CRF analogs

Analogs of CRF, which are based upon hCRF, oCRF and alpha-helical CRF, are disclosed that can be administered to achieve a substantial elevation of ACTH, .beta.-endorphin, .beta.-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone levels. Analogs include those having the formula (see SEQ ID NO:9): Y-Ser-Xaa.sub.2 -Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Xaa.sub.12 -His-Leu-Leu-Arg-Glu-Val-Leu-Xaa.sub.20 -Xaa.sub.21 -Xaa.sub.22 -Xaa.sub.23 -Xaa.sub.24 -Xaa.sub.25 -Gln-Leu-Ala-Gln-Gln-Ala-Xaa.sub.32 -Ser-Asn-Arg-Xaa.sub.36 -Leu-Xaa.sub.38 -Xaa.sub.39 -Ile-Xaa.sub.41 -NH.sub.2, wherein Y is an acyl group having 7 or fewer carbon atoms or hydrogen; Xaa.sub.2 is Glu or Gln; Xaa.sub.12 is Phe or D-Phe; Xaa.sub.20 is Ala or Glu; Xaa.sub.21 is Met or Nle; Xaa.sub.22 is Ala or Thr; Xaa.sub.23 is Arg or Lys; Xaa.sub.24 is D-Ala or Ala; Xaa.sub.25 is Glu or Asp; Xaa.sub.32 is D-His or His; Xaa.sub.36 is Lys or Arg; Xaa.sub.38 is Met, Nle or Leu; Xaa.sub.39 is Ala, Glu or Asp; Xaa.sub.41 is Ile or Ala; provided however that at least one of Xaa.sub.20 and Xaa.sub.39 is Ala and that the N-terminus may be shortened by a sequence of up to about 5 residues. By shortening the N-terminus by 11 residues, particularly potent CRF antagonists are created. These analogs or their pharmaceutically acceptable salts, dispersed in an acceptable liquid or solid carrier, can be administered to humans.

This invention is directed to peptides and to methods for pharmaceutical 
treatment of mammals using such peptides. More specifically, the invention 
relates to analogs of the hentetracontapeptide CRF, to pharmaceutical 
compositions containing such CRF analogs and to methods of treatment of 
mammals using such CRF analogs. 
BACKGROUND OF THE INVENTION 
Experimental and clinical observations have supported the concept that the 
hypothalamus plays a key role in the regulation of adenohypophysial 
corticotropic cells secretory functions. Although over 25 years ago, it 
was demonstrated that factors present in the hypothalamus would increase 
the rate of ACTH secretion by the pituitary gland, when 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. As disclosed in U.S. Pat. No. 4,415,558, oCRF was 
found to have the formula (SEQ ID NO: 1): 
Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-L 
eu-Asp-Ile-Ala wherein the C-terminus is amidated. oCRF lowers blood 
pressure in mammals and stimulates the secretion of ACTH and 
.beta.-endorphin. 
Rat CRF(rCRF) was later isolated, purified and characterized as a 
hentetracontapeptide having the formula (SEQ ID NO: 2): 
Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-M 
et-Glu-Ile-Ile, wherein the C-terminus is amidated, as described in U.S. 
Pat. No. 4,489,163. It is sometimes referred to as rat amunine. The 
formula of human CRF has now been determined to be the same as that of 
rCRF, and the terms rCRF and hCRF are used interchangeably. A CRF analog 
having a high alpha-helical forming potential and the formula (SEQ ID NO: 
3): 
Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-phe-His-Leu-Leu-Arg-Glu-Met-Le 
u-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-L 
eu-Glu-Glu-Ala, wherein the C-terminus is amidated, has been developed; it 
is referred to as AHC (alpha- helical CRF) and is described in U.S. Pat. 
No. 4,594,329. 
Synthetic rCRF, oCRF and AHC stimulate ACTH and .beta.-endorphin-like 
activities (.beta.-END-LI) in vitro and in vivo and substantially lower 
blood pressure. 
SUMMARY OF THE INVENTION 
Analogs of these 41-residue CRF peptides have been discovered which exhibit 
greater biological activity in vitro than the native peptides and thus are 
termed CRF agonists. These peptides have at least one Ala substitution in 
the 20- or the 39-position, and the peptides may optionally also have 
D-Phe in the 12-position, D-Ala in the 24-position and/or D-His in the 
32-position, and Norleucine may be substituted in the 18, 21 and/or 38 
positions. The Lys residue in the 36-position can be substituted by Arg. 
The Leu residue in the 37-position can be substituted with a methyl group 
on its .alpha.-carbon atom, as can be other Leu residues as well as the 
Ala residues, and such are considered to be equivalents for purposes of 
this application. Beginning at the N-terminus, the peptide can be 
optionally shortened by the deletion of 1 to about 5 residues in sequence, 
and is preferably shortened by deletion of about the first 4 residues. The 
N-terminus of the peptide is optionally acylated. 
Pharmaceutical compositions in accordance with the invention include such 
CRF analogs, or nontoxic addition salts thereof, dispersed in a 
pharmaceutically or veterinarily acceptable liquid or solid carrier. The 
administration of such peptides or pharmaceutically or veterinarily 
acceptable addition salts thereof to mammals, particularly humans, in 
accordance with the invention may be carried out for the regulation of 
secretion of ACTH, .beta.-endorphin, .beta.-lipotropin, other products of 
the pro-opiomelanocortin gene and corticosterone and/or for lowering 
systemic blood pressure when given intravenously and/or for affecting 
mood, behavioral and gastrointestinal functions and autonomic nervous 
system activities. Furthermore CRF analogs may be used for the evaluation 
of the status of pituitary, cardiovascular, gastrointestinal or central 
nervous system functions. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
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 right. The standard 3-letter abbreviations are used 
to identify the alpha-amino acid residues, and where the amino acid 
residue has isomeric forms, it is the L-form of the amino acid that is 
represented unless otherwise expressly indicated, e.g. Ser=L-serine, 
Orn=L-ornithine, Nle=L-norleucine, Nva=L-norvaline and Har=L-homoarginine. 
In addition the following abbreviations are used: CML=C.sup..alpha. 
CH.sub.3 -L-leucine; Aib=C.sup..alpha. CH.sub.3 -L-alanine or 
2-aminoisobutyric acid. 
The invention provides analogs of CRF having the following formula (SEQ ID 
NO:9): 
Ser-Xaa-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Xaa-His-Leu-Leu-Arg-Glu-Val-Le 
u-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Gln-Leu-Ala-Gln-Gln-Ala-Xaa-Ser-Asn-Arg-Xaa-Leu-X 
aa-Xaa-Ile-Xaa, wherein Y is present at the N-terminus and is an acyl group 
having 7 or fewer carbon atoms or hydrogen and the C-terminus is amidated; 
with the Xaa groups being defined using subscripts that indicate their 
positions relative to the N-terminus, as follows: Xaa.sub.2 is Gln or Glu; 
Xaa.sub.12 is Phe or D-Phe; Xaa.sub.20 is Ala or Glu; Xaa.sub.21 is Met or 
Nle; Xaa.sub.22 is Ala or Thr; Xaa.sub.23 is Arg or Lys; Xaa.sub.24 is 
D-Ala or Ala; Xaa.sub.25 is Glu or Asp; Xaa.sub.32 is D-His or His; 
Xaa.sub.36 is Lys or Arg; Xaa.sub.38 is Met, Nle or Leu; Xaa.sub.39 is 
Ala, Glu or Asp; Xaa.sub.41 is Ile or Ala; provided however that at least 
one of Xaa.sub.20 and Xaa.sub.39 is Ala. In some analogs based more 
closely on the native peptides, Xaa.sub.12 is Phe and Xaa.sub.36 is Lys. 
Nontoxic addition salts of these peptides can be used as well. These CRF 
agonist analogs remain potent even if slightly shortened at the 
N-terminus, i.e., by removal of a sequence of up to about 5 residues. By 
deleting residues 1-11, potent CRF antagonists are created. 
In a broader sense, the invention provides analogs of CRF of the following 
formula (SEQ ID NO: 10): 
Xaa-Xaa-Xaa-Xaa-Pro-Ile-Ser-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Leu-Arg-Xaa-Xaa-Xa 
a-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Gln-Ala-Xaa-Xaa-Asn-Arg-Xaa-Xaa-X 
aa-Xaa-Xaa-Xaa wherein Y is present at the N-terminus and is an acyl group 
having 7 or fewer carbon atoms or hydrogen and the C-terminus is amidated; 
Xaa.sub.1 is Ser or D-Ser; Xaa.sub.2 is Glu, Gln, pGlu, or D-pGlu; 
Xaa.sub.3 is Glu, Gly or D-Tyr; Xaa.sub.4 is Pro or D-Pro; Xaa.sub.8 and 
Xaa.sub.19 are selected from the group consisting of Leu, Ile, Ala, Gly, 
Val, Nle, Phe and Gln; Xaa.sub.9 is Asp or Glu; Xaa.sub.11 is Thr or Ser; 
Xaa.sub.12 is Phe, D-Phe, Leu, Ala, Ile, Gly, Val, Nle or Gln; Xaa.sub.13 
is His, Tyr or Glu; Xaa.sub.14 is Leu or Met; Xaa.sub.17 is Glu or Lys; 
Xaa.sub.18 is Val, Nle or Met; Xaa.sub.20 is Ala or Glu; Xaa.sub.21 is 
Arg, Met, Nva, Ile, Ala, Leu, Nle, Val, Phe or Gln; Xaa.sub.22 is Ala, 
Thr, Asp or Glu; Xaa.sub.23 is Arg, Orn, Har or Lys; Xaa.sub.24 is Ala, 
D-Ala, Met, Leu, Ile, Gly, Val, Nle, Phe and Gln; Xaa.sub.25 is Glu, Ala 
or Asp; Xaa.sub.26 is Gly, Gln, Asn or Lys; Xaa.sub.27 is Leu, Ile, Ala, 
Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; Xaa.sub.28 is Ala, Arg or 
Lys; Xaa.sub.29 is Gln, Ala or Glu; Xaa.sub.32 is Leu, His, D-His, Gly, 
Tyr or Ala; Xaa.sub.33 is Ile, Ser, Asn, Leu, Thr or Ala; Xaa.sub.36 is 
Asn, Lys, Orn, Arg, Har or Leu; Xaa.sub.37 is Leu or Tyr; Xaa.sub.38 is 
Met, Nle or Leu; Xaa.sub.39 is Ala, Glu or Asp; Xaa.sub.40 is Ile, Thr, 
Glu, Ala, Val, Leu, Nle, Phe, Nva, Gly, Asn or Gln; Xaa.sub.41 is Ile, 
Ala, Gly, Val, Leu, Nle, Phe or Gln, provided however that at least one of 
Xaa.sub.20 or Xaa.sub.39 is Ala, as well as nontoxic salts thereof. Again, 
a sequence of up to about 5 residues can be eliminated from the N-terminus 
without destroying biopotency, and the elimination of a sequence of 11 
residues from the N-terminus creates potent CRF antagonists. 
A subgroup of these analogs which particularly include residues having a 
high alpha-helical forming potential are those having the following 
formula (SEQ ID NO: 11): 
Ser-Xaa-Glu-Pro-Pro-Ile-Ser-Leu-Xaa-Leu-Thr-Xaa-Xaa-Xaa-Leu-Arg-Glu-Xaa-Le 
u-Xaa-Xaa-Ala-Lys-Xaa-Glu-Gln-Xaa-Ala-Glu-Gln-Ala-Xaa-Xaa-Asn-Arg-Xaa-Xaa-X 
aa-Xaa-Xaa-Xaa wherein Y is present at the N-terminus and is an acyl group 
having 7 or fewer carbon atoms or hydrogen and the C-terminus is amidated; 
Xaa.sub.2 is Glu or Gln; Xaa.sub.9 is Asp or Glu; Xaa.sub.12 is Phe, D-Phe 
or Leu; Xaa.sub.13 is His or Glu; Xaa.sub.14 is Leu or Met; Xaa.sub.18 is 
Nle or Met; Xaaz.sub.20 is Ala or Glu; Xaa.sub.21 is Met, Nle or Ile; 
Xaa.sub.24 is Ala or D-Ala; Xaa.sub.27 is Glu or Leu; Xaa.sub.32 is His, 
D-His or Ala; Xaa.sub.33 is Ser or Leu; Xaa.sub.36 is Leu or Lys; 
Xaa.sub.37 is Leu or Tyr; Xaa.sub.38 is Leu or Nle; Xaa.sub.39 is Ala, Glu 
or Asp; Xaa.sub.40 is Ile or Glu and Xaa.sub.41 is Ile, Ala or Val; 
provided however that at least one of Xaa.sub.20 and Xaa.sub.39 is Ala. 
Again, a sequence of up to about 5 residues can be eliminated from the 
N-terminus. 
Antagonists of CRF are provided having the following formula which is an 
N-terminally shortened version of SEQ ID NO: 9: H-Asp-Leu-Thr-Xaa.sub.12 
-His-Leu-Leu-Arg-Glu-Val-Leu-Xaa.sub.20 -Xaa.sub.21 -Xaa.sub.22 
-Xaa.sub.23 -Xaa.sub.24 -Xaa.sub.25 -Gln-Leu-Ala-Gln-Gln-Ala-Xaa.sub.32 
-Ser-Asn-Arg-Xaa.sub.36 -Leu-Xaa.sub.38 -Xaa.sub.39 -Ile-Xaa.sub.41, 
wherein 1, 2 or 3 residues can be deleted beginning at the N-terminus and 
the C-terminus is amidated; with the Xaa groups being defined using 
subscripts that indicate their positions relative to the N-terminus of the 
native CRF molecule, as follows: Xaa.sub.12 is Phe or D-Phe; Xaa.sub.20 is 
Ala or Glu; Xaa.sub.21 is Met or Nle; Xaa.sub.22 is Ala or Thr; Xaa.sub.23 
is Arg or Lys; Xaa.sub.24 is D-Ala or Ala; Xaa.sub.25 is Glu or Asp; 
Xaa.sub.32 is D-His or His; Xaa.sub.36 is Lys or Arg; Xaa.sub.38 is Met, 
Nle or Leu; Xaa.sub.39 is Ala, Glu or Asp; Xaa.sub.41 is Ile or Ala; 
provided however that at least one of Xaa.sub.20 and Xaa.sub.39 is Ala. In 
some analogs based more closely on the native peptides, Xaa.sub.36 is Lys. 
Nontoxic addition salts of these peptides can be used. 
A preferred group of CRF antagonists have the following formula: 
H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Xaa.sub.20 -Nle-Xaa.sub.22 -Xaa.sub.23 
-Xaa.sub.24 -Xaa.sub.25 -Gln-Leu-Ala-Gln-Gln-Ala-Xaa.sub.32 
-Ser-Asn-Arg-Arg-Leu-Nle-Xaa.sub.39 -Ile-Xaa.sub.41 -NH.sub.2 wherein 
Xaa.sub.20 is Ala or Glu; Xaa.sub.22 is Ala or Thr; Xaa.sub.23 is Arg or 
Lys; Xaa.sub.24 is D-Ala or Ala; Xaa.sub.25 is Glu or Asp; Xaa.sub.32 is 
D-His or His; Xaa.sub.39 is Ala, Glu or Asp; Xaa.sub.41 is Ile or Ala; 
provided however that at least one of Xaa.sub.20 and Xaa.sub.39 is Ala. 
The peptides are synthesized by a suitable method, such as by exclusively 
solid-phase techniques, by partial solid-phase techniques, by fragment 
condensation or by classical solution addition. Common to 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 occurring at that site until the group is 
ultimately removed. Usually also common is the protection of an 
alpha-amino group on an amino acid or a fragment while that entity reacts 
at the carboxyl group, followed by the selective removal of the 
alpha-amino protecting group to allow subsequent reaction to take place at 
that location. 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 various 
of these residues having side-chain protecting groups. 
Thus, chemical synthesis of such a peptide analog may result in the 
formation of an intermediate of the Formula (IA), which is based on SEQ ID 
NO: 9: X.sup.1 -Ser(X.sup.2)-Xaa.sub.2 (X.sup.4 or 
X.sup.5)-Glu(X.sup.5)-Pro-Pro-Ile-Ser(X.sup.2)-Leu-Asp(X.sup.5)-Leu-Thr(X. 
sup.2)-Xaa.sub.12 
-His(X.sup.7)-Leu-Leu-Arg(X.sup.3)-Glu(X.sup.5)-Val-Leu-Xaa.sub.20 
(X.sup.5)-Xaa.sub.21 -Xaa.sub.22 (X.sup.2)-Xaa.sub.23 (X.sup.3 or 
X.sup.6)-Xaa.sub.24 -Xaa.sub.25 
(X.sup.5)-Gln(X.sup.4)-Leu-Ala-Gln(X.sup.4)-Gln(X.sup.4)-Ala-Xaa.sub.32 
(X.sup.7)- Ser(X.sup.2)-Asn(X.sup.4)-Arg(X.sup.3)-Xaa.sub.36 (X.sup.3 or 
X.sup.6)-Leu-Xaa.sub.38 -Xaa.sub.39 (X.sup.5)-Ile-Xaa.sub.41 -X.sup.8 
wherein: the Xaa-groups are as hereinbefore defined. 
X.sup.1 is either hydrogen or an alpha-amino protecting group. The 
alpha-amino protecting groups contemplated by X.sup.1 are those known to 
be useful in the art in the step-wise synthesis of polypeptides. Among the 
classes of alpha-amino protecting groups covered by X.sup.1 are (1) 
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, such as p-chlorobenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such 
as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, 
isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl 
urethan-type protecting groups, such as fluorenylmethyl- 
oxycarbonyl(FMOC), cyclopentyloxycarbonyl, adamantyloxycarbonyl,and 
cyclohexyloxycarbonyl; and (5) thiourethan-type protecting groups, such as 
phenylthiocarbonyl. The preferred alpha-amino protecting group is BOC if 
the synthesis employs acid-catalyzed removal 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 
protecting groups can be used, including t-Butyl esters or ethers as well 
as BOC. 
X.sup.2 is a protecting group for the hydroxyl group of Thr and Ser and is 
generally selected from the class containing acetyl(Ac), benzoyl(Bz), 
tert-butyl(t-Bu), triphenylmethyl(trityl), tetrahydropyranyl, benzyl 
ether(Bzl) and 2,6-dichlorobenzyl(DCB) when a BOC strategy is employed. 
The preferred protecting group is Bzl for a BOC strategy and t-Bu for FMOC 
strategy. X.sup.2 can also be hydrogen, which means there is no protecting 
group on the hydroxyl group. 
X.sup.3 is a protecting group for the guanidino group of Arg generally 
selected from the class containing nitro, p-toluenesulfonyl(Tos), Z, 
adamantyloxycarbonyl and BOC, or is hydrogen. Tos is preferred for a BOC 
strategy and 4-methoxy-2,3,6- trimethyl benzene sulfonyl (MTR) or 
pentamethylchroman-6-sulfonyl(PMC) for FMOC strategy. 
X.sup.4 is hydrogen or a suitable protecting group, preferably 
xanthyl(Xan), for the side chain amido group of Asn or Gln. Asn or Gln is 
preferably coupled without side chain protection in the presence of 
hydroxybenzotriazole (HOBt). 
X.sup.5 is hydrogen or an ester-forming protecting group for the .beta.- or 
.gamma.-carboxyl group of Asp or Glu, and is generally selected from the 
class containing the esters of cyclohexyl(OChx), benzyl(OBzl), 
2,6-dichlorobenzyl, methyl, ethyl and t-butyl(To-Bu). OChx is preferred 
for a BOC strategy and To-Bu for FMOC strategy. 
X.sup.6 is hydrogen or a protecting group for the side chain amino 
substituent of Lys. Illustrative of suitable side chain amino protecting 
groups are Z, 2-chlorobenzyloxycarbonyl(2-Cl-Z), Tos, 
t-amyloxycarbonyl(Aoc), BOC and aromatic or aliphatic urethan-type 
protecting groups as specified hereinbefore. 2-Cl-Z is preferred for a BOC 
strategy and BOC for FMOC strategy. 
X.sup.7 is hydrogen or a protecting group for the imidazole nitrogen of His 
such as Tos or 2,4-dinitrophenyl(DNP). 
When Met is present, the sulfur may be protected, if desired, with oxygen. 
The selection of a side chain amino protecting group is not critical except 
that it should must 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. 
X.sup.8 is NH.sub.2, a protecting group such as an ester or an anchoring 
bond used in solid phase synthesis for linking to a solid resin support, 
preferably one represented by the formulae: 
-NH-benzhydrylamine (BHA) resin support and -NH-paramethylbenzhydrylamine 
(MBHA) resin support. Cleavage from a BHA or MBHA resin directly gives the 
CRF analog amide. By employing an N-methyl-derivative of such a resin, a 
methyl-substituted amide can be created 
In the formula for the intermediate, at least one of X.sup.1, X.sup.2, 
X.sup.3, X.sup.4, X.sup.5, X.sup.6 and X.sup.7 is a protecting group The 
particular amino acid chosen for each the R-group determines whether there 
will also be a protecting group attached as specified hereinbefore and as 
generally known in the art. In selecting a particular side chain 
protecting group to be used in the synthesis of the peptides, the 
following rules are followed: (a) the protecting group should be stable to 
the reagent and under the reaction conditions selected 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 side chain protecting group must 
be removable, upon the completion of the synthesis containing the desired 
amino acid sequence, under reaction conditions that will not alter the 
peptide chain. 
For the acyl group at the N-terminus represented by Y, acetyl, formyl, 
acrylyl and benzoyl are preferred. Moreover, as indicated hereinbefore, 
the N-terminus can be slightly shortened without significantly affecting 
biological potency. 
Thus, there is also disclosed herein processes for the manufacture of 
compounds defined by SEQ ID NO: 9 comprising (a) forming a peptide 
intermediate having at least one protective group and having the Formula 
(IA) wherein: X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6 and 
X.sup.7 are each either hydrogen or a protective group, and X.sup.8 is 
either a protective group or an anchoring bond to resin support or 
NH.sub.2 and (b) splitting off the protective group or groups or anchoring 
bond from said peptide intermediate of the Formula (IA) and (c) if 
desired, converting a resulting peptide into a nontoxic addition salt 
thereof. 
When the peptides 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 other 
equivalent chemical syntheses known in the art can also be used as 
previously mentioned. Solid-phase synthesis is 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. Pat. No. 4,244,946 issued 
Jan. 21, 1981 to Rivier et al. Such a starting material for rCRF analogs 
can be prepared by attaching alpha-amino-protected Ile to a BHA or MBHA 
resin. 
Ile protected by BOC is coupled to the BHA or MBHA resin using methylene 
chloride and dimethylformamide (DMF). Following the coupling of BOC-Ile to 
the resin support, the alpha-amino protecting group is removed, as by 
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 0.degree. C. and room temperature. Other 
standard cleaving reagents and conditions for removal of specific 
alpha-amino protecting groups may be used as described in Schroder & 
Lubke, "The Peptides", Vol 1 pp. 72-75 (Academic Press 1965). 
After removal of the alpha-amino protecting group of Ile, the remaining 
alpha-amino- and side chain-protected amino acids are coupled step-wise in 
the desired order to obtain the intermediate compound defined 
hereinbefore. As an alternative to adding each amino acid separately in 
the synthesis, some of them may be coupled to one another prior to 
addition to the solid phase reactor. The selection of an appropriate 
coupling reagent is within the skill of the art. Particularly suitable as 
coupling reagents are N,N'-dicyclohexyl carbodiimide(DCC) and 
N,N'-diisopropyl carbodiimide(DICI). 
The activating reagents used in the solid phase synthesis of the peptides 
are well known in the peptide art. Examples of suitable activating 
reagents are carbodiimides, such as DCC, DICI and 
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide. Other 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 127 (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 be coupled 
overnight using one equivalent of HOBt in a 50% mixture of DMF and 
methylene chloride, in which case no DCC is added. 
Each protected amino acid or amino acid sequence is introduced into the 
solid phase reactor in about a fourfold excess, and the coupling is 
carried out in a medium of dimethylformamide(DMF):CH.sub.2 Cl.sub.2 (1:1) 
or in DMF or CH.sub.2 Cl.sub.2 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 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 automatically, as 
on a BECKMAN 990 automatic synthesizer, using a program such as that 
reported in Rivier et al., Biopolymers, 17, pp. 1927-1938, (1978). 
After the desired amino acid sequence has been completed, the intermediate 
peptide is removed from the resin support by treatment with a reagent, 
such as liquid hydrogen fluoride, which not only cleaves the peptide from 
the resin but also cleaves all remaining side chain protecting groups 
X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6 and X.sup.7 and the 
alpha-amino protecting group X.sup.1 (unless it is an acyl group which is 
intended to be present in the final peptide) to obtain the peptide. When 
using hydrogen fluoride for cleaving, anisole or cresole and methylethyl 
sulfide are included in the reaction vessel as scavengers. When Met is 
present in the sequence, the BOC protecting group may be cleaved with 
trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving the peptide from 
the resin to eliminate potential S-alkylation.

The following Example sets forth the preferred method for synthesizing CRF 
analogs by the solid-phase technique. 
EXAMPLE I 
The synthesis of [Ala.sup.20 ]-oCRF having the formula (SEQ ID NO: 4 ): 
Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Ala-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-L 
eu-Asp-Ile-Ala, wherein the C-terminus is amidated, is 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, preferably as follows: 
______________________________________ 
MIN. MIX 
STEP REAGENTS AND OPERATIONS TIMES 
______________________________________ 
1 CH.sub.2 Cl.sub.2 wash-80 ml. (2 times) 
3 
2 Methanol(MeOH) wash-30 ml. (2 times) 
3 
3 CH.sub.2 Cl.sub.2 wash-80 ml. (3 times) 
3 
4 50 percent TFA plus 5 percent 1,2-ethane- 
12 
dithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times) 
5 Isopropanol wash-80 ml. (2 times) 
3 
6 TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml. 
5 
(2 times) 
7 MEOH wash-40 ml. (2 times) 
2 
8 CH.sub.2 Cl.sub.2 wash-80 ml. (3 times) 
3 
9 Boc-amino acid (10 mmoles) in 30 ml. of 
30-300 
either DMF or CH.sub.2 Cl.sub.2, depending upon the 
solubility of the particular protected 
amino acid, (1 time) plus DCC (10 mmoles) 
in CH.sub.2 Cl.sub.2 
______________________________________ 
Coupling of BOC-Ile results in the substitution of about 0.35 mmol. Ile 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 that might undesirably oxidize the sulfur of 
the Met residue. 
After deprotection and neutralization, the peptide 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 
equivalent of 2 molar DCC in methylene chloride, for two hours. When 
BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methylene chloride 
is used. Bzl is used as the hydroxyl side-chain protecting group for Ser 
and Thr. BOC-Asn or BOC-Gln is coupled in the presence of using one 
equivalent of DCC and two equivalents of HOBt in a 50% mixture of DMF and 
methylene chloride. 2-Cl-Z is used as the protecting group for the Lys 
side chain. Tos is used to protect the guanidino group of Arg and the 
imidazole group of His, and the side-chain carboxyl group of Glu or Asp is 
protected by OChx. At the end of the synthesis, the following intermediate 
composition is obtained: 
BOC-Ser(Bzl)-Gln-Glu(OChx)-Pro-Pro-Ile-Ser(Bzl)-Leu-Asp(OChx)-Leu-Thr(Bzl) 
-Phe-His(Tos)-Leu-Leu-Arg(Tos)-Glu(OChx)-Val-Leu-Ala-Met-Thr(Bzl)-Lys(2-Cl- 
Z)-Ala-Asp(OChx)-Gln-Leu-Ala-Gln-Gln-Ala-His(Tos)-Ser(Bzl)-Asn-Arg(Tos)-Lys 
(2-Cl-Z)-Leu-Leu-Asp(OChx)-Ile-Ala-resin support. 
In order to cleave and deprotect the resulting protected peptide-resin, it 
is treated with 1.5 ml. anisole, 0.5 ml. of methylethylsulfide or 
dimethylsulfide and 15 ml. hydrogen fluoride (HF) per gram of 
peptide-resin, first at -20.degree. C. for 20 min. and then at 0.degree. 
C. for one and one-half hours. After elimination of the HF under high 
vacuum, the resin-peptide mixture is washed with dry diethyl ether, and 
the peptide amide is then extracted with de-gassed 2N aqueous acetic acid 
or a 1:1 mixture of acetonitrile and water, separated from the resin by 
filtration, and lyophilized. 
The lyophilized peptide amide is then purified by preparative or 
semi-preparative HPLC as described in Rivier, et al., J. Chromatography, 
288, 303-328 (1984); and Hoeger, et al., BioChromatography, 2, 3, 134-142 
(1987). The chromatographic fractions are carefully monitored by HPLC, and 
only the fractions showing substantial purity are pooled. 
The peptide is judged to be homogeneous by reversed-phase high performance 
liquid chromatography using a Waters HPLC system with a 0.46.times.25 cm. 
column packed with 5 .mu.m C.sub.18 silica, 300 .ANG. pore size. The 
determination is run at room temperature using gradient conditions with 2 
buffers. Buffer A is an aqueous trifluoroacetic acid (TFA) solution 
consisting of 1.0 ml. of TFA per 1000 ml. of solution. Buffer B is 1 ml 
TFA diluted to 400 ml with H.sub.2 O which is added to 600 ml. of 
acetonitrile. The analytical HPLC was run under gradient condition of 55 
vol. % Buffer B to 85 vol. % Buffer B over 30 minutes. At a flow rate of 2 
ml. per minute, the retention time is 17.0 minutes. If 2.25 molar 
triethylammonium phosphate (TEAP) is used Buffer A and Buffer B consists 
of 60% acetonitrile in Buffer A, under gradient conditions of 50% Buffer B 
to 80% Buffer B over a 30-minute period, a retention time of 16.2 minutes 
is obtained. 
Specific optical rotation of the CRF analog peptide, which is synthesized 
and purified in the foregoing manner, is measured on a Perkin Elmer Model 
241 Polarimeter as [.alpha.].sub.D.sup.22 =-91 
8.degree..+-.1.0.degree.(c=1 in 1% acetic acid, without correction for the 
presence of H.sub.2 O and TFA); it has a purity of greater than about 95%. 
Purity is further confirmed by mass spectroscopy (MS) and capillary zone 
electrophoresis. 
To check whether the precise sequence is achieved, the CRF analog is 
hydrolyzed in sealed evacuated tubes containing 4 molar methane sulfonic 
acid, 3 .mu.l of thioglycol/mi. and 1 nmol of Nle (as an internal 
standard) for 9 hours at 140.degree. C. Amino acid analysis of the 
hydrolysates using a BECKMAN 121 MB amino acid analyzer shows amino acid 
ratios which confirm that the 41-residue peptide structure has been 
obtained 
EXAMPLE II 
The peptide [Ala.sup.39 ]-oCRF having the formula (SEQ ID NO: 5): 
Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-L 
eu-Ala-Ile-Ala wherein the C-terminus is amidated is synthesized using a 
procedure generally as set forth in Example I. 
The peptide is judged to be homogeneous by reversed-phase high performance 
liquid chromatography using a Waters HPLC system with a 0.46.times.25 cm. 
column packed with 5 .mu.m C.sub.18 silica, 300 .ANG. pore size. The 
determination is run at the same conditions as in Example I with the 
retention time for the TFA buffer system being 16.6 minutes. When the 
triethylammonium phosphate (TEAP) buffer system is used, the retention 
time is 17.4 minutes. 
Specific optical rotation of the CRF peptide, which is synthesized and 
purified in the foregoing manner, is measured on a Perkin Elmer Model 241 
Polarimeter as [.alpha.].sub.D.sup.22 =-81 1.degree..+-.1.0.degree.(c=0.5 
in 1% acetic acid, without correction for the presence of H.sub.2 O and 
TFA); it has a purity of greater than about 90%. 
Amino acid analysis of the resultant, purified peptide is consistent with 
the formula for the prepared peptide and confirms that the 41-residue 
peptide structure is obtained. 
The synthetic CRF agonist peptides [Ala.sup.20 ]-oCRF and [Ala.sup.39 
]-oCRF are examined for their effects on the secretion of ACTH and 
.beta.-endorphin in vitro and also in vivo. The potency of synthetic oCRF 
analogs to stimulate the secretion of ACTH and .beta.-endorphin by 
cultured rat pituitary cells is measured using the procedure as generally 
set forth in Endocrinology, 91, 562 (1972) and compared against synthetic 
oCRF. [Ala.sup.20 ]-oCRF is considered to be about 2 to 4 times as potent 
as the native hormone. Similar tests of [Ala.sup.39 ]-oCRF showed about an 
85% increase in biopotency in vitro over the native hormone. In vivo 
testing which can be carried out using the general procedure set forth in 
C. Rivier et al., Science, 218, 377 (1982) shows biopotency to stimulate 
the secretion of ACTH and .beta.-END-LI and a significant lowering of 
blood pressure when injected peripherally, e.g. intravenously. 
EXAMPLE III 
The peptide [Ala.sup.20,39 ]-oCRF having the formula (SEQ ID NO: 6): 
Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Ala-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-L 
eu-Ala-Ile-Ala is synthesized using a procedure generally as set forth in 
Example I. 
The peptide is purified and judged to be homogeneous using MS. Amino acid 
analysis of the resultant, purified peptide is consistent with the formula 
for the prepared peptide. The 41-residue peptide is biopotent and lowers 
blood pressure when injected peripherally. 
EXAMPLE IV 
The peptide [D-Phe.sup.12, Ala.sup.20 ]-rCRF(3-41) having the formula: 
H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Al 
a-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-G 
lu-Ile-Ile-NH.sub.2 is synthesized. Specific optical rotation of the CRF 
analog peptide, which is synthesized and purified in the foregoing manner, 
is measured on a Perkin Elmer Model 241 Polarimeter as 
[.alpha.].sub.D.sup.22 =-63.2.degree..+-.1.0.degree.(C=1 in 1% acetic 
acid, without correction for the presence of H.sub.2 O and TFA); it has a 
purity greater than about 90%. The peptide is likewise biopotent and 
causes significant lowering of blood pressure when injected peripherally. 
EXAMPLE V 
The peptide [Ala.sup.20 ]-rCRF having the formula (SEQ ID NO: 7): 
Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Ala-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-M 
et-Glu-Ile-Ile wherein the C-terminus is amidated is synthesized using a 
procedure generally as set forth in Example I. The peptide is likewise 
biopotent and stimulates the secretion of ACTH and .beta.-END-LI and 
causes significant lowering of blood pressure when injected peripherally. 
EXAMPLE VI 
The peptide [Ala.sup.39 ]-rCRF, having the formula (SEQ ID NO: 8): 
Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Le 
u-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-M 
et-Ala-Ile-Ile wherein the C-terminus is amidated is synthesized using a 
procedure generally as set forth in Example I. The peptide is likewise 
biopotent, stimulates the secretion of ACTH and .beta.-END-LI and causes 
significant lowering of blood pressure when injected peripherally. 
EXAMPLE VIA 
The peptide [D-Phe.sup.12, Ala.sup.20,39, Nle.sup.21,38, Arg.sup.36 ]-rCRF, 
having the formula: 
H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-phe-His-Leu-Leu-Arg-Glu-Va 
l-Leu-Ala-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-L 
eu-Nle-Ala-Ile-Ile-NH.sub.2 is synthesized using a procedure generally as 
set forth in Example I. 
The peptide is judged to be homogeneous by reversed-phase high performance 
liquid chromatography using a Waters HPLC system with a 0.46.times.25 cm. 
column packed with 5 .mu.m C.sub.18 silica, 300 .ANG. pore size. Specific 
optical rotation of the CRF analog peptide, which is synthesized and 
purified in the foregoing manner, is measured on a Perkin Elmer Model 241 
Polarimeter as [.alpha.].sub.D.sup.22 =-57.degree..+-.1.0.degree.(c=10.5 
in 1% acetic acid, without correction for the presence of H.sub.2 O and 
TFA); it has a purity of greater than about 95%. Purity is further 
confirmed by mass spectroscopy (MS) and capillary zone electrophoresis. 
The peptide is likewise biopotent, stimulates the secretion of ACTH and 
.beta.-END-LI and causes significant lowering of blood pressure when 
injected peripherally. 
EXAMPLE VII 
Using the procedure set forth in Example I, the following CRF agonist 
peptides are also prepared: 
[Acetyl-Ser.sup.1, D-phe.sup.12, Ala.sup.20, Nle.sup.21,38 -rCRF 
[D-Phe.sup.12, Ala.sup.20,22 ]-oCRF 
[D-Phe.sup.12, Ala.sup.20,32, D-Ala.sup.24 ]-rCRF(4-41) 
[D-Phe.sup.12, Nle.sup.21, Ala.sup.39 ]-oCRF 
[Formyl-Ser.sup.1, D-phe.sup.12, Ala.sup.20, Nle.sup.21,38, D-His.sup.32 
]-rCRF 
[Ala.sup.20,25, D-Ala.sup.24 ]-oCRF 
[D-phe.sup.12, Ala.sup.20, D-Ala.sup.24 ]-rCRF(2-41) 
[Ala.sup.20,39, D-Ala.sup.24, Nle.sup.21,38 ]-oCRF 
[Ala.sup.20,39, D-Ala.sup.24, Nle.sup.21,38, Arg.sup.36 ]-oCRF 
[Benzoyl-Ser.sup.7, D-phe.sup.12, Ala.sup.20, Nle.sup.21,38, D-His.sup.32 
]-rCRF 
[D-His.sup.32, Ala.sup.39 ]-oCRF 
[D-Phe.sup.12, Ala.sup.20,33, D-Ala.sup.24, D-His.sup.32 ]-rCRF(6-41) 
[Ala.sup.20,29, Nle.sup.21, D-His.sup.32 ]-oCRF 
[Acrylyl-Glu.sup.2, Ala.sup.20, Nle.sup.21,38, D-His.sup.32 ]-rCRF(2-41) 
[Nle.sup.18,21, Ala.sup.20,29, D-His.sup.32 ]-AHC 
[D-Pro.sup.4, D-Phe.sup.12, Nle18,21, Ala.sup.20,32, Ile.sup.33, Asn.sup.36 
]-AHC 
[D-Tyr.sup.3, Nle.sup.18, Nva.sup.21, Ala.sup.20,33, D-Ala.sup.24 ]-AHC 
[Glu.sup.2,13,22, D-phe.sup.12, Nle.sup.18, Orn.sup.23, Ala.sup.39 ]-AHC 
[D-Phe.sup.12, Glu.sup.13, Ala.sup.20, Ile.sup.21, Lys.sup.36, Tyr.sup.37, 
Val.sup.41 ]-AHC 
[D-Phe.sup.12, Ala.sup.20,39,40, Arg.sup.21 ]-AHC 
[Nle.sup.18,21, Ala.sup.20,39 ]-AHC 
[Ala.sup.20 ]-AHC 
[Ala.sup.39 ]-AHC 
[Ala.sup.20,39, Nle.sup.21, CML.sup.37 ]-oCRF 
[D-phe.sup.12, Ala.sup.20,32, Nle.sup.21,38, CML.sup.37 ]-oCRF 
[Ala.sup.20, Nle.sup.21,38,D-His.sup.32, CML.sup.37 ]-oCRF 
These peptides are biopotent in stimulating the secretion of ACTH and 
.beta.-END-LI. 
EXAMPLE VIII 
Using the procedure set forth in Example I, the following peptides are also 
prepared which are CRF antagonists: 
[Ala.sup.20 ]-AHC(9-41) 
[Ala.sup.39 ]-AHC(12-41) 
[Ala.sup.20 ]-oCRF(10-41) 
[D-Phe.sup.12, Ala.sup.20, Nle.sup.21,38 ]-rCRF(12-41) 
[D-Phe.sup.12, Ala.sup.20 ]-oCRF(12-41) 
[Nle.sup.18,21, Ala.sup.20,39 ]-AHC(10-41) 
[D-Phe.sup.12, Ala.sup.20 ]-rCRF(12-41) 
[D-Phe.sup.12, Nle.sup.21, Ala.sup.39 ]-oCRF(12-41) 
[D-Phe.sup.12, Ala.sup.20,39 ]-AHC(12-41) 
[D-Phe.sup.12, Ala.sup.20, Nle.sup.21,38 ]-rCRF(12-41) 
[D-phe.sup.12, Ala.sup.20, Nle.sup.21,38, Arg.sup.36 ]-rCRF(12-41) 
[Ala.sup.20, D-Ala.sup.24 ]-oCRF(11-41) 
[Nle.sup.18,21, Ala.sup.20, D-His.sup.32 ]-AHC(11-41) 
[D-phe.sup.12, Ala.sup.20, D-Ala.sup.24 ]-rCRF(12-41) 
[Ala.sup.20, Nle.sup.21,38, D-His.sup.32 ]-rCRF(10-41) 
[D-His.sup.32, Ala.sup.39 ]-oCRF(9-41) 
[D-Phe.sup.12, Ala.sup.20, D-His.sup.32 ]-rCRF(12-41) 
[Ala.sup.20, Nle.sup.21,38, Ala.sup.39 ]-oCRF(9-41) 
[Ala.sup.20, Nle.sup.21,38, Arg.sup.36, Ala.sup.39 ]-oCRF(9-41) 
[Ala.sup.20, Nle.sup.21 ]-oCRF(10-41) 
[Ala.sup.20, Nle.sup.21,38, D-His.sup.39 ]-rCRF(9-41) 
[Nle.sup.18, Ala.sup.20, D-Ala.sup.24 ]-AHC(10-41) 
[D-Phe.sup.12, Nle.sup.18, Ala.sup.39 ]-AHC(12-41) 
[D-Phe.sup.12, Nle.sup.18,21, Ala.sup.20 ]-AHC(12-41) 
[D-Phe.sup.12, Ala.sup.20, Lys.sup.36 ]-AHC(12-41) 
[Ala.sup.20, Nle.sup.21, D-His.sup.32, CML.sup.37 ]-oCRF(11-41) 
[Ala.sup.20, Nle.sup.21,38 CML.sup.37 ]-oCRF(10-41) 
[D-Phe.sup.12, Ala.sup.20, Nle.sup.21,38, CML.sup.37 ]-oCRF(12-41) 
These CRF antagonist peptides are all considered to inhibit the secretion 
of ACTH and .beta.-END-LI in response to various stimuli. 
EXAMPLE IX 
The peptide [D-Phe.sup.12, Ala.sup.20,39, Nle.sup.21,38, Arg.sup.36 
]-rCRF(12-41) having the formula: 
H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Ala-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gl 
n-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nle-Ala-Ile-Ile-NH.sub.2 is synthesized 
using a procedure generally as set forth in Example I. 
The peptide is judged to be homogeneous by reversed-phase high performance 
liquid chromatography using a Waters HPLC system with a 0.46.times.25 cm. 
column packed with 5 .mu.m C.sub.18 silica, 300 .ANG. pore size. Specific 
optical rotation of the CRF analog peptide, which is synthesized and 
purified in the foregoing manner, is measured on a Perkin Elmer Model 241 
Polarimeter as [.alpha.].sub.D.sup.22 =-67.2.degree..+-.1.0.degree.(c=1 in 
1% acetic acid, without correction for the presence of H.sub.2 O and TFA); 
it has a purity of greater than about 95%. Purity is further confirmed by 
mass spectroscopy (MS) and capillary zone electrophoresis. 
The peptide is biopotent and inhibits the secretion of ACTH and 
.beta.-END-LI when injected peripherally. 
EXAMPLE X 
The peptide [D-Phe.sup.12, Nle.sup.21,38, Arg.sup.36, Ala.sup.39 
]-rCRF(12-41) having the formula: 
H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gl 
n-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nle-Ala-Ile-Ile-NH.sub.2 is synthesized 
using a procedure generally as set forth in Example I. 
The peptide is judged to be homogeneous by reversed-phase high performance 
liquid chromatography using a Waters HPLC system with a 0.46.times.25 cm. 
column packed with 5 .mu.m C.sub.18 silica, 300 .ANG. pore size. Specific 
optical rotation of the CRF analog peptide, which is synthesized and 
purified in the foregoing manner, is measured on a Perkin Elmer Model 241 
Polarimeter as [.alpha.].sub.D.sup.22 =-66.0.degree..+-.1.0.degree.(c=1 in 
1% acetic acid, without correction for the presence of H.sub.2 O and TFA); 
it has a purity of greater than about 95%. Purity is further confirmed by 
mass spectroscopy (MS) and capillary zone electrophoresis. 
The peptide is biopotent and inhibits the secretion of ACTH and 
.beta.-END-LI when injected peripherally. 
CRF profoundly stimulates the pituitary-adrenalcortical axis, and CRF 
agonists should be useful to stimulate the functions of this axis in some 
types of patients with low endogenous glucocorticoid production. For 
example, CRF and its agonists should be useful in restoring 
pituitary-adrenal function in patients having received exogenous 
glucocorticoid therapy whose pituitary-adrenalcortical functions remain 
suppressed. 
Most other regulatory peptides have been found to have effects upon the 
central nervous system and upon the gastrointestinal tract. Because ACTH 
and .beta.-END secretion is the "sine qua non" of mammal's response to 
stress, it was not surprising that CRF has significant effects on the 
brain as a mediator of the body's stress response. For example, CRF in the 
brain appears to increase respiratory rate and may be useful in treating 
respiratory depression. CRF and its analogs may also find application in 
modifying the mood, learning and behavior of normal and mentally 
disordered individuals. Because CRF agonist analogs elevate the levels of 
ACTH, .beta.-END, .beta.-lipotropin, other pro-opiomelanocortin gene 
products and corticosterone, administration can be used to induce their 
effects on the brain and periphery to thereby influence memory, mood, pain 
appreciation, etc., and more specifically, alertness, depression and/or 
anxiety. For example, when administered into the ventricles, CRF increases 
activity and improves learning performance in rats and thus may function 
as a natural stimulant. 
CRF agonist analogs when given intravenously should also be of use for 
increasing blood flow to the gastrointestinal tract of mammals, 
particularly humans and other mammals. All CRF agonist peptides when given 
intravenously have been shown to dilate the mesenteric vascular bed. Also, 
oCRF inhibits gastric acid production, and CRF agonist analogs are 
expected to also be effective in the treatment of gastric ulcers by 
reducing gastric acid production and/or inhibiting gastrointestinal 
functions in a mammal. 
CRF analogs or the nontoxic addition salts thereof, combined with a 
pharmaceutically or veterinarily acceptable carrier to form a 
pharmaceutical composition, may be administered to mammals, including 
humans, either intravenously, subcutaneously, intramuscularly, 
percutaneously, e.g. intranasally, intracerebrospinally or orally. The 
peptides should be at least about 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 percent of all like 
peptides and peptide fragments present. Administration to humans may be 
employed by a physician to lower blood pressure or to stimulate endogenous 
glucocorticoid production. The required dosage will vary with the 
particular condition being treated, with the severity of the condition and 
with the duration of desired treatment. 
These 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 body 
functions. For example, administration may be used as a diagnostic tool to 
evaluate Cushing's disease and affective disorders, such as depressive 
illness. 
CRF antagonists should be useful to inhibit the functions of the 
pituitary-adrenalcortical axis in some types of patients with high ACTH 
and endogenous glucocorticoid production. For example, CRF antagonists may 
be useful in regulating pituitary-adrenal function in patients having 
pituitary Cushings disease or any CRF-sensitive tumor. 
Most other regulatory peptides have been found to have effects upon the 
endocrine system, the central nervous system and upon the gastrointestinal 
tract. Because ACTH and .beta.-END-LI secretion is the "sine qua non" of 
mammal's response to stress, it was not surprising that CRF has 
significant effects on the brain as a mediator of many of the body's 
stress responses. Accordingly, CRF antagonists delivered to the brain 
should also find application in modifying the mood, learning and behavior 
of normal and mentally disordered individuals. Furthermore, CRF 
antagonists in the brain could ameliorate stress-induced conditions to 
which endogenous CRF might contribute, including some types of 
hypertension, infertility, decreased libido, impotency and hyperglycemia. 
Because peripherally administered CRF antagonists reduce the levels of 
ACTH, .beta.-END, .beta.-lipotropin, other pro-opiomelanocortin gene 
products and corticosterone, administration of the antagonists may be used 
to reduce the effects of all of these substances on the brain to thereby 
influence memory, mood, pain appreciation, etc., and more specifically, 
alertness, depression and/or anxiety, as well as to modulate the immune 
system, gastrointestinal tract and adrenalcortical growth and function. 
For example, CRF antagonists may be of use for decreasing blood flow to 
the gastrointestinal tract of mammals, particularly humans, and are 
expected to also be effective to modulate gastrointestinal functions. 
Administration of CRF antagonists to humans may be employed by a physician 
to inhibit endogenous glucocorticoid production or for possible uses 
outlined above. The required dosage will vary with the particular 
condition being treated, with the severity of the condition and with the 
duration of desired treatment. In order to block the stress-related 
effects of endogenous CRF within the central nervous system, it may be 
necessary to deliver the CRF antagonists into the cerebral ventricle or 
spinal fluid. Alternatively, a means of modifying the antagonists so that 
they could penetrate the blood-brain barrier should be found. 
Such peptides are often administered 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, 
aluminum or the like (which are considered as addition salts for purposes 
of this application). Illustrative of such acid addition salts are 
hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, 
fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, 
succinate, malate, ascorbate, tartrate and the like. If the active 
ingredient is to be administered in tablet form, the tablet may contain a 
binder, such as tragacanth, corn starch or gelatin; a disintegrating 
agent, such as alginic acid; and a lubricant, such as magnesium stearate. 
If administration in liquid form is desired, sweetening and/or flavoring 
may be used, and intravenous administration in isotonic saline, phosphate 
buffer solutions or the like may be effected. 
The peptides should be administered under the guidance of a physician, and 
pharmaceutical compositions will usually contain the peptide in 
conjunction with a conventional, pharmaceutically or 
veterinarily-acceptable carrier. Usually, the dosage will be from about 1 
to about 200 micrograms of the peptide per kilogram of the body weight of 
the host animal. In some instances, 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. As used herein, all temperatures are 
0.degree. C. and all ratios are by volume. Percentages of liquid materials 
are also by volume. 
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 may be 
made without departing from the scope of the invention which is set forth 
in the claims appended hereto. In the examples given, substitutions at 
positions in the CRF peptide chain as known in this art, or with commonly 
accepted comparable residues, other than at the specified position-20 and 
position-39 can be made without detracting from the potency of the 
analogs, and peptides having such substitutions are considered to be 
equivalents. It appears important that the amino acid sequence, or 
equivalents thereof, from about position-7 through the C-terminus be 
present in the synthetic peptide to assure biopotency as a CRF agonist, 
whereas the remainder of the molecule does not appear as critical. For 
instance, instead of the simple amide at the C-terminus, a lower 
alkyl-substituted amide, e.g. methylamide, ethylamide, etc, may be 
incorporated in a peptide without adversely affecting biological potency, 
and such peptides are also considered as equivalents. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
SerGlnGluProPro IleSerLeuAspLeuThrPheHisLeuLeuArg 
151015 
GluValLeuGluMetThrLysAlaAspGlnLeuAlaGlnGlnAlaHis 
20 2530 
SerAsnArgLysLeuLeuAspIleAla 
3540 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
SerGluGluProProIleSerLeuAspLeuThrPheHisLeuLeuArg 
151015 
GluValLeuGluMetAlaArg AlaGluGlnLeuAlaGlnGlnAlaHis 
202530 
SerAsnArgLysLeuMetGluIleIle 
3540 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
SerGlnGluProProIleSerLeuAspLeuThrPheHisLeuLeuArg 
15 1015 
GluMetLeuGluMetAlaLysAlaGluGlnGluAlaGluGlnAlaAla 
202530 
LeuAsnArgLeuLeuLeuGluGluAla 
3540 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
SerGlnGluProProIleSerLeuAspLeuThrPheH isLeuLeuArg 
151015 
GluValLeuAlaMetThrLysAlaAspGlnLeuAlaGlnGlnAlaHis 
2025 30 
SerAsnArgLysLeuLeuAspIleAla 
3540 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
SerGlnGluProProIleSerLeuAspLeuThrPheHisLeuLeuArg 
151015 
GluValLeuGluMetThrLysAlaAspGlnLeuAlaGlnGln AlaHis 
202530 
SerAsnArgLysLeuLeuAlaIleAla 
3540 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
SerGlnGluProProIleSerLeuAspLeuThrPheHisLeuLeuArg 
151015 
GluValLeuAlaMetThrLysAlaAspGlnLeuAlaGlnGlnAlaHis 
202530 
SerAsnArgLysLeuLeuAlaIleAla 
35 40 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
SerGluGluProProIleSerLeuAspLeuThrPheHisLeuLeuArg 
1 51015 
GluValLeuAlaMetAlaArgAlaGluGlnLeuAlaGlnGlnAlaHis 
202530 
SerAsnAr gLysLeuMetGluIleIle 
3540 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
SerGluGluProProI leSerLeuAspLeuThrPheHisLeuLeuArg 
151015 
GluValLeuGluMetAlaArgAlaGluGlnLeuAlaGlnGlnAlaHis 
20 2530 
SerAsnArgLysLeuMetAlaIleIle 
3540 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
SerXaaGluProProIleSerLeuAspLeuThrXaaHisLeuLeuArg 
151015 
GluValLeuXaaXaaXaaXaa XaaXaaGlnLeuAlaGlnGlnAlaXaa 
202530 
SerAsnArgXaaLeuXaaXaaIleXaa 
3540 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
XaaXaaXaaXaaProIleSerXaaXaaLeuXaaXaaXaaXaaLeuArg 
151 015 
XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaGlnAlaXaa 
202530 
XaaAsnArgXaaXaaXaaXaaXaaXaa 
3540 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
SerXaaGluProProIleSerLeuXaaLeuThrXaaXa aXaaLeuArg 
151015 
GluXaaLeuXaaXaaAlaLysXaaGluGlnXaaAlaGluGlnAlaXaa 
2025 30 
XaaAsnArgXaaXaaXaaXaaXaaXaa 
3540