A~g Ile Ile Phe A~p Ser Val Synthesis by the use of recombinant DNA t~hn;ques, for purposes of this application, should be understood to include the suitable employment isolated nucleic acid encoding for Ucn or an appropriate analog, as is well known 45 in the art at the present time. As explained in detail hereinafter, synthetic Ucn peptides may be obtained by transforming a microorganism using an expression vector CA 02223792 1997-12-0~

including appropriate regulatory sequences associated with nucleic acid encoding a Ucn-like peptide and causing such transformed microorganism to express the Ucn peptide.
Because of the relative shortness of the Ucn-like 5 peptides, about 40 residues or less, chemical or chain elongation synthesis is presently felt to be the method of choice. Analogs of hUcn or rUcn having one or more substitutions can be readily synthesized in this manner and then tested for biological activity in a straightforward 10 manner to determine the specific biological effect of such substitution(s). Common to such chemical syntheses of peptides is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from 15 occurring at that site until the group is ultimately removed. Also common is the protection of an alpha-amino group on an amino acid or a short peptide fragment while that entity reacts at the free carboxyl group to effect chain elongation, followed by the selective removal of the 20 alpha-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with 25 various of these residues having side-chain protecting ~ L OU~ "; .
Thus, in such chemical syntheses, intermediates are formed having a protected amino acid sequence such as the following which is based on hUcn SEQ ID NO:15 (Formula II):
30 X1-Asp( X5)-Asn( X4)-- Pro-Ser(X2)-Leu-Ser(X2)-Ile-Asp( X5)-Leu-Thr(X2)--Phe-His(X6)-Leu-Leu-Arg (X3) -Thr(X2)-Leu-Leu-Glu (X5)-Leu-Ala--Arg (X3) -Thr(X2)-Gln (X4) -Ser(X2)-Gln (X4) -Arg (X3)-Glu (X5) -Arg (X3) -Ala-Glu (X5) -Gln (X4) -Asn (X4) -Arg (X3) -Ile-Ile-Phe-Asp(X5)-Ser(X2)-Val-X7 (or suitably N-terminally 35 shortened versions thereof) wherein:
e CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 Xl is either hydrogen or an alpha-amino protecting group. The alpha-amino protecting groups contemplated by X
are those known to be useful in the art in the step-wise synthesi~ of polypeptides. Among the classes of 5 alpha-amino protecting groups covered by X1 are (l) acyl-type protecting groups, such as formyl, acrylyl(Acr), benzoyl(Bz) and acetyl(Ac) which are preferably used only at the N-terminal; (2) aromatic urethan-type protecting groups, such as benzyloxycarbonyl(Z) and substituted Z, 10 such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting y~OU~S~ such as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, iso~Lcs~yloxycarbon 15 ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as fluorenylmethyloxycarbonyl(FMOC), cyclopentyloxycarbonyl, adamantyloxycarbonyl,and cyclohexyloxycarbonyl; and (5) thiourethan-type protecting groups, such as 20 phenylthiocarbonyl. The preferred alpha-amino protecting group is BOC if the synthesis employs acid-catalyzed ~ ~l of the alpha-amino protecting groups; however, for syntheses employing a base-catalyzed removal strategy, FMOC
is preferred, in which case, more acid-labile side chain 25 protecting yLou~ can be used, including t-Butyl esters or ethers as well as BOC.
X2 is h-ydLGyen or a protecting group for the hydroxyl group of Thr and Ser and is preferably acetyl(Ac), benzoyl(Bz), tert-butyl, triphenylmethyl(trityl), 30 tetrahydLu~ylanyl~ benzyl ether(Bzl) or 2,6-dichlorobenzyl tDCB). The most preferred protecting group is Bzl.
X3 iS hyd~oyen or a protecting group for the guanidino group of Arg, preferably selected from nitro, p-toluenesulfonyl(Tos), Z, adamantyloxycarbonyl and BOC.
35 Tos is preferred for a BOC strategy, and 4-methoxy-2,3,6-CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 trimethyl benzenesulfonyl (MTR) or pentamethyl chroman-6-sulfonyl (PMC) is preferred for FMOC strategies.
X4 is hydrogen or a protecting group for the side chain amido group of Asn or Gln, preferably xanthyl(Xan).
5 Asn or Gln is preferably coupled without side chain protection in the presence of hydroxybenzotriazole (HOBt).
X5 iS hydrogen or an ester-forming protecting group for the ~- or ~-carboxyl group of Asp or Glu, preferably cyclohexyl (OChx), benzyl (OBzl), 2,6-dichlorobenzyl, 10 methyl, ethyl and t-butyl ester (Ot-Bu). Chx is preferred for a BOC strategy and Ot-Bu for FMOC strategy.
X6 is hydrogen or a protecting group for the side chain imidazole nitrogen of His, such as Tos.
The selection of a side chain amino protecting group 15 is not critical except that the protecting group should be one which is not removed during deprotection of the alpha-amino groups during the synthesis. Hence, the alpha-amino protecting group and the side-chain-amino protecting group cannot be the same.
X7 is NH2, a protecting group, such as an ester, or is an anchoring bond of the type used in solid phase synthesis for linking the peptide being synthesized to a solid resin support, preferably one represented by the formulae:
-NH-benzhydrylamine tBHA) resin support and -NH-paramethylbenzhydrylamine (MBHA) resin support.
Cleavage from a BHA or MBHA resin directly gives the Ucn-like peptide in amidated form. By employing a methyl-derivative of such a resin, if desired, the 30 corresponding, equivalent, methyl-substituted amide can be created. Alternatively, using an appropriate resin support, the ethylamide, which is also considered to be an equivalent, can be created as well known in this art.
In the formula for the intermediate, at least one of x1, X2, X3, X4, X5 and x6 is a protecting group. In CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following rules are ~ollowed: (a) the protecting group should be stable to the reagent, and under the reaction conditions, selected 5 for removing the alpha-amino protecting group at each step of the synthesis, (b) the protecting group should retain its protecting properties and not be split off under coupling conditions and (c) the protecting group must be removable upon completion of the synthesis under reaction 10 conditions that will not alter the peptide chain.
For the acyl group at the N-terminus of a Ucn-like agonist peptide, which is represented by Y, acetyl, formyl, acrylyl and benzoyl are preferred. Moreover, as indicated earlier, the N-terminus can be slightly shortened 15 by removal of the N-terminal residue or the first two N-tel ;nAl residues without significantly affecting biological potency of the peptide to function as a Ucn agonist, and when such shortening occurs, acylation of the residue at the shortened N-terminus may be preferred. More 20 extensive shortening of the N-terminus by a sequence of 7 to 11 residues results in the creation of Ucn antagonists which strongly bind CRF-R without activating the receptor as discllc~e~ hereinafter.
Overall, there is broadly provided a process for the 25 manufacture of peptides defined by Formula I or analogs thereof comprising (a) forming an intermediate peptide according to Formula II or an analog thereof wherein there is at least one protective group, with X1, X2, X3, X4, X5 and X6 being each either hydrogen or a protective group, and X7 30 being either a protective group or an anchoring bond to resin ~uy~o~- or NH2 and (b) splitting off the protective group or groups or anchoring bond from the intermediate peptide of the Formula II and (c) if desired, converting the resulting peptide into a nontoxic sal~ thereof.

CA 02223792 1997-12-0~

WO 97/00063 . PCT~US96/10240 When the peptides of the invention are prepared by chemical synthesis, they are preferably prepared using solid phase synthesis, such as that described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1964), although 5 other equivalent chemical syntheses known in the art can also be used as previously mentioned. Thus, Ucn-like peptides can be prepared in a straightforward manner and then simply tested for biological activity. This facilitates the ready preparation and evaluation of Ucn-10 like peptides which are analogs of hUcn or rUcn.Solid-phase synthesis is preferably commenced from the C-terminus of the peptide by coupling a protected alpha-amino acid to a suitable resin as generally set forth in U.S. Patent No. 4,244,946 issued Jan. 21, 1981 to Rivier 15 et al. by coupling with the free carboxyl group. The synthesis of Ucn can be initiated by coupling alpha-amino-protected Val to a BHA resin using methylene chloride and dimethylformamide(DMF). Following the coupling of BOC-Val to the resin support, the alpha-amino 20 protecting group may be removed using trifluoroacetic acid(TFA) in methylene chloride, TFA alone or with HCl in dioxane. Preferably 50 volume % TFA in methylene chloride is used with 0-5 weight % 1,2 ethanedithiol. The deprotection is carried out at a temperature between about 25 0~C and room temperature. Other stAn~rd cleaving reagents and conditions for removal of specific alpha-amino protecting groups may be used as described in Schroder &
Lubke, "The Peptides", 1, pp 72-75 (Academic Press 1965).
After removal of the alpha-amino protecting group of 30 Val, the remaining alpha-amino- and side chain-protected amino acids are coupled stepwise in the desired order to obtain the intermediate compound of Formula II. As an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior 35 to addition to the solid phase reactor. The selection of CA 02223792 1997-12-0~

W O 97/00~63 ~CTrUS96/10240 an a~ru~Liate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N,N'-dicyclohexyl carbodiimide(DCCI) and N,N'-diisopropyl carbodiimide(DICI).
Activating reagents used in the solid phase synthesis of the peptides of the invention are-well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,N~-diisopropyl carbodiimide and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide. Other 10 activating reagents and their use in peptide coupling are described by Schroder & Lubke, supra, in Chapter III and by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970). P-nitrophenyl ester (ONp) may also be used to activate the carboxyl end of Asn or Gln for coupling. For example, BOC-Asn (ONp) can 15 be coupled overnight using one equivalent of HOBt in a 50%
mixture of DMF and methylene chloride, in which case no DCCI is ad~ed.
Each protected amino acid or amino acid sequence is il~L~oduced into the solid phase reactor in about a fourfold 20 eYcecs~ and the coupling is carried out in a medium of dimethylformamide(DMF):CH2Cl2 (l:l) or in DMF or CH2C12 alone. In instances where the coupling is carried out manually, the success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin 25 reaction, as described by E. Kaiser et al., Anal. Biochem.
34, 595 (1970). In cases where incomplete coupling occurs, the coupling procedure is repeated before removal of the alpha amino protecting group prior to the coupling of the next amino acid. The coupling reactions can be performed 30 automatically, as on a Beckman 990 automatic synthesizer, using a ~o~Lam such as that reported in Rivier et al., Rio~olYmers, 1978, 17, pp.l927-1938.
After the desired amino acid sequence has been completed, the intermediate peptide is removed from the 35 resin support by treatment with a suitable clearing agent, CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all r~ ~;ning side chain protecting groups x2, X3, X4, X5 and X6 and the alpha-amino protecting group X1 (unless it is an acyl group 5 which is inten~ to be present in the final peptide). When using hydrogen fluoride for cleaving, anisole or cresole and methylethyl sulfide are preferably included in the reaction vessel as scavengers. The BOC protecting group at the N-terminus is preferably cleaved with trifluoroacetic 10 acid(TFA)/ethanedithiol prior to the cleaving of the peptide from the resin. I
The determination of whether any Ucn-like peptide of about 40 residues in length, or a fragment thereof, or an antagonist version thereof, will have desirable 15 pharmacological properties can be made in a straightforward manner. First, assays are run to determine the effect of a candidate agonist or antagonist peptide on the different CRF receptors; then, the ability of the peptide to promote or inhibit production of ACTH is determined. Fragments 20 which function as CRF-BP blockers can be likewise similarly assayed using an inhibitory binding assay with hCRF-BP and a known labelled ligand.
The candidate peptide is easily evaluated in binding assays with the various CRF receptors~earlier discussed 25 using assays as described in Perrin, M., et al., Endocrlnology, 118, 1171-1179 (1986). A bin~ing assay with human CRF-R1 is preferably carried out using a radioligand oCRF analog; such a binding assay utilizing CRF-R1 receptor is described in Chen, et al., P.N.A.S. , 90 , supra .
A straightforward assay using rat anterior pituitary cells in monolayer culture can be carried out to determine whether a candidate peptide thereof will function as a CRF
agonist and stimulate ACTH secretion by activating CRF
receptors on such cells. The procedure which is used is 35 that generally set forth in Endocrinology, 91, supra. A

CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 very similar assay is used to test for antagonistic properties, using a challenge dose of oCRF or the like.
By the in vivo administration to mammals of peptides which have a high affinity to human CRF binding protein and 5 which thus compete with endogenous C~F and Ucn for binding to hCRF-BP, CRF-BP is effectively blocked. This leaves endogenous CRF and Ucn available in higher concentrations to carry out their usual biological functions throughout the body, particularly in localized areas where the peptide 10 is administered and/or where CRF-BP is present.
More specifically, fragments of Ucn, or analogs of Ucn, between about 19 and 28 residues in length have a very high affinity to hCRF-BP, but generally exhibit relatively low propensity for binding CRF receptors. As a result, 15 these blocking fragments can be administered to prevent the clearance of endogenous CRF and/or Ucn from particular regions in the body and thereby stimulate the biological effect of CRF and/or Ucn in vivo. In certain in~tances, it may be advantageous to administer such peptides along wi~h 20 CRF or Ucn or an agonist thereof. The very nature of these fragments is such that potentially undesirable immunogenic side effects are minimized or totally obviated. They might also be administered along with antagonists to prevent the clearance of antagonists having a fairly high binding 25 affi~ity to hCRF-BP from a target region. These blocking fragments are useful for therapeutic treatment to ~ - Le parturition in pregnancy, to stimulate the respiratory system, to combat obesity, and to counteract the effects of Alzheimer's ~;co~ce, and of chronic fatigue syndrome; for 30 the latter four indications, the blocking fragments are preferably administered in a manner so as to be delivered to the brain.
Ucn peptides can be used in diagnostic methods to detect the level of Ucn present in a body sample as well as 35 in an inoculum for the preparation of antibodies that CA 02223792 1997-12-0~

immunoreact with epitopes on Ucn. Ant; ho~l; es generated against Ucn can be employed for diagnostic applications, therapeutic applications, and the like. Such antibodies can be prepared employing st~n~rd t~c~niques, as are well 5 known to those of skill in the art, using Ucn or a fragment thereof, as an antigen. Antibodies of the present invention are typically produced by ; ln;zing a mammal, e.g. rabbit, sheep, goat, etc., with an inoculum containing Ucn or fragment thereof thereby inducing the production of 10 antibody molecules having immunospecificity for the immunizing agent.
Ant;ho~;es which recognize Ucn are raised against either the entire 40-residue amino acid sequence or against a synthetic fragment of a sequence of at least about 5 or 15 preferably 6 residues. For example, such antibodies can be raised against the 6 N-terminal residues, or against the 6 C-terminal residues, or against an interior sequence, such as the sequence embracing residues 18-23. Such antibodies will bind to and thus can be employed to indicate the 20 presence of Ucn; they are therefore useful in assays.
Moreover, certain of these antibodies will bind to and biologically inactivate Ucn, and such antibodies can be administered to ~n; ~1 s for the purpose of neutralizing endogenous Ucn. In an instance where endogenous antibodies 25 are being created that would bind Ucn, short amino acid se~uences from Ucn might be al ; n i~tered as antibody blockers. To generate such blocking antibodies, either the entire 40-residue sequence can be used, or a short peptide sequence can be synthesized constituting one region of 30 particular interest. Such a synthetic short chain peptide is generally conjugated to a large carrier molecule, and the conjugate is then used as inoculum to induce a mammalian immune system in rabbits or sheep or the like.
Details of the production of such polyclonal antibodies are 35 set forth in U.S. Patent No. 4,864hO19 (9/5/89). If CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 instead of polyclonal antibodies, it is desire~ to produce monoclonal antibodies~ such can be made ln a straightforward r~nne~ using similar inoculum by employing hybridoma t~chni ques now well established in this art.
5 Details of exemplary monoclonal antibody production are set forth in U.S. Patent No. 5,032,521 (7/16/91), and U.S.
Patent No. 5,051,364 (9/24/91).
Antibodies so produced can be used in diagnostic methods and systems to detect the level of Ucn present in a 10 human or other mammalian body sample, such as tissue or flui~. The anti-Ucn antibodies may also be used for immunoaffinity or affinity chromatography purification of Ucn, the details of which are well known in this art. In addition, an anti-Ucn antibody can be used in human lS therapeutic methods. Moreover, it is contemplated that DNA
encoding such antibodies may be injected via gene therapy methods to raise desired antibodies within a patient or alternatively to provide antibody blockers in an a~r~iate situation.
The lack of evolutionary digression with respect to the amino acid sequences of the 41-residue CRF biological messenger in rats and humans (which sequences are identical) is fairly indicative of the probability of conserved regions in the corresponding amino acid sequences 25 of Ucn in mammalian species, such as human, bovine, porcine, ovine, caprine, murine, canine, feline, baboon, monkey, rabbit, etc. The corollary is that, once one has a significant portion of the Ucn nucleic acid seq~ence of one mammalian species, i.e. the rat sequence as disclosed 30 herein, it is a straightforward exercise to obtain naturally G~ ing variant homolog nucleic acid sequences of other animal species which encode homologs, such as CRF-bin~ng proteins (see e.g., Potter et al., Nature, 349, 423-426 (1991), where it was shown that the cDN~ coding 35 region for human serum-derived CRF binding protein was of CA 02223792 1997-12-0~

WO 97/00063 PCTrUS96/10240 suf~icient homology in the rat cDNA coding region to permit identification of the latter. The first disclosed nucleic acid sequence (SEQ ID NO: 9) is of the native rat species;
in addition to its being useful in an expression vector to 5 express a Ucn peptide, it is also useful to obtain the DNA
of other mammalian species encoding the respective counterpart Ucn peptides. In this respect, either the entire nucleic acid sequence or nucleic acid sequences at least about 14 nucleotides in length can be used as 10 hybridization probes to obtain and clone counterpart mammalian sequences, as is presently well known in this art. Similarly, primers based upon the foregoing nucleic acid sequence can be used along with PCR (Polymerase Chain Reaction) t~c~hrl;ques to amplify nucleic acid sequences of 15 other mammalian species using suitable sources of DNA.
As used herein, a nucleic acid "probe" may be a single-stranded DNA or RNA that has a nucleotide sequence of at least 14, and preferably at least 20 or more, contiguous bases that are the same as (or the complement 20 of) any 14 or more contiguous bases set forth in SEQ ID
NO:g.
Labeled nucleic acid enro~ing Ucn, or fragments thereof, can be employed to probe cDNA libraries, genomic libraries and the like for additional nucleotide sequences 25 encoding other novel mammalian members of the Ucn family.
Such scr~en;ng may be initially carried out under stringency conditions employing a temperature of about 42.5~C, a formamide concentration of about 20%, and a salt conc~ntration of about 5X st~n~Ard saline citrate (SSC; 20X
30 SSC contains 3M sodium chloride, 0.3M sodium citrate, pH
7.5). Such conditions will allow the identification of seqn~nr~s having substantial similarity with the probe sequence, without requiring perfect homology. By "substantial similarity" is meant nucleotide sequences 35 which share at least about 50% homology. It may be ;

CA 02223792 1997-12-0~

W O 97/00063 ~CTrUS96/10240 desirable to select hybridization conditions which will identify only se~lenc~ having at ieast 70% homology with the probe, while discriminating against seq~nr~C which have a lower degree of homology with the probe; such is 5 effected by increasing the stringency used to exceed the above-stated conditions as is well known in thi~ art.
For example, using established methods well known to those skilled in the art (see e.g., Molecular Cloning, A
Laboratory ~nlJ~ 7 2Ed, Chapter 8, Construction and Analysis 10 of cDNA Libraries, J. Sambrook et al. (1989)), either the entire SEQ ID NO:9 or portions thereof of at least about 14 or 17 or 20 nucleotides in length may be used to screen mammalian genomic or cDNA libraries to identify and isolate homologous nucleic acids ~nco~ing Ucn from human and other 15 mammalian species. Oligonucleotide seguences of about 14 or 17 or 20 nucleotides or longer can be prepared by conventional in vitro synthesis terhniques. Screening with such oligonucleotides as probes is preferably carried out under high stringency conditions as defined in Sambrook et 20 al., suDra, Chapter 11, pp. 11.45-ll.S7.
As indicated above, possession of a native DNA
sequence enco~ing a specific peptide hormone of one mammalian species allows one to obtain the homologous DNA
sequence of other mammalian species. Isolation of 25 nucleotide sequences encoding Ucn peptides of other species often involves utilization of either a genomic library or a cDNA library made from RNA isolated from tissue containing Ucn. If such a source is available, it will generally be preferable to create a cDNA library for isolation of 30 nucleotide sequences encoding Ucn so as to avoid any possible problems arising ~rom attempts to determine intron/exon borders. Libraries can be made in either eukaryotic or prokaryotic host cells. Widely available cloning vectors, such as plasmids, cosmids, phage and the 35 like, can be used to generate genetic libraries suitable CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 for the isolation of nucleotide sequences encoding Ucn peptides.
Methods for screening genetic libraries for the presence of target nucleotide sequences include using such 5 probes based on the sequence of a known nucleotide sequence are described in detail in Chapter ll of Sambrook et al., supra. In the present situation, it may be preferable to use the entire length of the rat nucleotide sequence SEQ ID
N0:9 labeled with radionuclides, enzymes, biotin, lO fluoresces, or the like, as a probe for screening such genetic libraries. I
Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense:antisense strands or probe:target-DNA) to each other through hydrogen bonds, 15 similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-nucleic acid can be readily varied by those of skill in the art. As used herein, the phrase "high stringency" hybridization refers to conditions that permit 20 target-nucleic acid to bind a complementary nucleic acid that has at least about 80% homology to the target-nucleic acid. An example of such stringency conditions would be conditions that are minimally equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS
25 at 42~C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65~C. Denhart's solution and SSPE are desirable in Sambrook et al., Molecular Cloning, A Laboratory M~n~A 7, Cold Spring Harbor Laboratory Press (1989) and are well known to those of skill in this art; there are other 30 suitable hybridization buffers that may be used. It may be preferred to use stringency conditions requiring greater than about 9~% homology to target-DNA.
As an example, the human form of Ucn was successfully cloned from a human placental genomic library.
35 Approximately 0.6 x 106 phage plaques of a human placental b CA 02223792 1997-12-0~

W O 97/00063 ~CTrUS9~10240 genomic library in the EMBL3 SP6/T7 vector (Cloffl ech) were screened by hybridization using a probe corresponding to the mature peptide region of rat Ucn. The 160 bp probe encoding the rat U~n mature peptide was synthesized by PCR
5 using the following oligos (sense: 5'-TGCAGGCGAGCGGCAACGAC~r-ACGA-3') and (antisense: 5'-ATACGGGGCCGATCACTTGCCCACCGAG-3') and t~32P-dCTP].
Hybridization was carried out at 42~C in stA~A~d buffers with 20% formamide. Final washes were at 42~C in 2X
10 SSC/0.1% SDS. The phage DNA from an individual positive plaque for the clone was purified and then subcloned into pBluescrip~ (Strategene). Dideoxy sequencing was done using the Sequenase kit (US Biochemical).
The genomic clone isolated from the library which 15 contains the gene for human Ucn has an insert size of approximately 15 kb. A large number of base pairs of the insert have been sequenced (see SEQ ID N0:16) in the region corresponding to the precursor and the mature peptide region of human Ucn. The portion of nucleotide sequence 20 initially sequenced encoding the mature human Ucn peptide, as c-_lp~red to the mature rat Ucn peptide, was set forth in TABLE 2A hereinbefore. The nucleotide sequence for the mature human Ucn peptide is 88% similar to the nucleotide sequence for the rat Ucn peptide. The amino acid sequence 25 encoded by this region shows 95% similarity between human Ucn and rat Ucn. The mature human peptide (see residues 83-122 of SEQ ID N0:15) is 40 residues of the total of 124.
~ nother suitable techni que which may be used in the present situation involves the use of primers based on 30 sequences derived from rat Ucn nucleic acid and the polymerase chain reaction (PCR) to amplify target nucleic acid. The target can then be isolated using a specific hybridization probe based on the amplified segment, which is then analyzed for its o~erall sequence and the 35 polypeptide which it encodes.

CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 To synthesize a peptide of the invention by recombinant DNA, a double-stranded DNA which encodes the desired peptide can be synthetically constructed. Although PCR t~c-h~;ques might nowadays be the method of choice to 5 produce a DNA sequence, for example, SEQ ID N0:9, DNA
encoding Ucn can be designed using certain codons that are particularly efficient for polypeptide expression in a certain organism, i.e. selection might employ those codons which are most efficient for expression in the type of 10 organism which is to serve as the host for the recombinant vector. However, any correct set of codons will encode a desired product, although perhaps slightly less efficiently. Codon selection may also depend upon vector construction considerations; for example, it may be 15 n~c~s~Ary to avoid placing a particular restriction site in the DNA coding sequence if, subsequent to inserting the coding sequence, the vector is to be manipulated using a restriction enzyme that cleaves at such a site. Of course one would avoid placing restriction sites in the DNA coding 20 sequence if the host organism, which is to be transformed with the recombinant vector, is known to produce a restriction enzyme that would cleave at such a site within the DNA chain.
Isolated nucleotide sequences encloding Ucn and analogs 25 thereof can be used to produce purified Ucn by either recombinant DNA methodology or by in vitro polypeptide synthesis t~chniques. The term "isolated" refers to a nucleotide sequence or a polypeptide sequence that has been manually produced and is separated from its native, in 30 vivo, cellular environment and is present in the substantial absence of other biological molecules of the same type. As a result of this human intervention, the recombinant, isolated and/or substantially pure DNAs, RNAs, polypeptides and proteins of the invention can be produced 35 in large quantities and are useful in ways that the DNAs, - CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 RNAs, polypeptides or proteins as they naturally occur are not, such as identification of selective drugs or compounds. The term "purified" as used herein for nucleotide sequences preferably means at least 95% by 5 weight, and most preferably at least 99% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, can be present).
To assemble a synthetic, nonchromosomal nucleic acid sequence, oligonucleotides are constructed by conventional 10 procedures such as those described in Sambrook et al., ~u~ra. Sense and antisense oligonucleotide chains, up to about 70 nucleotide residues long, are synthesized, preferably on automated synthesizers well known in this art. The oligonucleotide chains are constructed so that 15 portions of the sense and antisense oligonucleotides overlap, associating with each other through hydrogen bonding between complementary base pairs and thereby forming double-stranded chains, in most cases wlth gaps in the strands. Subsequently, the gaps in the strands are 20 filled in, and oligonucleotides of each strand are joined end to end with nucleotide triphosphates in the presence of appropriate DNA polymerases and/or with ligases. As an alternative to such stepwise construction of a synthetic nucleic acid sequence, DNA or cDNA ~nco~ing the complete 25 structure of a native polypeptide as obtained by scr~nin~
a library is used. As indicated hereinbefore, amplification is preferably carried out by using PCR, and the isolated and purified DNA is then incorporated into recombinant molecules.
The desired nucleic acid coding sequence to be inserted into a vector preferably has linkers at its end~
to facilitate insertion into restriction sites within the cloning vector. Optionally, the nucleic acid coding sequence may be constructed so as to encode the desired 35 peptide as a portion of a fusion polypeptide; and if so, CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 the coding sequence will generally contain terminal se~lenceC that encode amino acid residue sequences that serve as proteolytic processing sites, whereby the encoded polypeptide may be proteolytically cleaved from the 5 ~ ~;n~er of the fusion polypeptide. The terminal portions of the nucleic acid coding sequence may also contain appropriate start and stop signals.
The desired peptide is then expressed by recombinant techn;ques after the nucleic acid coding sequence is 10 functionally inserted into a vector. By "functionally inserted" is meant in proper reading frame and orientation, as is well understood by those skilled in this art. For example, when producing a genetic construction containing a complete Ucn reading frame, the preferred starting material 15 is a cDNA library isolate encoding Ucn rather than a genomic library isolate. Typically, the Ucn-encoding sequence will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if 20 desired. In general, host-cell-specific sequences which improve the production yield of Ucn will be used, and appropriate control sequences will be added to the expression vector, such as enhancer sequences, polyadenylation sequences, and ribosome binding sites.
The production of Ucn can be carried out in both prokaryotic and eukaryotic cell lines to provide protein for biological and therapeutic use. While Ucn synthesis is easily demonstrated using either bacteria or yeast cell lines, the synthetic genes should also be insertable for 30 expression in cells of higher animals, such as Chinese hamster ovary (CHO) cells or mammalian tumor cells as described in detail in Sambrook et al, supra. Some mammalian cells may be grown, for example, as peritoneal tumors in host animals, and Ucn harvested from the 35 peritoneal fluid. Descriptions of mammalian expression -W O 97/00063 PCT~US96/10240 systems, baculovirus expression systems, bacterial expression systems and yeast expression systems are set forth in U.S. Patent No. 5,212,074 (5/18/93).
The following Examples set forth preferred chemical 5 methods for synthesizing Ucn, Ucn analogs, Ucn fragments, and Ucn antagonists, by the solid-phase chain elongation te~h~;que.

~ANPLE I
The synthesis of rat Ucn having the formula (SEQ ID
NO:8):
Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val NH2 is 15 conducted in a stepwise manner on a MBHA hydrochloride resin, such as available from Bachem, Inc., having a substitution range of about 0.1 to 0.5 mmoles/gm. resin.
The synthesis is performed on an automatic Beckman 990B
peptide synthesizer using a suitable program, such as the 20 following:
STEP REAGENTS AND OPERATIONS MIX TIMES MIN.
1 CH2Cl2 wash-80 ml. (2 times) 3 2 Methanol (MeOH) wash-30 ml. 3 (2 times) 3 CH2Cl2 wash-80 ml. ~3 times) 3

4 50 percent TFA plus 5 percent ~2 1,2-eth~ne~ithiol in CH2C12-70 ml. (2 times) Isopropanol wash-80 ml. 3 (2 times) 6 TEA 12.5 percent in CH2Cl2-70 ml. 5 (2 times) 7 MeOH wash-40 ml. (2 times) 2 CH2Cl2 wash-80 ml. (3 times) 3 CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 g Boc-amino acid (10 mmoles in 30-300 30 ml. of either DMF or CHzCI2, depending upon the solu~ ty of the particular protected amino acid, (1 time), plus DCCI
(10 mmoles) in CH2Cl2 Coupling of BOC-Val results in the substitution of about

5 0.35 mmol Val per gram of resin. All solvents that are used are carefully degassed, preferably by sparging with an inert gas, e.g. helium or nitrogen, to insure the absence of oxygen.
After deprotection and neutralization, the peptide 10 chain is built step-by-step on the resin. Generally, one to two mmol. of BOC-protected amino acid in methylene chloride is used per gram of resin, plus one e~uivalent of 2 molar DCCI in methylene chloride, for two hours for the coupling of each additional residue. When BOC-Arg(Tos) is 15 being coupled, a mixture of 50% DMF and methylene chloride i8 used. Bzl is used as the hydroxyl side-chain protecting group for Ser and Thr. The amido group of Asn or Gln can be protected by Xan but need not be. ;BOC-Asn or BOC-Gln is coupled overnight using one equivalent of DCC and two 20 equivalents of HOBt in a 50% mixture of DMF and methylene chloride. Tos is used to protect the guanidino group of Arg and the imidazole nitrogen of His. The side chain carboxyl group of Glu or Asp is protec~e~ by OChx. At the end of the synthesis, the following composition is 25 obtained:
BOC-Asp(OChx)-Asp(OChx)-Pro-Pro-Leu-Ser(Bzl)-Ile-Asp(OChx)-Leu-Thr(Bzl)-Phe-His(Tos)-Leu-Leu-Arg(Tos)-Thr(Bzl)-Leu-Leu-Glu(OChx)-Leu-Ala-Arg(Tos)-Thr(Bzl)-Gln-Ser(Bzl)-Gln-Arg(Tos)-Glu(OChx)-Arg(Tos)-Ala-Glu(OChx)-Gln-Asn-Arg(Tos)-30 Ile-Ile-Phe-Asp(OChx)-Ser(Bzl)-Val-NH-resin support.
In order to cleave and deprotect the resulting peptide, the peptide-resin is treated with 1.5 ml. anisole, 0.5 ml. of methylethylsulfide and 15 ml. hydrogen fluoride -.
CA 02223792 1997-12-0~

(HF) per gram of peptide-resin, first at -20~C. for 20 min.
and then at ooc for one and one-half hours. After elimination of HF under high vacuum, the resin-peptide is washed with dry diethyl ether, and the peptide amide is 5 then extracted with de-gassed 2N aqueous acetic acid or a 1:1 mixture of acetonitrile and water. The extract is separated from the resin by filtration, and then lyophilized.
The lyophilized peptide amide is purified by 10 preparative or semi-preparative HPLC as described in Rivier et al., J. ChromatoqraPhy, 288, 303-328 (1984); and Hoeger et al., ~iochromatoqra~hv, 2, 3, 134-142 (1987). The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity are 15 pooled.
Specific optical rotation of the isolated and purified Ucn peptide is measured on a Perkin Elmer Model 241 Polarimeter as t~]D = -62.5~ + 1.0 (c=1 in 1% acetic acid, without ~o~e~-tion for the presence of H2O and TFA); it has 20 a purity of greater than about 95%. Purity is further confirmed by mass spectroscopy and capillary zone electrophoresis (CZE). Liquid secondary ion mass spectrometry (LSIMS) mass spectra are measured with a JEOL
model JMS-HX110 double-focusing mass spectrometer fitted 25 with a Cs' gun. An accelerating voltage of 10 kV and Cs~
gun voltage between 25 and 30 kV are employed. The measured value of 4705.36 Da obtained using LSIMS is in agreement with the calculated value of 4705.52 Da.
To verify the precise sequence, the Ucn peptide is 30 hydrolyzed in sealed evacuated tubes containing constant boiling HCl, 3 ~1 of ~hioglycol/ml. and 1 nmol of Nle (as an internal standard) for 9 hours at 140~C. Amino acid analyses of the hydrolysates using a stAn~A~d amino acid analyzer shows the expected amino acid ratios, which 35 confi~ms that a 40-residue peptide structure is obtained '~ CA 02223792 1997-12-0~

W 0 97/00063 PCT~US96/10240 with the expected amino acid residues which constitute the inten~e~ sequence.
To provide a labelled Ucn peptide for use in assays, including binding assays and the likel, the synthesis is 5 ext~n~e~ to link Tyr to Asp at the N-terminus, producing the 41-residue peptide referred to as tTyr~]-Ucn, which can be readily iodinated with 1Z5I and then used in diagnostic assays and in drug-screening assays.
To test the ability of purified Ucn or another 10 candidate Ucn-like peptide to function as a CRF agonist and to promote ACTH production and/or secretion, cultures of dispersed primary murine anterior pituitary cells (0.15 x 106 cells/well are used), as generally described in Vale et al. Meth . ~n7,ym . , 103 , 565-577 (1983), and in 15 Endocrinology, 91, 562 (1972). These cultures are maintained in 0.5 ml/well of ~-PJ (a reagent available from nc~5 City BiochemicalS) containing 2% fetal bovine serum.
On the morning of culture day 5, the cells are washed 3 times with ~-PJ containing 0.1% bovine serum albumin and 20 then incubated for 1 hour at 37~C in the same medium. The medium is then replaced with Ucn, or an analog thereof, diluted in ~-PJ containing 0.1% bovine serum albumin.
TncllhAtions are terminated after 3 hours, at which time the medium is removed and stored at -20~C until ACTH
25 radioimmunoassays are performed using a suitable kit, such as that commercially available from Diagnostic Products Corporation of Los Angeles, CA. Secreted ACTH is measured using Allfit computer program with results expressed as the average + s.e.m. of 3 replicate bioassays.
The rUcn peptide strongly stimulates the secretion of ACTH and ~-endorphin in cultured rodent pituitary cells.
It is more biopotent than either r/hCRF or sauvagine, having an EC50 of about 0.006 + 0.003 nanomolar, co ~red to about 0.043 + 0.012 nM and about 0.033 + 0.010 nM, 35 respectively; it is also more potent than oCRF. In c W O 97/00063 ~CTrUS96/10240 addition, it is more potent than suckerfishUI (sfUI) which has an EC50 of 0.017 + 0.003. rUcn also stimulates ACTH
and ~-END-LI secretion in vivo in rats to a greater extent than r/hCRF; in fact, at 30 minutes, a 1 ~g/kg dose of rUcn 5 elevates ACTH level in plasma to a substantially greater extent (659 + S3pg/ml) than does a 5 ~g/kg dose of r/hCRF
(422 + 66 pg/ml), using an assay as generally described in Science, 218, 377 (1982). Such a greater effect continues at 1 hour and at 2 hours. The peptide when administered 10 peripherally, e.g. iv, also causes a marked fall in mean arterial blood pressure in rats at a dose as low as 250 ng for a st~ rd laboratory rat of about 250 to 275 grams.
At a dose of about 3.77 ~g/kg, it lowers blood pressure 2-3 times as much as either sfUI or r/hCRF and for a longer 15 duration.
Testing is also carried out for the ability of Ucn to cause elevation of the level of intracellular cAMP in cells which express murine CRF-Rl and also in cells which express murine CRF-R2~ using an assay as generally described in 20 Chen et al., Expression Cloning of a Human CoritcoLrGpin Releasing Factor (CRF) Receptor, P.N.A.S., 90, 8967-8971 (1993). rUcn is slightly more potent than either r/hCRF or sfUI in elevating cAMP levels in cells expressing mCRF-Rl.
However, the effect is even more dramatic in assays 25 utilizing cells expressing mCRF-R2~ wherein the ED50 for rUcn is 0.18 + 0.04 nanomolar, c~ pAred to an ED50 for r/hCRF of 1.7 + O.4 nM and an ED50 for urotensin of 0.74 0.1 ~M. It is also more potent than sauvagine which exhibits an ED50 of 0.5 + 0.2.
Binding assays with cells expressing human CRF-Rl are carried out as described in the Chen et al. P.N.A.S., supra. The affinities of test peptides for CRF R1 and CRF-R2~ stably expressed in CH0 cells were determined by competitive displacement of 125I-(Nle2~, Tyr32) ovine CRF
35 (for CRF-R1) or of [l25~I-Tyr~]Ucn (for CRF-R2~) as CA 02223792 1997-12-0~

W097/000~ PCT~S96/1o~0 described. Data from at lest 3 experiments were pooled and inhibitory dissociation constant (Kj) values (95%
confidence limits) were calculated using the LIGAND program of Munson and Ro~hArd (1980), Anal. Biochem, 107:220-239.
5 The cloned hCRF-R1 binds Ucn with high affinity as determined by the competitive displacement of bound radioligand. The Kj for rUcn was determined to be about 0.16(0.08 - 0.32)nM, compared to r/hCRF of about 0.95(0.47 - 2.0)nM, sfUI of about 0.43(0.23 - 0.81)nM, and sauvagine 10 of about 1.2(0.54 - 2.5)nM. Again, the difference is even more dramatic for similar stably transfected CH0 cells expressing human CRF-R2~ where the respective results were 0.41(0.26 - 0.66)nM, 17(10 - 29)nM, 3.0(1.8 - 4.8)nM and 2.0(1.1 - 3.6)nM. Testing also shows that rUcn binds more 15 strongly than does r/hCRF to human CRF~ binding protein (hCRF-BP) by a factor of about 2, using a competitive hCRF-BP ligand binding assay with the radioligand 12sI[Nle21, Tyr32]-oCRF, much more strongly than does sauvagine, but less strongly than sfUI.
ESANRLE IA
Human Ucn(1-40) having the formula (see SEQ ID NO:lS):
H-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-25 Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. LSIMS
shows a value of 4694.31 Da which agrees with the calculated value of 4694.51 Da. In vi_ro testing for ACTH
secretion using anterior pituitary cell cultures as set 30 forth in Example I shows that the peptide is about 3 times as effective as r/hCRF, i.e. 3.10 (1.41 - 6.65). The peptide also has significant mammalian vasodilatory-hypotensive activity, including lowering systemic blood pressure and stimulating the secretion of 35 ACTH.

W O 97/00063 ~CTrUS96/10240 ~aa~P~ IB
The peptide [Tyr~]rUcn(1-40) having the amino acid sequence (see SEQ ID N0:8):
H-Tyr-Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-S Leu Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. LSIMS
shows a value of 4868.58 Da which agrees perfectly with the calculated value of 4868.58 Da. In vitro testing for ACTH
10 secretion using anterior pituitary cell cultures as set forth in Example I shows that the peptide is about twice as effective as r/hCRF, i.e. 2.20 (1.28 - 3.88). The peptide al80 has significant mammalian vasodilatory-hypotensive activity, including lowering systemic blood pressure and 15 stimulating the secretion of ACTH.

EXAMP~ IC
The peptide tD-Tyr~]hUcn(l-40) having the formula:
H-D-Tyr-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-20 Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala--Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. LSIMS
shows a value of 4857.37 Da which agrees with the calculated value of 4857.58 Da. In vitro testing for ACTH
25 secretion using anterior pituitary cell cultures as set forth in Example I sho~ws that the peptide is about 1.25 times as effective as r/hCRF, i.e. 1.23 (0.60 - 2.54). The peptide also has significant mammalian vasodilatory-hypotensive activity, including lowering systemic blood 30 pressure and stimulating the secretion of ACTH.

E~AMP~
The peptide [Ac-Pro3]-hUcn(3-40) having the amino acid sequence (see SEQ ID N0:15):

-CA 02223792 1997-12-0~ 1 W O 97/00063 PCT~US96/10240 Ac-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the ~nn~r described in Example I but, in addition, the S N-terminus is subjected to acetylation by treatment with acetic anhydride after removal of the BOC-protecting group.
The resultant peptide likewise stimulates the secretion of ACTH and ~-END-LI and causes vasodilatory-hypotensive activity, including lowering of systemic blood pressure.
E~AM2LE IIA
The peptide (cyclo 29-32) [Ac-Pro3,D-Phe~1, Glu29, D-Glu31, Lys32]-hUcn(3-40) having the amino acid sequence:
Ac-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-15 Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner as generally described in Example I but, in addition, the N-terminus is subjected to acetylation by treatment with acetic anhydride after 20 removal of the BOC-protecting group. The cyclizing lactam bond is created as described in Example I of U.S. Patent No. S,064,939. LSIMS shows a value of 4562.36 Da which agrees with the calculated value of 4462.42 Da. In vitro testing for ACTH secretion using anterior pituitary cell 25 cultures as set forth in Example I shows that the peptide is about 6 times as effective as r/hCRF, i.e. 6.14 (2.83 -14.05). The resultant peptide likewise stimulates the secretion of ACTH and ~-END-LI and causes vasodilatory-hypotensive activity, including lowering of 30 systemic blood pressure.

ESAMPLB IIB
The peptide (cyclo 29-32) tAc-Pro3,D-Pro4,D-Phe11, Glu29, Lys32]-hUcn(3-40) having the amino acid sequence:

-CA 02223792 1997-12-0~

Ac-Pro-D-Pro4-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val NH2 is synthesized in the manner as generally described in Example 5 I but, in addition, the N-terminus is subjected to acetylation by treatment with acetic anhydride after ~ 1 of the BOC-protecting group. The cyclizing lactam bond is created as described in Example I of U.S. Patent No. 5,064,939. LSIMS shows a value of 4472.40 Da which 10 agrees with the calculated value of 4472.44 Da. In vitro testing for ACTH secretion using anterior pituitary cell cultures as set forth in Example I shows that the peptide is about 10 times as effective as r/hCRF, i.e. 9.90 (4.48 -22.85). The resultant peptide likewise stimulates the 15 secretion of ACTH and ~-END-LI and causes vasodilatory-hypotensive activity, including lowering of systemic blood pressure.

ESAMPLE IIC
The peptide (cyclo 29-32) [Ac-Pro3,D Ser4,D Phe11, Glu~, Lys32]-hUcn(3 40) ha~ing the amino acid sequence:
Ac-Pro-D-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is 25 synthesized in the manner as generally described in Example I but, in addition, the N-terminus is subjected to acetylation by treatment with acetic anhydride after removal of the BOC-protecting group. The cyclizing lactam bond is created as described in Example I of U.S. Patent 30 No. 5,064,939. LSIMS shows a value of 4462.33 Da which agrees with the calculated value of 4462.42. In vitro testi~g for ACTH secretion using anterior pituitary cell cultures as set forth in E~ample I shows that the peptide is about 5.75 times as effective as r/hCRF, i.e. 5.69 (2.43 35 - 14.49). The resultant peptide likewise stimulates the CA 02223792 1997-12-0~

WO 97/00063 PCT~US96/10240 secretion of ACTH and ~-END-LI and causes vasodilatory-hypotensive activity, including lowering of systemic blood pressure.

EXAMPLE III
The peptide hUcn(2-40) having the amino acid sequence (see SEQ ID NO:15):
H-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-10 Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NHz is synthesized in the manner described in Example I. The peptide has significant mammalian vasodilatory- hypotensive activity, including lowering systemic blood pressure and st; ~l~ting the secretion of ACTH.
B~AMPLE IV
The peptide [D-Phell]-hUcn having~the amino acid sequence: H-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-20 Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. The peptide has significant mammalian vasodilatory-hypotensive activity, including lowering systemic blood pressure and stimulating the secretion of ACTH.
~XAMPLB V
The peptide agonist analogs of hUcn as set forth hereinafter, which are considered to have substantially the same amino acid sequence as hUcn, are synthesized:
tGlUl]-hUcn tLeU36]-hUcn tIle5]-hUcn tIle1s]_hUcn tVal17~-hUcn tIIe20]-hUcn tLYs22]-hUcn tLysZ7]-hUcn tLeu35]-hUcn tLeu37]-rUcn W O 97/00063 ~CTrUS96/10240 [Glu38]-hUcn [Ile40]-rUcn [Ser10]-hUcn ~Thr39]-rUcn tLeU~]-hucn tLeu7]-rUcn [Glu2]-hUcn tLeu711]-rUcn tGln29]-hUcn tLys2Z~27]-rUcn [Asn32]-hUcn [Ile5, GlnZ9]-rUcn Each of the foregoing Ucn like agonist peptides has significant mammalian vasodilatory-hypotensive activity, 10 including lowering systemic blood pressure and stimulating the secretion of ACTH.

EXANPLB VI
A further group of Ucn-like agonist peptides are 15 synthesized which fall within the following amino acid ~equence:
Y-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-R~9-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu--R29-Ala--Glu-R32--Asn--Arg-Ile-R36--Phe--R38--Ser-Val--NH2, 20 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R19 is Glu or Ala; ~ is Arg, Glu, Lys or Orn; R32 is Gln, Lys, Orn or Glu; R36 iS Ile, CaMeIle or ~MeLeu; R~8 is Asp or Ala; provided that when R29 is Glu, R32 is either Lys or Orn and the side chains thereof are linked by an 25 amide bond, and that when R29 is either Lys or Orn, R32 is Glu, and the side ch;~ in~: thereof are linked by an amide bond. In this group, D-Phe11 can be replaced by D-Leu or by a D-isomer of another natural ~-amino acid; Glu in the 31-position can be replaced by a D-isomer, e.g. D-Glu, D-30 Arg, imBzlD-His, etc.; and the N-terminus can be shortened by 1 or 2 residues.

The specific peptides are as follows:
tAc-Pro3,D-Phe11]-hUcn(3-40) [D-Leul1, Ala19]-hUcn CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 [D-Phe1~, Ala38]-hUcn ~D-Tyr1~, CaMeIle36]-hUcn [D-Phe1~, CaMeLeu36]-hUcn [Ac-Asp1, D-Phe11, Ala19~38]-hUcn (cyclo 29-32)tD-Leu11, Glu29, Lys3Z]-hUcn (cyclo 29-32)[D-Phe11, Glu29, Orn32]-hUcn(2-40) (cyclo 29-32)tAc-Pro3~D-Phe11 Lys29 Glu32] hU (3 (cyclo 29-32)[D-Tyr11, Orn29, Glu32]-hUcn(3-40) (cyclo 29-32)[D-Phe11~ Glu29, D-Glu31 Lys32]-hUcn (cyclo 29-32)[D-phell~ Glu29, D-Arg31 Lys32] hUc (cyclo 29-32)tD-Tyr11, Glu29, imBzD-His31, Lys32~-hUcn Each of the foregoing Ucn-like agonist peptides has significant mammalian vasodilatory-hypotensive activity, 15 including lowering systemic blood pressure and stimulating the secretion of ACTH.

E~AMPLE VII
A still further group of Ucn-like agonist peptides 20 are synthesized which fall within the following amino acid sequence (SEQ ID N0: 14):
Y--Xaa1--Xaa2--Pro-Xaa4-Xaas-Ser-Xaa7-Asp-Leu-Xaa10-Xaal1-Xaal2-Xaa --Leu-Arg-xaal6-xaal7-xaal8-xaalg-xaa2o Xaa21 Xa 22 Z3 Xaa24~Xaa25~Xaa26~Xaa27~Xaa28-Xaa29-Ala-Xaa31 Xaa3z Asn Arg 35 36 Xaa37 Xaa38-Xaa39-Xaa40-NH2, wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; Xaa1 is Asp, Glu or Gln; Xaa2 is Asn, Asp, Glu or Gly; Xaa4 is Ser or Pro;
Xaa5 is Leu, Ile or Met; Xaa7 is Ile or Leu; Xaa10 is Thr or Ser; Xaa11 is Phe or Leu; Xaa12 is His or Glu; Xaa13 is Leu 30 or Met; Xaa16 is Thr, Asn, Glu, or Lys; Xaa17 is Leu, Met or Val; Xaa~8 is Leu or Ile; Xaa19 is Glu or His; Xaa20 is Leu, Met, Ile or Arg; Xaa21 is Ala, Glu or Thr; Xaa22 is Arg or Lys; Xaa23 is any natural amino acid and preferably Thr, Ser, Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu 3S or Asp; Xaa24 is Gln, Glu or Asp; Xaa25 is any natural amino CA 02223792 1997-12-0~

W O 97/00063 ~CT~US96/10240 acid and preferably Ser, Thr, Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp; Xaa26 is Gln, Leu or Glu;
Xaa27 is Arg, Ala or Lys; Xaaz8 is Glu or Gln; Xaa29 is Arg or Gln; Xaa31 is any natural amino acid aTId preferably Ala, 5 Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp;
Xaa3z is any natural amino acid and preferably Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp; Xaa35 is Ile, Lys, Leu or Asn; Xaa36 is Ile, Tyr, Met or Leu;
Xaa37 is Phe, Leu or Met; Xaa38 is Asp or Glu; Xaa39 is Ser, 10 Ile, Glu or Thr; and xaa40 is Val, Ile, Phe or Ala;
provided that there are no more than 3 residues different from Ucn, and that the N-terminus may be shortened by 1 or 2 residues.
The following specific peptides are synthesized:
[Ac-Pro3,Met5]-hUcn(3-40) tGlu~2]-hUcn tMet13]-hUcn tAsn16~ Ala23]-hUcn [Glu16, Ala25~31]-hUcn [Lysl6, Ile23, Ala32]-hUcn [Vall7, Ile25, Met3~]-hUcn tMet17, Ile3l~32]_hUcn [Serl~, Ilel~, Hisl4]-hUcn tGlul, Met20, Lys35]--hUcn tIle20, Met32, Glu38]--hucn tArg20~ A8n23, Thr25]--hUcn tThr2l Gly23~32]_hucn tGlu21, Lys23, Gln28]-hUcn tLys22~ His23, Leu31]--hUcn tVal23, Glu24, Met36]--hUcn tGln23, Asp25, Glu39]-hUcn tMet2, Gly25, Leu35]-hUcn [Glu23, Tyr36, Phe40]-hucn [Asp23 Lys27, Leu3Z]-hUcn tSer23, Asp24, Met25]-hUcn tAc-pro3,Leu23~25, Gln29]--hUcn(3--40) tAsn25~32, Ile39]_hucll tGln25~3l, Asn35]--hUcn tLys25, Val31, Ala32]--hUcn tGlu2, Asn31, His32]-hUcn tGlnl, Ile5, Val32]--hUcn tGly2, Leul1, Thr3~]--hUcn tGlu25, Leu37]-hUcn tIle5, HisZ5, Ala40]-hUcn tLeu7, His31, Asp32]-hUcn tGln32, Leu36, Ala40]-hUcn tHis25, Ala31, Thr39]-hUcn tLeu26, Lys31, Thr32]-hUcn tGlu26~ Ala27, Lys32]-hUcn tLys27, Asp31, Met37]-hUcn tAc-Pro3,Val25, Leu36]-hUcn(3-40) 35 tAla25, Agp3l~ Ile40]-hUcn tVal32, Leu37, Ile39]-hUcn _ CA 02223792 1997-12-0~
W O 97/00063 ' PCT~US96/10240 tLeu11~ Ile18, Thr39]-hUcn Each of the foregoing Ucn-like agonist peptides has significant mammalian vasodilatory-hypotensive activity, 5 including lowering systemic blood pressure and stimulating the production of ACTH.
The following group of Examples are directed to N-terminally shortened versions (e.g. shortened by 7-10 residues) of the Ucn-like peptides which have antagonistic 10 properties. All of the statements made hereinbefore with respect to the chemical character and/or the synthesis of Ucn analogs are considered to apply equally to the antagonists and are not thus repeated; the antagonists are merely N-terminally shortened versions of the agonists.
15 The specific peptides set forth in the following Examples exhibit antagonistic biological properties with respect to the effect of Ucn on at least the CRF receptors, CRF-Rl, and CRF-R2. In this respect these Ucn-antagonists are considered to generally at least have characteristics and 20 uses similar to those described for CRF antagonists in U.S. Patent No. 5,245,009.

EXANPLE VIII
The peptide, Ucn(11-40), having the amino acid 25 sequence (see SEQ ID N0:8):
H-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. To evaluate the biological activity of a 30 candidate peptide as a Ucn antagonist ,(which will be indicative of its effective binding to CRF receptors), the previously mentioned assay from Endocrinology, 91, supra, is run in the presence of a challenge dose of ovine CRF.
The performance of such candidate in this assay is 35 routinely c~ ,-~ed to the performance of a highly potent CA 02223792 1997-12-0~

WO 97/00063 ~CTrUS96/lOZ40 linear CRF antagonist, i.e. [D-Phel2, Nle21-38]-r/hCRF~12-41) which is hereinafter referred to as the StAn~Ard Antagonist. An in V1VO test measuring elevation of mean arterial blood pressure as a result of iv injection i8 5 also simply and straightforwardly performed. The peptide Ucn(11-40) exhibits significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, as do known CRF antagonists.
The synthesis is repeated twice to produce Ucn (10-10 40) and tAc-Thr10]Ucn(10-40); both show bioactivity as Ucn antagonists.

EZAMPL~ IX
The peptide, tD-Phe1~-Ucn(11-40), having ~he amino 15 acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile Ile-Phe-Asp-Ser-Val-NHz is synthesized in the manner described in Example I. Its specific optical rotation is measured 20 under the conditions set forth hereinbefore as C~]D = -62~
+ 1Ø LSIMS shows a value of 3638.88 Da which agrees with the calculated value of 3639.00 Da. In vitro testing is carried out as described in Example VIII, which demonstrates the peptide is bioactive, exhibiting a value 25 of 0.551 (0.333 - 0.857) compared to this highly biopotent St~n~rd Antagonist. The peptide has significant ma alian vasoconstrictive activity, causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.
~SAMPLB X
The peptide, [D-Tyr11]-Ucn(ll-40), having the amino acid sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala Arg-Thr-35 Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile Ile-Phe-CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 Asp-Ser-Val-NH2 is synthesized in the manner described in Example I. The peptide has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn 5 antagonist. It is also effectively iodinated to provide tl25I-D-Tyr1~]-Ucn(11-40) for use in scrP~i~q assays and the like.

~XANPL~ XI
The peptide, (cyclo 29-32)[D-Phe11, Glu29, Lys32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in 15 Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. 5,064,939, issued Nov. 12, 1991. Its specific optical rotation is measured under the conditions set forth hereinbefore as [~]D : - 49~ + 1Ø LSIMS shows a value of 3593.89 Da which 20 agrees with the calculated value of 3593.97 Da. In vitro testing for ACTH secretion using anterior pituitary cell cultures as set forth in Example VlII shows that the peptide is about 10 times as effective as the StAn~Ard Antagonist, i.e. 10.34 (4.27 - 25.58). The peptide also 25 has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.

E~AMPL~ XII
The peptide, (cyclo 29-32)[D-Tyr11, Glu29, Lys3Z]-Ucn(11-40), having the amino acid sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in W O 97/0~063 PCTAUS96/10240 Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. 5,064,939.
LSIMS shows a value of 3609.82 Da which agrees with the calculated value of 3609.96 Da. In vitro testing for ACTH
5 secretion using anterior pituitary cell cultures as set forth in Example VIII shows that the peptide is about 4 times as effective as the StAn~rd Antagonist, i.e. 4.01 (2.32 - 7.05). The peptide has significant mammalian vasoconstrictive activity causing elevation of mean 10 arterial blood pressure, indicative of its being a Ucn antagonist. It is also iodinated to provide 1Z5~-D-Tyr cyclic analog for use in screening assays and the like.

~XAMP~B SIIA
15 The peptide, (cyclo 29-32)tD-Tyr~, Glu29, D-Glu31, Lys32J-Ucn(11-40), having the amino acid sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-N82 is synthesized in the ~n~r described in 20 Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. 5,064,939. The peptide has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist. It is 25 also iodinated to provide 1Z5I-D-Tyrll cyclic analog for uce in screening assay and the like.

EXAMPLE XIII
The peptide, (cyclo 29-32)[D-Phe11, GluZ9, D-Glu31, 30 Lys32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-~hr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I, with the cyclizing lactam bond being created as 35 described in Example I of U.S. Patent No. 5,064,939.

CA 02223792 1997-12-0~

WO 97100063 PCT~US96/10240 LSIMS shows a value of 3593.80 Da which agrees with the calculated value of 3593.97. In vitro testing for ACTH
secretion using anterior pituitary cell cultures as set forth in Example VIII shows that the peptide is about 4.75 5 times as effective as the Standard Antagonist, i.e. 4.72 (2.19 - 10.00). The peptide also has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.
The synthesis is repeated twice to substitute D-Leu and D-His for D-Phe. The peptides show similar bioactivity as Ucn antagonists.

EXAMPLE XIIIA
The peptide, (cyclo 29-32)[Pro1~,D-Phe11, Glu29, D-Glu31, Lys32]-Ucn(10-40), having the amino acid sequence:
H-Pro-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described 20 in Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. 5,064,939.
LSIMS shows a value of 3690.91 Da which agrees with the calculated value of 3691.02 Da. In vitro testing for ACTH
secretion using anterior pituitary cell cultures as set 2S forth in Example VIII shows that the peptide is about 2.75 times as effective as the Standard Antagonist, i.e. 2.74 (1.02 - 8.02). The peptide also has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn 30 antagonist. This synthesis and testing show that the inclusion of an additional L-isomer at the N-terminus does not significantly alter its bioactivity as a Ucn antagonist.

2XAMPL~ XIIIB

I

CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 The peptide, (cyclo 29-32)tD-Pro10,D-Phe11, Glu29, D-Glu31r Lys32]-Ucn(10-40), having the amino acid sequence:
H-D-Pro-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-5 Phe-~sp-Ser-Val-NH2 is synthesized in the manner described in Example I, with the cyclizing lactam bond being created a~ described in Example I of U.S. Patent No. 5,064,939.
LSIMS shows a value of 3691.00 Da which agrees with the calculated value of 3691.02 Da. In vitro testing for ACTH
10 secretion using anterior pituitary cell cultures as set forth in Example VIII shows that the peptide is about 5 times as effective as the St~n~rd Antagonist, i.e. 4.99 (2.26 - 11.55). The peptide also has significant mammalian vasoconstrictive activity causing elevation of 15 mean arterial blood pressure, indicative of its being a Ucn antagonist. This synthesis and testing show that the inclusion of an additional D-isomer at the N-terminus does not significantly alter the bioactivity of a Ucn antagonist, as by comparison to Peptide XIII.
EXAMP~ SIV
The peptide, ~cyclo 29-32)tD-Phe1l, Glu29, D-Arg31, Orn32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-25 Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Arg-Orn-Asn Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. S,064,939. The peptide has significant mammalian vasoconstrictive 30 activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.
The synthesis is repeated twice substituting imBzlD
His and D-2Nal for D-Arg and twice again to also add Ac-Thr a~ the N-terminus with these substitutions. All four 35 peptides show good bioactivity as Ucn antagonists.

-CA 02223792 1997-12-0~

Ea~P~E arv The peptide, (cyclo 29-32)tD-Phe11, Lys29, Glu32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-5 Gln-Ser-Gln-Arg-Glu-Lys-Ala-Glu-Glu-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the manner described in Example I, with the cyclizing lactam bond being created as described in Example I of U.S. Patent No. 5,064,939. The peptide has significant mammalian vasoconstrictive 10 activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.

ESAMP E XVI
The peptide, (cyclo 29-32)tD-Phel~, Lys29, D-Glu31, 15 Glu32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Lys-Ala-D-Glu-Glu-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 is synthesized in the ~nnPr described in Example I, with the cyclizing lactam bond being created as 20 described in Example I of U.S. Patent No. 5,064,939. The peptide has significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.

~AMPLE XVII
Additional Ucn antagonist peptides, as set forth as follows, many of which are considered to be substantially 30 the same as Ucn(11-40), are synthesized in the manner described in Example I:

[Vall7]--rUcn(11--40) tCaMeLeu36]-rUcn(11-40) [Lys2Z]-rUcn(8-40) [D-Phel1, Ilel8]-rUcn(1l-40) 35 [Ac-Asp8,Lys22]-Ucn(8-40) [Leu35]-rUcn(11-40) CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 [Ile2~]-rUcn(10-40) [Ac-Thr10,Ile20]-Ucn(10-40) [Glu38]--rUcn(11--40) tD-Phe11, Lys27]--rUcn(11-40) [Serl~]-rUcn(10-40) [C~MeIle36, Leu37]-rUcn(11-40) [D-Leu~1]-rUcn(11-40) [Ala38,Ile40]-rUcn(11-40) 5 tGln29]-rUcn(9-40) [Ala19, T~r39]-rUcn(11-40) [Asn32]-rUcn(11-40) [D-Phe~1, Lys22~27]-rUcn(11-40) [Ac-Leu9,Gln29]-Ucn(9-40) [Lys22, Gln29] rUcn(11-40) Each of the foregoing Ucn like antagonist peptides has 10 significant mammalian vasoconstrictive activity causing elevation of mean arterial blood pressure, indicative of its being a Ucn antagonist.
The following group of Examples are directed to the synthesis of CRF-BP blockers which increase the available 15 amount of endogenous CRF and Ucn by complexing with CRF-BP.
~SAMPLE XVIII
A peptide, Ucn(5-32), having the amino acid sequence (see SEQ ID NO:8):
20 Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala~Glu-Gln is synthesized in the manner described in Example I.
A prospective peptide blocker is evaluated using a competitive hCRF-BP ligand binding assay. Binding of t1Z5I-25 DTyr~]hCRF to soluble hCRF-BP is performed in p~osphate-buffered saline (PBS) containing 25 mM EDTA, 0.25% bovine serum albumin, and .01% Triton X-100, using medium enriched by recombinant CHO cells as a source of hCRF-BP.
Reactions are performed in a total volume of 400 ~l 30 including 50,000 CPMt125I-DTyr0]hCRF. A constant amount of radioactive hCRF and hCRF-BP and varying amounts of the sample peptide are used to carry out competitive binding assays. After an overnight ;n~llh~tion at room temperature, precipitation is accomplished using rabbit 35 anti-hCRF-BP antiserum (1:9000 final dilution) and 200 ~l CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 of sheep anti-rabbit IgG solution. After incubating with the primary and secondary antisera for 30 minutes each, 1 ml of saline wash is added, and the test tubes are centrifuged at 2000 x g for 20 minutes at 4~C.
5 Precipitates are counted in a gamma counter. Inhibitory b;n~;ng affinity constant (Kj) values are determined using parameters calculated by the LIGAND computer program, Munson et al., Anal. Biochem., 107, 220 (1980), and a Vax/VMS computer system.
The CRF-BP blocker Ucn(5-32) has a Kj lower than that of hCRF(9-33) and thus is a potentially superior blocking agent for increasing the available amount of CRF and/or Ucn.

E~AMPLE SIX
A peptide, Ucn(8-32), having the amino acid sequence (see SEQ ID NO:8):
Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln is synthesized 20 in the manner described in Example I. LDMS shows a value of 3023.48 Da which agrees with the calculated value of 3023.65 Da. Testing shows that the peptide has a Kj lower than that of hCRF(9-33) and thus is a potentially superior blocking agent for increasing the available amount of CRF
25 and/or Ucn.

~AHP~2 XX
A peptide, Ucn(3-27), having the amino acid sequence (see SEQ ID N0:8):
30 Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg is synthesized in the manner described in Example I. The peptide has a K
lower than that of hCRF(9-33) and thus is a potentially superior blocking agent for increasing the available 35 amount of CRF and/or Ucn. I

CA 02223792 1997-12-0~

WO 97/00063 ~CTAUS96/10240 EaI~CP~E ~ I
A peptide, tIle~8]-Ucn(6-29), having the amino acid sequence (see SEQ ID NO:8):
Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu Ile-Glu-5 Leu-~la-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg is synthesized in the manner described in Example I. The peptide has a Kl lower than that of hCRF(9-33) and thus is a potentially superior blocking agent for increasing the available amount of CRF and/or Ucn.
Ucn profoundly stimulates the pituitary-adrenalcortical axis and is considered to be useful to stimulate the func~ions of this axis in some types of patients with low endogenous glucocorticoid production.
For example, Ucn and its analogs should be useful in 15 restoring pituitary-adrenal function in patients having received exogenous glucocorticoid therapy whose pituitary-adrenalcortical functions remain su~essed.
Most other regulatory peptides have been found to have effects upon the central nervous system and upon the 20 gastrointestinal tract. Because ACTH and sympathetic nervous system activation secretion is the "sine qua non"
of mammal'c response to stress, it was not surprising that CRFs have significant effects on the brain as a mediator of the body's stress response. Accordingly, Ucn and its 25 analogs are considered to also find application in modifying the mood, learning, memory and behavior of normal and mentally disordered individuals. Because Ucn elevates the levels of ACTH, ~-END, ~-lipotropin, other pro-opiomelanocortin gene products and corticosterone, its 30 administration can be used to induce the effects of the foregoing POMC-derived peptides on the brain to thereby influence memory, mood, pain appreciation, etc., and more specifically, alertness, depression and/or anxiety, and also their effects peripherally. For example, when 35 administered directly into the ventricles, CRFs increase CA 02223792 1997-12-0~

W O 97/00063 ; PCTrUS96/10240 physical activity and improve learning performance in rats and thus may function as a natural stimulant; because Ucn similarly activates the CRF receptors, it will function similarly.
Because CRF-R2 has been found to be abundantly expressed in the heart, especially in association with blood vessels, and because it is known that the addition of CRF into the left atrium of an isol;ated perfused heart induces a prolonged dilatory effect on coronary arteries, 10 transiently produces a positive inotropic effect and stimulates the secretion of atrial natriuretic peptide, it is now believed that Ucn is responsible, at least in part, for regulating cardiac perfusion because of its particularly high affinity for the CRF-R2s. It is also 15 expected that other vascular beds, such as the superior mesenteric, will be dilated by Ucn and its analogs.
Because of these biological effects in the heart, Ucn and agonists/antagonists thereof (as well as anti-Ucn antihoA;es), can be effectively used to selectively 20 modulate cardiac perfusion.
Moreover, because of the localization of CRF-R2 on blood vessels, it is considered that Ucn-like agonist and antagonist peptides of the invention aFe therapeutically useful to modulate blood flow in many various vascular 25 beds, and particularly in desired tissues and organs. Ucn and its agonist analogs are considered to be of use for increasing blood flow to the gastrointestinal tract of animals, particularly humans and other mammals, because all CRF-related peptides have been shown to dilate the 30 mesenteric vascular bed. CRF and certain fragments have been shown to modulate vascular permeability (Wei E.T. et al., "Peripheral anti-inflammatory actions of cortic~Llopin-releasing factor", pp. 258-276, Corticotropin-~eleasing Factor (Ciba Foundation Symposium 35 172) John Wiley & Sons, 1993). Ucn and its fragments will CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 also reduce va~-c~ r leakage and have a salutary effect on injury- or surgery-in~l~c~ tissue swelling and inflammation. Therefore, Ucn and its analogs and fragments that are agonists can be a~' ; ni ~tered 5 parenterally to decrease inflammation, swelling and oedema and to reduce fluid loss following heat injury.
oCRF, r/hCRF, urotensin I and sauvagine have been shown to inhibit gastric acid production, and Ucn and its analogs are considered to also be effective in the 10 treatment of gastric ulcers by reducing gastric acid production and/or inhibiting certain gastrointestinal functions in a mammal. Ucn and its analogs will be effective in increasing intestinal transit rate and useful in the treatment of acute constipation.
A number of direct stimulatory effects of CRF on the GI tract have earlier been described. For example, CRF
acts on the gut in vitro to depolarize myenteric neurons in the small intestine. The results of in vivo studies with intravenously administered CRF and CRF antagonists 20 have been consistent with the observed effect of CRF to control gastric emptying and intestinal motility. The Ucn-like peptides of the invention are considered useful in treating intestinal and gastrointestinal disorders, such as irritable bowel syndrome. CRF antagonists have 25 previously been used to therapeutically treat irritable bowel syndrome, and antagonists based upon Ucn (which would be selective for CRF-R2) are considered to be even more useful. These antagonists may also be used to treat spastic colon and Crohn's disease.
These Ucn-like peptides may also be used to evaluate hypothalamic pituitary adrenal function in mammals with suspected endocrine or central nervous system pathology by suitable administration followed by monitoring bodily functions. For example, administration may be used as a CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 diagnostic tool to evaluate Cllching~s disease and affective disorders, such as depressiye illness.
Ucn, an analog or a nontoxic salt thereof, combined with a pharmaceutically or veterinarily acceptable carrier 5 to form a pharmaceutical composition, may be a~ inistered to animals, including humans and other mammals, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g. intranasally, intracerebrospinally or orally. The isolated peptides should;be at least about 10 90% pure and preferably should have a purity of at least about 98%; however, lower purities are effective and may well be used with mammals other than humans. This purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments 15 present. Administration to humans may be employed by a physician to lower blood pressure or to stimulate endogenous gluco-corticoid production. The required dosage will vary with the particular condition being treated, with the severity of the condition and with the 20 duration of desired treatment. Ucn or Ucn analogs can also be administered, e.g., icv, to cause an increase of Ucn in the brain and thereby cause (a) improvement in short to medium term memory in a subject afflicted with Al7h~ 'S disease; (b) relief from chronic fatigue 25 syndrome; (c) suppression of appetite, (d) stimulation of the respiratory system, (e) improvement in learning performance; (f) improvement in memory; (g) im~uv~- ?nt in alertness; (h) reduction of depression and/or (i) lessening of anxiety. Particular effectiveness is shown 30 in appetite suppression.
Such peptides are often a~ ;ni~tered in the form of pharmaceutically or veterinarily acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, 35 aluminum or the like. Illustrative of such nontoxic salts - CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 are hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, ~umarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be 5 administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gela~in; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may 10 be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
It may also be desirable to deliver Ucn or analogs thereof over prolonged periods of time, for exa~ple, for 15 periods of one week to one year from a single administration, and slow release, depot or implant dosage forms may be utilixed as well known in this art~ For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low 20 degree of solubility in body fluids, for example, an acid addi~ion salt with a polybasic acid; a salt with a polyvalent metal cation; or combination of the two salts.
A relatively insoluble salt may also be formula~ed in a gel, for example, an aluminum stearate gel. A suitable, 25 sloworelease depot formulation for injection may also contain Ucn or an analog or a salt thereof dispersed or ~ncArculated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described 30 in U.S. Pat. No. 3,773,919.
Therapeutically effective amounts of the peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, 35 pharmaceutically or veterinarily-acceptable carrier. A

CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 therapeutically effective amount is considered to be a predetermined amount calculated to achieve the desired effect, e.g. to increase or decrease the amount of ACTH, in a patient. The required dosage will vary with the 5 particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milli,gram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to 10 administer such compounds in depot or long-lasting form as earlier described. A therapeutically effective amount is typically an amount of a Ucn or an analog thereof that, when administered peripherally in a physiologically acceptable composition, is sufficient to achieve a plasma 15 concentration thereof from about 0.1 ~g/ml to about 100 ~g/ml, preferably from about 1 ~g/ml to about 50 ~g/ml, more preferably at least about 2 ~g/mh and usually 5 to 10 ~g/ml. Antibodies or antisense polynucleotides are also administered in proportionately appropriate amounts in 20 accordance with known practices in this art. The level of ACTH present in a patient, particularly in the plasma, can be readily determined by routine clinical analysis.
Changes in ACTH levels can be monitored during a treatment regimen to determine the effectiveness of the administered 25 Ucn-like peptide over time. In some inst~nc~, treatment of subjects with these peptides can be carried out in lieu of the administration of ACTH or corticosteroids, in such instances a dosage as low as about 10 ng/Kg of body weight may be employed.
Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art 35 may be made without departing from the scope of the CA 02223792 1997-12-0~

W O 97/00063 ~CTAUS96/10240 invention which is set forth in the claims appended hereto. Although the claims variously define the invention in terms of a peptide sequence, it should be understood that such is intended to include nontoxic salts 5 thereof which are well known to be the full equivalent thereof and which are most frequently a~;nictered.
Instead of the simple ami~e at the C-terminus, a lower alkyl-substituted amide, e.g. methylamide, ethylamide, etc, may be incorporated or the C-terminus may be 10 otherwise blocked as well known in the peptide art.
Polypeptides having an amino acid residue sequence substantially identical to the sequence of Ucn specifically shown herein, in which one or more residues have been conservatively substituted with a functionally 15 similar residue, are considered to be equivalents of Ucn so long as they mimic a biological function of CRF. Such peptides and salts thereof are considered as being within the scope of the claimed invention.
The disclosures of all patents and publications set 20 forth hereinbefore, as well as of the two priority applications, are expressly incorporated herein by reference. As used herein, all temperatures are ~C., and all ratios and percentages of liquid materials are by volume.

CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 Sequence Listing 8ummary SEQ ID NO:l, when the C-terminus is amidated, is the amino acid sequence of ovine CRF.
SEQ ID NO:2, when pGlu is at the N-terminus and the 5 C-terminus is amidated, is the amino acid sequence of frog sauvagine.
SEQ ID NO:3, when the C-terminus is amidated, is the amino acid sequence of rat/human CRF.
SEQ ID NO:4, when the C-terminus is amidated, is the 10 amino acid sequence of suckerfish urotensin.
SEQ ID NO:5, when the c-terminus is amidated, is the amino acid sequence of carp urotensin.
SEQ ID NO:6, when the C-terminus is amidated, is the amino acid sequence of flathead sole (Maggy).
SEQ ID NO:7, when the C-terminus is amidated, is the amino acid sequence of fish CRF.
SEQ ID NO:8, when the C-terminus is amidated, is the amino acid sequence of rat-derived urocortin(Ucn).
SEQ ID NO:9 is the nucleic acid sequence from which 20 SEQ ID NO:8 was deduced.
SEQ ID NO:10 is the amino acid sequence of the rat-derived CRF receptor referred to as "rCRF-Rl".
SEQ ID NO:11 is the amino acid sequence of a mouse-derived CRF receptor referred to as "mCRF-R2~".
SEQ ID No:12 is the amino acid sequence of a rat-derived CRF receptor referred to as "rCRF-R2~".
SEQ ID NO:13 is the amino acid sequence of a rat-derived CRF receptor referred to as "rCRF-R2~".
SEQ ID NO:14 is the amino acid sequence of a 40-30 residue peptide defining certain analogs of Ucn.
SEQ ID NO:15 is the amino acid sequence of the precursor plus the mature human Ucn peptide.
SEQ ID NO:16 is the nucleic acid sequence from which SEQ ID NO:15 was deduced.

W O 97/00063 PCTrUS96/10240 SEQUENCE LISTING

(1) r~N~RAT- lNr OR~ATION:
(i) APPLICANT:
~AI NAME: THE SALR lwx~l~u~ FOR BIOLOGICAL STUDIES
BI STREET: 10010 North Torrey Pine~ Road ,C, CITY: La Jolla ~Dl STATE: Q
E CUuh~r: USA
~Fl POSTAL CODE (ZIP): 92037 Al NAME: VALE Jr., Wylie W.
IB STREET: 1643 Valdez ,C, CITY: La Jolla ,DI STATE: CA
El C~uh~Y: USA
l;F POSTAL CODE (ZIP): 92037 ,AI NAME: VAUGHAN, Jo~n Bl STREET: 729 Mo~aic Circle ,C CITY: Ocean~ide D STATE: CA
EI COUhl~r: USA
FJ POSTAL CODE (ZIP): 92057 ~Al NAME: DONALDSON, Cynthi~ J.
(Bl STREET: 1767 T-i ao~ Street ,CJ CITY: San Diego D STATE: CA
EI Co~h~Kr: USA
F POSTAL CODE (ZIP~: 92110 rAl NAME: LEWIS, Xathy A.
BI STREET: 1760 Montecito Way ,C CITY: S~n Diego Dl STATE: CA
El CUUh~Y: USA
,F~ POSTAL CODE (ZIP): 92103 rAJ NAME: SAWCHENXO, Paul Bl STREET: 1820 Autumn Place ,C, CITY: Encinitas D STATE: CA
~E COu.~Y: USA
~F, POSTAL CODE (ZIP): 92024 ~Al NAME: RIVIER, Jean E.F.
B~ STREET: 9674 Blackgold Road C CITY: La Jolla Dl STATE: CA
~E! COu.~.~r: USA
F POSTAL CODE (ZIP): 92037 ~Al NAME: PERRIN, Marilyn H.
B STREET: 8844 Robinhood Lane ,C CITY: La Jolla DI STATE: CA
El COUhl~Y: USA
~F POSTAL CODE (ZIP): 92037 CA 02223792 l997-l2-05 W O 97/00063 PCT~US96/10240 (ii) TITLE OF lNVh~lON: U~OC~r lN PEPTIDES
(iii) NUMBER OF ~yu S~q: 16 (iv) COk~ rONDENCE ADDRESS:
AJ AnDRF.cs~: FITCH, EVEN, TABIN & FT~N~Y
8 STREET: 135 S. LaSalle Street, Suite 900 ,CJ CITY: Chi CA~0 (D STATE: Illinois rE, COUh~Y: USA
~F ZIP: 60603 (v) COMPUTER R~AnART.T! FORM:
A~ MEDIUM TYPE: Floppy di~k B COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
DI SOFTWARE: PatentIn Relea~e #1.0, Version ~1.30 (EPO) (V) ~U~RLh~ APPLICATION DATA:
APPLICATION NUMBER: PCT/US96/
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/490,314 (B) FILING DATE: 13-JUN-1995 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/002,223 (B) FILING DATE: 11-AUG-1995 (vili) A~ORNY;Y/AGENT lNyoRMATIoN:
(A) NAME: S ' -nn, J~mes J.
(B) REGISTRATION NUMBER: 20,856 (C) h~rKkh~L/DOCXET NUMBER: 57611-PC
(iX) ~TCT-~ '~I CATION INFORMATION:
(A) TELEPHONE: 619-552-1311 (B) TELEFAX: 619-552-0095 (2) INFORMATION FOR SEQ ID NO:1:
($) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) M~T~FcuT~ TYPE: p~ptide (Xi) ~:QUL.._~ DESCRIPTION: SEQ ID NO:l:
Ser Gln Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe Hi~ Leu Leu Arg 1 5 10 ~ 15 Glu Val Leu Glu Met Thr Lys Ala A~p Gln Leu Ala Gln Gln Ala His Ser A~n Arg Ly~ Leu Leu A~p Ile Ala (2) IN~-~R~ATIoN FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:

.

W O 97/00063 PCTrUS96/10240 (A) LENGTH: 40 amino acids (B) TYPE: amino acLd (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~yu ~ DESCRIPTION: SEQ ID No:2:
Glu Gly Pro Pro Ile Ser Ile A~p Leu Ser Leu Glu Leu Leu Arg Ly~

Met Ile Glu Ile Glu Lys Gln Glu LYB Glu Ly~ Gln Gln Ala Ala A~n A~n Arg Leu Leu Leu A~p Thr Ile (2) l~hATIoN FOR SEQ ID NO:3:
(L) ~y~ ~~ CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ~OT.TCCTJT.TC TYPE: peptide (~i) SEQUEN OE DESCRIPTION: SEQ ID NO:3:
S~r Glu Glu Pro Pro Ile Ser Leu Anp Leu Thr Phe His Leu Leu Arg 1 5 lO 15 Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala Hi~

Ser A~n Arg Ly~ Leu Met Glu Ile Ile (2) l~OR~ATION FOR SEQ ID NO:4:
T~' CHARACTERISTICS:
(A) LENGTH: 41 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (Li) ~T ~C~T TC TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
A~n A~p ABP Pro Pro Ile Ser Ile A~p Leu Thr Phe Hi~ Leu Leu Arg A~n Met Ile Glu Met Ala Arg Ile Glu A~n Glu Arg Glu Gln Ala Gly Leu A~n Arg Ly~ Tyr Leu A~p Glu Val (2) lN~ofi~ATIoN FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
c (A) LENGTH: 41 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear CA 02223792 l997-l2-0~

W O 97/00063 , PCT~US96/10240 ( ii ) M~T-~CTJr ~ TYPE: peptide (xi) SEQUENCE rT~sr~TpTIoN: SEQ ID NO:5:
A~n A~p Asp Pro Pro Ile Ser Ile A~p Leu Thr Phe His LQU Leu Arg l 5 10 15 A~n Met Ile Glu Met Ala Arg AQn Glu A~n Gln Arg Glu Gln Ala Gly Leu A~n Arg Lys Tyr Leu Asp Glu Val (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acidn (B) TYPE: amino acid (D) TOPOLOGY: linear ( 1i ) MOT T~CUr T~' TYPE: peptide (xi) ~Luu~N~ DESCRIPTION: SEQ ID NO:6:
Ser Glu Glu Pro Pro Met Ser Ile Asp Leu Thr Phe Hi~ Met Leu Arg l 5 10 15 Arn M~t I1Q HL~ Arg Ala Ly~ Met Glu Gly Glu Arg Glu Gln Ala Leu Ile A-n Arg Asn Leu Leu Asp Glu Val (2) INFORMATION FOR SEQ ID NO:7:
(i) ~Qu~ E CHARACTERISTICS:
(A) LENGTH: 41 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~UL._~: DESCRIPTION: SEQ ID NO:7:
Ser Glu Glu Pro Pro Ile Ser Leu A~p Leu Thr Phe His Leu Leu Arg 1 5 10 ' 15 Glu Val Lnu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala ~in Ser A~n Ary Lys Met Met Glu Ile Phe (2) lNruKMATION FOR SEQ ID NO:8:
(i) ~EUU~N~ CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) M~T-FCUT T~ TYPE: peptide (xi) ~E~u~r._~ D~CrRTPTION: SEQ ID NO:8:

W O 97/00063 PCTrUS96/10240 A~p A~p Pro Pro Leu Ser Ile A~p Leu Thr Phe Hi~ Leu Leu Arg Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln Asn Arg Ile Ile Phe A~p Ser Val (2) I~OX~ATION FOR SEQ ID NO:9:
( i ) ~UL.._~ CHARACTERISTICS:
A~l LENGTH: 129 ba~e pairP
~Bl TYPE: nucleic acid C, STRANDEDNESS: ~ingle ~D TOPOLOGY: linear ( ii ) ~T~T~'~UT T~! TYPE: cDNA to mRNA
(iii) n~ro~n~lcAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rat Brain (vii) IMMEDIATE SOURCE:
(B) CLONE: CR21 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..120 (xi) ~Qu ~ DESCRIPTION: SEQ ID NO:9:
GAC GAC CCG CCG TTG TCC ATC GAC CTC ACC TTC CAC CTG CTG CGG ACC 48A~p A~p Pro Pro Leu Ser Ile A~p Leu Thr Phe His Leu Leu Arg Thr CTG CTA GAG CTA GCT CGG ACA CAG AGC CAG CGC GAG CGC GCA GAG CAG 96Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln A~n Arg Ile Ile Phe A~p Ser Val (2) lNrO~MATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 415 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOT~T~'CUT~T~' TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..415 (D) OTHER INFORMATION: /note= "Human Pituitary CRF-Receptor-Rl"

CA 02223792 l997-l2-0~

W O 97/00063 PCT~US96/10240 ~x) PUBLICATION INFORMATION:
(A) AUTHORS: Perrin, Marilyn H
Donald~on, Cynthia J
Chen, Ruoping Lewis, Kathy A
Vale, Wylie W
(B) TITLE: Cloning and Functional Expression of a Rat Brain Corticotropin Releasing Factor (CRF) Receptor ~C' JOURNAL: Endocrinology D VOLUME: 133 E, ISSUE: 6 F PAGES: 3058-3061 ,GJ DATE: 1993 (Xi) ~QU~N~ DESCRIPTION: SEQ ID NO:10:
Met Gly Arg Arg Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu Gly Leu Asn Pro Val Ser Thr Ser Leu Gln Asp Gln Arg Cy8 Glu Asn Leu Ser Leu Thr Ser Asn Val Ser Gly Leu Gln Cys Ann Ala Ser Val A~p Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val Val Arg Pro cyn Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr A~n A~n Gly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg Val A~n Tyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 lOS 110 Lys Val His Tyr Hia Val Ala Val Ile Ile Ann Tyr Leu Gly Hi~ Cy~

Ile Ser Lcu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu Arg Ser Ile Arg Cy~ Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser Ala Phe Ile Leu Arg A~n Ala Thr Trp Phe Val Val Gln Leu Thr Val Ser Pro Glu Val His Gln Ser A~n Val Ala Trp Cys Arg Leu Val Thr Ala Ala Tyr Asn Tyr Phe His Val Thr Aan Phe Phe Trp Met Phe Gly Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp Arg Leu Arg Ly~ Trp Met Phe Val Cys Ile Gly Trp Gly Val Pro Phe W O 97/00063 PCTrUS96/10240 Pro Ile Ile Val Ala Trp Ala Ile Gly Ly~ Leu Hi~ Tyr ARP A~n Glu Ly~ cy8 Trp Phe Gly Ly~ Arg Pro Gly Val Tyr Thr A~p Tyr Ile Tyr Gln Gly Pro Met Ile Leu Val Leu Leu Ile A~n Phe Ile Phe LQU Phe A~n Ile Val Arg Ile Leu Met Thr Ly~ L~u Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala Val Ly~ Ala Thr Leu Val Leu Leu Pro Leu Leu ~ly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu A~p Glu Val Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cy~ Phe Leu Asn Ser Glu Val Arg Ser Ala Il~ Arg Ly~ Arg Trp Arg Arg Trp Gln Asp Ly~

Hi~ Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Val Ser Phe Hi~ Ser I 1Q Ly~ Gln Ser Thr Ala Val (2) INFOR~ATION FOR SEQ ID NO~
(i) ~u~._~ CHARACTERISTICS:
(A) LENGTH: 431 amino acid~
(B~ TYPL: amino acid (D) TOPOLOGY: linear (ii) MOr~CUrr~' TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..431 ~D) OTHER INFORMATION: /note= ~Product-mou~e heart derived CRF-R2 Long Form"
(x) PUBLICATION lNrORhATION:
(A) AUTHORS: Perrin, Marilyn Donaldson, Cynthia Chen, Ruoping Blount, Amy Be,~en, Travi~
Rilezikjian, Loui~e Sawchenko, Paul Vale, Wylie (B) TITLE: Identification of a ~econd corticotropin-relea~ing factor ,ecepLoL gene and characterization of a cDNA expre~ed in heart (C) JOURNAL: Proc. Natl. Acad. Sci. U.S~A.
(D) VOLUME: 92 CA 02223792 1997-12-0~

W O 97/00063 PCT~US96/10240 (F) PAGES: 2969--2973 ~G) DATE: March-1995 (xi) SEQUENCE DFSr-~TPTION: SEQ ID NO:11:
Met Gly Thr Pro Gly Ser Leu Pro Ser Ala Gln Leu Leu Leu Cys Leu Phe Ser Leu Leu Pro Val Leu Gln Val Ala Gln Pro Gly Gln Ala Pro Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu Gln Tyr Cy~ Hi3 Arg Thr Thr Ile Gly Asn Phe Ser Gly Pro Tyr Thr Tyr Cys Ann Thr Thr Leu A~p Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala Pro Gly Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe A~n Gly I le Ly~ Tyr A~n Thr Thr Arg Ann Ala Tyr Arg Glu Cy~ Leu Glu Asn Gly Thr Trp Ala Ser Arg Val A~n Tyr Ser Hi~ Cy~ Glu Pro I le Leu Asp Asp Lys Gln Arg Lys Tyr A~p Leu HLs Tyr Arg Ile Ala Leu Ile Val Asn Tyr Leu Gly Hin Cy- Val Ser Val Val Ala Leu Val Ala Ala Phe Leu Leu Phe Leu Val Leu Arg Snr Ile Arg Cy~ Leu Arg Ann Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg A~n Ile Ala Trp Phe Leu Leu Gln Leu Ile A~p His Glu Val His Glu Gly Ann Glu Val Trp Cy~ Arg Cy~ Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr A~n Phe Phe Trp Met Phe Val Glu Gly Cy~ Tyr Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu His Leu Arg Lys Trp Leu Phe Leu Phe I le Gly Trp Cys I le Pro Cys Pro Ile Ile Ile Ala Trp Ala Val Gly Ly~ Leu Tyr Tyr Glu A~n Glu Gln Cys Trp Phe Gly Lys Glu Ala Gly A~p Leu Val Asp Tyr Ile Tyr Gln Gly Pro Val Met Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe W O g7/00~63 ~CTrUS96/10240 Asn Ile Val Arg Ile Leu Met Thr LYR Leu Arg Ala Ser Thr Thr Ser 305 310 31~ 320 Glu Thr Ile Gln Tyr Arg Ly~ Ala Val Ly~ Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val A~n Pro Gly Glu A3p A~p Leu Ser Gln Ile Val Phe Ile Tyr Phe A~n Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cy~ Phe Phe Asn Gly Glu Val Arg Ala Ala Leu Arg Lyn Arg Trp Hi~ Arg Trp Gln A~p Hi~

Hi~ Ala Leu Arg Val Pro Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe Hi~ Ser Ile Ly~ Gln Thr Ala Ala Val (2) 1N~OR~AT1ON FOR SEQ ID NO:12:
i ) ~L_. _K CHARACTERISTICS:
(A) LENGTH: 411 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) MOLECULE TYPE: protein (lx) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..411 (D) OTHER INFORMATION: /note- nRat CRF-R2 Short Form"
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Lovenberg, Timothy W
Liaw, Chen W
Grigoriadi~, Dimitri E
ClevenyeL, William Ch-l - ~, D~r~k T
DeSouza, Errol B
Olter~dorf, Tilman (B) TITLE: Cls~i ng and characterization of a functionally di~tinct corticotropin-releasing factor receptor ~ubtype from rat brain C', JOURNAL: Proc. Natl. Acad. Sci. U.S.A.
Dl VOLUME: 92 ~F, PAGES: 836-840 ~G, DATE: January-1995 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met A~p Ala Ala Leu Leu Leu Ser Leu Leu Glu Ala Asn Cy~ Ser Leu Ala Leu Ala Glu Glu Leu Leu Leu A~p Gly Trp Gly Glu Pro Pro A~p , CA 02223792 1997-12-0~

WO 97/00063 PCTrUS96/10240 Pro Glu Gly Pro Tyr Ser Tyr Cys Asn Thr Thr Leu Asp Gln I le Gly Thr Cy~ Trp Pro Gln Ser Ala Pro Gly Ala Leu Val Glu Arg Pro Cy8 Pro Glu Tyr Phe Asn Gly Ile Ly~ Tyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cy~ Leu Glu A~n Gly Thr Trp Ala Ser Arg Ile Asn Tyr Ser H$s Cys Glu Pro Ile Leu A~p A~p Lys Gln Arg Ly~ Tyr A~p Leu HLs Tyr Arg Ile Ala Leu Ile Ile Asn Tyr Leu Gly His Cys Val Ser Val Val Ala Leu Val Ala Ala Phe Leu Leu Phe Leu Val Leu Arg Ser Ile Arg Cy~ Leu Arg Asn Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile L~u Arg Asn Ile Thr Trp Phe Leu Leu Gln Leu Ile Asp HL~ Glu Val 165 1~0 i 175 H$~ Glu Gly Asn Glu Val Trp Cys Arg Cy8 Val Thr Thr Ile Phe Arn Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr L~u H$~ Thr Ala IlQ Val Met Thr Tyr Ser Thr Glu Hi~ Leu Arg Ly~

Trp Leu Phe Leu Phe Ile Gly Trp Cy~ Ile Pro Cy8 Pro Ile Ile Val Ala Trp Ala Val Gly Lys Leu Tyr Tyr Glu A~n Glu aln Cy~ Trp Phe Gly Lys Glu Pro Gly A~p Leu Val Anp Tyr Ile Tyr Gln Gly Pro Ile Ile Leu Val Leu Leu Ile A~n Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr LyE~ Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Ly~ Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val A~n Pro Gly Glu Asp A~p Leu Ser Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Phe Asn Gly Glu Val Arg Ser --78 ~

CA 02223792 l997-l2-05 W O 97/00063 PCTrUS96/10240 Ala Leu Arg Ly~ Arg Trp ~i~ Arg Trp Gln A~p Hi3 Hi~ Ala Leu Arg Val Pro Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe Hi~ Ser Ile Ly~ Gln Thr Ala Ala Val (2) lNr-~R~ATION FOR SEQ ID NO:13:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 431 _mino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ~T-~CuT~ TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B~ LOCATION: 1..431 (D) OTHER lN~ATION: /note= "Rat CRF-R2 Long Form"
(x) PUBLICATION l~Ofi~ATION:
(A) AUTHORS: Lovenberg, Timothy W
Liaw, Chcn W
Grigoriadi~, Dimitri E
Cl~venger, William rha ~ - 6, Derek T
DeSouza, Errol B
Olter~dorf, Tllman (B~ TlTLE: ~l~ni n7 and characterization of a functio~Ally dL~tinct corticotropin-relea~ing factor rcceptor subtype from rat brain IC JOURNAL: Proc. Natl. Acad. Sci. U.S.A.
~D VOLUME: 92 Fl PAGES: 836-840 ~G) DATE: January-1995 (xi) SEQUEN OE D~SCRTPTION: SEQ ID NO:13:
Met Gly HiJ Pro Gly Ser Leu Pro Ser Ala Gln Leu Leu Leu Cy~ Leu l 5 10 15 Tyr Ser Leu Leu Pro Leu Leu Gln Val Ala Gln Pro Gly Arg Pro Leu Gln A~p Gln Pro Leu Trp Thr Leu Leu Glu Gln Tyr Cy~ Hin Arg Thr ~5 Thr Thr Arg Asn Phe Ser Gly Pro Tyr Ser Tyr Cy~ A~n Thr Thr Leu A~p Gln Ile Gly Thr cy8 Trp Pro Gln Ser Ala Pro Gly Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe A~n Gly Ile Ly~ Tyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cys Leu Glu A~n Gly Thr Trp Ala Ser Arg CA 02223792 1997-12-0~

W O 97/00063 PCTrUS96/10240 Ile Asn Tyr Ser Hin CYQ Glu Pro Ile Leu A~p Aup Ly~ Gln Arg Ly~

Tyr A~p Leu Hin Tyr Arg Ile Ala Leu Ile Ile A~n Tyr Leu Gly Hi~

CYE~ Val Ser Val Val Ala Leu Val Ala Ala Phe Leu Leu Phe Leu Val Leu Arg Ser Ile ArS7 CYQ Leu Arg Af~n Val Ile Hi~ Trp A~n Leu Ile Thr Thr Phe Ile Leu Arg A~an Ile Thr Trp Phe Leu Leu Gln Leu Ile Anp Hin Glu Val Hi~ Glu Gly A~n Glu Val Trp Cys Arg Cy~ Val Thr Thr Ile Phe Asn Tyr Phe Val Val Thr A~n Phe Phe Trp Met Phe Val Glu Gly CYE~ Tyr LQU Hi~ Thr Ala Ile Val Met Thr Tyr Ser Thr Glu Hi~ Leu Arg Lys Trp Leu Phe Leu Phe Ile Gly Trp Cy~ Ile Pro Cyf Pro Ile Ile Val Ala Trp Ala Val Gly Ly~ Leu Tyr Tyr Glu A~n Glu Gln Cys Trp Phe Gly LYQ Glu Pro Gly Anp Leu Val Af5p Tyr Ile Tyr Gln Gly Pro Ile I1Q Leu Val Leu Leu Ile Ann Phe V2~1 Phe Leu Phe A-n I1R Val Arg Ile Leu Met Thr Ly~ Leu Arg Ala Ser Thr Thr Ser 305 310 315; 320 Glu Thr Ile Gln Tyr Arg Lys Ala Val LyE~ Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val A~n Pro Gly Glu Anp AHP Leu Ser Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cy~ Phe Phe A~n Gly Glu Val Arg Ser Ala Leu Arg Ly~ Arg Trp Hi~ Arg Trp Gln Anp Hi~

Hi~ Ala Leu Arg Val Pro Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe HiQ Ser Ile Ly~ Gln Thr Ala Aia Val (2) 1NrUR~ATION FOR SEQ ID NO: 14:
( i ) SEQUENCE CHARACTERISTICS:

CA 02223792 l997-l2-05 W O 97/000~3 PCT~US96/10240 (A) LENGTH: 40 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (Li) M~T-T~ctlT-~ TYPE: peptide (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:14:
Xaa Xaa Pro Xaa Xaa Ser Xaa A~p Leu Xaa Xaa Xaa Xaa Leu Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa A~n Arg Xaa Xaa Xaa Xaa Xaa Xaa (2) lN~OR~ATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 125 amino acid~
(8) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~T~T'!CrTT~ TYPE: prot~in (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
MQt Arg Gln Ala Gly Arg Ala Ala Leu Leu Ala Ala Leu Leu Leu Leu Val Gln Leu Cy~ Pro Gly Ser Ser Gln Arg Ser Pro Glu Ala Ala Gly Val Gln Aup Pro Ser Leu Arg Trp Ser Pro Gly Ala Arg Asn Gln Gly Gly Gly Ala Arg Ala Leu Leu Leu Leu Leu Ala Glu Arg Phe Pro Arg Arg Ala Gly Pro Gly Arg Leu Gly Leu Gly Thr Ala Gly Glu Arg Pro Arg Arg A~p A~n Pro Ser Leu Ser Ile A~p Leu Thr Phe Hi~ Leu Leu Arg Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arq Ala Glu Gln Ann Arg Ile Ile Phe Anp Ser Val Gly Ly~ *

(2) lN~vKMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
lA' LENGTH: 375 ba~e pair~
Bl TYPE: nucleic acid C I ST~A~ h~SS: ~ingle DJ TOPOLOGY: linear ( ii ) ~T ~CuT-T~ TYPE: DNA (~e~-- ;c) (iii) ~rO~Ah.lCAL: NO

X CA 02223792 1997-12-0~

W O 97/00063 ' PCTrUS96/10240 (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human Gcr i~ Placental Library (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..375 (xL) ~hyuh~_h DESCRIPTION: SEQ ID NO: 16:

Met Arg Gln Ala Gly Arg Ala Ala Leu Leu Ala Ala LQU Leu Leu Leu Val Gln Leu Cy8 Pro Gly Ser Ser Gln Arg Ser Pro Glu Ala Ala Gly Val Gln A~p Pro Ser Leu Arg Trp Ser Pro Gly Ala Arg Asn Gln Gly Gly Gly Ala Arg Ala Leu Leu Leu Leu Leu Ala Glu Arg Phe Pro Arg Arg Ala Gly Pro Gly Arg Leu Gly Leu Gly Thr Ala Gly Glu Arg Pro Arg Arg Asp A~n Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Aln Glu Gln Asn Arg Ile Ile Phe Asp Ser Val Gly Lys *

Claims (37)

WHAT IS CLAIMED IS:

1. An isolated urocortin (Ucn) peptide which is characterized by binding to corticotropin releasing factor (CRF) receptor 2 (CRF-R2) with a binding affinity (KD) of 10 nanomolar or less, increasing production of adrenocorticotropic hormone (ACTH), exhibiting higher affinity for the long form of CRF-R2 (see SEQ ID NO:11) than does rat/human CRF (r/hCRF), and having less than 80% homology with r/hCRF (SEQ ID
NO:3), ovine CRF (SEQ ID NO:1) or carp Urotensin (SEQ ID
NO:5).

2. A peptide according to Claim 1 comprising the amino acid sequence of residues 83-122 of SEQ ID NO: 15 or a sequence which has at least 66% homology therewith or a biologically functional fragment of either which increases production of ACTH.

3. A peptide according to Claim 1 comprising residues 83-122 of amino acid sequence SEQ ID NO:15 or a sequence which has at least 80% homology therewith or a biologically functional fragment of either which increases production of ACTH.

4. A peptide according to Claim 3 which is an N-terminally shortened functional fragment of the amino acid sequence of SEQ ID NO:15 which increases production of ACTH wherein the C-terminus of said fragment is amidated.

5. A peptide according to Claim 4 wherein the N-terminus is acylated.

6. Antibodies which bind specifically to a urocortin peptide according to Claim 3 or to a fragment thereof at least 5 residues in length.

7. Antibodies according to Claim 6 which specifically bind to and biologically inactivate said urocortin peptide so it no longer increases production of ACTH.

8. A peptide according to Claim 1 having at least 66% homology with residues 83-122 of SEQ ID NO:15 and wherein all said substitutions for residues in SEQ ID
NO:15 are conservative substitutions.

9. A peptide according to Claim 1 comprising an amino acid sequence having at least 80% homology with the following amino acid sequence:
Y-R1-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 (residues 85-122 of SEQ ID NO:15), wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; and R1 is Asn or desR1.

l0. A peptide according to Claim 1 comprising the following amino acid sequence:
Y-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-R19-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-R29-Ala-Glu-R32-Asn-Arg-Ile-R36-Phe-R38-Ser-Val-NH2, wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R19 is Glu or Ala; R29 is Arg, Glu, Lys or Orn;
R32 is Gln, Lys, Orn or Glu; R36 is Ile, C.alpha.MeIle or C.alpha.MeLeu;
R38 is Asp or Ala; provided that when R29 is Glu, R32 is either Lys or Orn and the side chains thereof are linked by an amide bond and that when R32 is Glu, R29 is either Lys or Orn and the side chains thereof are linked by an amide bond; and provided further that D-Phe in the 12-position can be D-Leu or another D-amino acid; that Glu in the 31-position can be D-Glu or another D-amino acid; and that the N-terminus can be shortened by 1 or 2 residues.

11. A peptide according to Claim 1 having the following amino acid sequence (SEQ ID NO: 14):
Y-Xaa1-Xaa2-Pro-Xaa4-Xaa5-Ser-Xaa7-Asp-Leu-Xaa10-Xaa11-Xaa12-Xaa13-Leu-Arg-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Ala-Xaa31-Xaa32-Asn-Arg-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-NH2, wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; Xaa1 is Asp, Glu or Gln; Xaa2 is Asn, Asp, Glu or Gly; Xaa4 is Ser or Pro;
Xaa5 is Leu, Ile or Met; Xaa7 is Ile or Leu; Xaa10 is Thr or Ser; Xaa11 is Phe or Leu; Xaa12 is His or Glu; Xaa13 is Leu or Met; Xaa16 is Thr, Asn, Glu, or Lys; Xaa17 is Leu, Met or Val; Xaa18 is Leu or Ile; Xaa19 is Glu or His; Xaa20 is Leu, Met, Ile or Arg; Xaa21 is Ala, Glu or Thr; Xaa22 is Arg or Lys; Xaa23 is any natural amino acid other than Cys; Xaa24 is Gln, Glu or Asp; Xaa25 is any natural amino acid other than Cys; Xaa26 is Gln, Leu or Glu; Xaa27 is Arg, Ala or Lys; Xaa28 is Glu or Gln; Xaa29 is Arg or Gln; Xaa31 is any natural amino acid other than Cys; Xaa32 is any natural amino acid other than Cys; Xaa35 is Ile, Lys, Leu or Asn;
Xaa36 is Ile, Tyr, Met or Leu; Xaa37 is Phe, Leu or Met;
Xaa38 is Asp or Glu; Xaa39 is Ser, Ile, Glu or Thr; and Xaa40 is Val, Ile, Phe or Ala; provided that there are no more than 3 residues different from hUcn and that the N-terminus can be shortened by 1 or 2 residues.

12. Isolated DNA which encodes a peptide according to Claim 1.

13. Isolated DNA according to Claim 12 comprising DNA which has the nucleotide sequence as shown in SEQ ID

NO:16 encoding residues 83-122 or a sequence that has 80 homology therewith.

14. Isolated DNA according to Claim 12 comprising nucleotide sequence SEQ ID NO:16 encoding residues 83-122.

15. A DNA probe comprising at least 14 contiguous nucleotides of said isolated DNA of Claim 14 or the complement thereof which would hybridize to native urocortins only and would not hybridize under high stringency conditions to DNA encoding known native CRFs, known native urotensins or native sauvagine.

16. Isolated DNA capable of hybridizing under high stringency conditions (such as would identify only sequences having at least 80% homology) to said isolated DNA of Claim 14 or to the complement thereof.

17. Isolated DNA capable of hybridizing under high stringency conditions (such as would identify only sequences having at least 80% homology) to DNA encoding a peptide of Claim 1 and which would not hybridize under high stringency conditions to DNA encoding known CRFs, known urotensins or sauvagine.

18. Isolated DNA which encodes a peptide according to Claim 2.

19. A method for screening for ligands for CRF
receptors, which method comprises carrying out a competitive binding assay with a CRF
receptor, a peptide according to Claim 1 which contains a suitable label, and a candidate ligand and determining the ability of said candidate ligand to displace said labelled peptide.

20. A screening method according to Claim 19 wherein said CRF receptor is CRF Receptor 2 and said labelled peptide is 125I-Tyr°-Ucn.

21. A method of diagnosing for hypothalamic pituitary disease comprising administering an effective dose of a peptide according to Claim 1 to a human subject and monitoring the bloodstream of said subject for an increase in ACTH level.

22. A method for stimulating secretion of ACTH and .beta.-endorphin-like-activities (.beta.-END-LI) in mammals comprising peripherally administering to said mammal an effective amount of a peptide according to Claim 1 or a nontoxic salt thereof and a pharmaceutically acceptable carrier therefor.

23. A method of modifying blood flow and/or blood pressure which comprises administering an effective amount of a peptide according to Claim 1 or of an antagonist peptide thereof.

24. A method according to Claim 23 for modulating blood flow in a desired vascular bed which comprises peripherally administering said effective peptide amount.

25. A method of increasing coronary blood flow which comprises peripherally administering an effective amount of a peptide according to Claim 1.

26. A method of decreasing swelling and/or inflammation andtor vascular permeability which comprises parenterally administering an effective amount of a peptide according to Claim 1.

27. A pharmaceutical composition which comprises an effective amount of a peptide according to Claim 1 in combination with a pharmaceutically acceptable carrier, which amount is effective to modulate the transactivation of CRF receptors.

28. A pharmaceutical composition according to Claim 27 wherein said amount is effective to increase intestinal transit rate.

29. A Ucn antagonist peptide which comprises the following amino acid sequence:
Asp-Leu-R10-R11-His-Leu-Leu-Arg-Thr-Leu-Leu-R19-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-R29-Ala-Glu-R32-Asn-Arg-Ile-R36-Phe-R38-Ser-Val-NH2, wherein R10 is Pro, D-Pro, Thr or D-Tyr; R11 is D Phe or D-Tyr or another D-amino acid; R19 is Glu or Ala; R29 is Arg, Glu, Lys or Orn; R32 is Gln, Lys, Orn or Glu; R36 is Ile, C.alpha.MeIle or C.alpha.MeLeu; R38 is Asp or Ala;
provided that when R29 is Glu, R32 is either Lys or Orn and the side chains thereof are linked by an amide bond and that when R32 is Glu, R29 is either Lys or Orn and the side chains thereof are linked by an amide bond; and provided further that Glu in the 31-position can be D-Glu or another D-amino acid and that the N-terminus can be shortened by 1, 2 or 3 residues.

30. A Ucn antagonist peptide according to Claim 29 which is (cyclo 29-32)D-Phe11,Glu29,Lys32-Ucn(11-40).

31. A method for screening for antagonists for CRF
receptors which bind with high affinity to such receptors which method comprises carrying out a competitive binding assay with a CRF
receptor, a peptide according to Claim 30 which contains a suitable label, and a candidate antagonist and determining the ability of said candidate antagonist to displace said labelled peptide.

32. A peptide useful for blocking CRF-binding protein (CRF-BP) to thereby increase availability of endogenous CRF and/or Ucn, which peptide has the amino acid sequence:
Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln (residues 85-114 of SEQ ID NO:15), or a biologically active fragment thereof which is formed by deleting 1 to 8 residues in sequence from the N-terminus, or 1 to 5 residues in sequence from the C-terminus, or both or which has at least 80% homology therewith and binds to CRF-BP.

33. A peptide according to Claim 32 selected from the group consisting of Ucn(5-32), Ucn(8-32) and hUcn(3-27).

34. A method for increasing the in vivo level of CRF
and/or Ucn, which method comprises administering an effective amount of a peptide according to Claim 32.

35. A method according to Claim 34 wherein said effective amount is sufficient to promote parturition in a pregnant female.

36. A method according to Claim 34 wherein said amount of said peptide administered is effective so as to result in an increase in free endogenous CRF and/or Ucn in the brain which causes (a) improvement in short to medium term memory in a subject afflicted with Alzheimer's disease; (b) relief from chronic fatigue syndrome; (c) suppression of appetite; (d) stimulation of the respiratory system, (e) improvement in learning performance; (f) improvement in memory; (g) improvement in alertness; (h) reduction of depression and/or (i) lessening of anxiety.

37. A method according to Claim 36 wherein said peptide is administered so that said peptide reaches the brain.