CRF ANALOGS

Abstract: Analogs of 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 and/or a lowering of blood pressure over an extended period of time. One analog which has been found to be particularly potent is: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Val-Leu-His-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Ar g-Lys-Leu-Met-Glu-Ile-Ile-NH2. In the analogs, one or more of the first five N-terminal residues may be deleted or may be substituted by a peptide up to 10 amino acids long and/or by an acylating agent containing up to 7 carbon atoms. A number of other substitutions may also be made throughout the chain. These analogs or pharmaceutically or veterinarily acceptable salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier, can be administered to mammals, including humans. These analogs may also be used as stimulants to elevate mood and improve memory and learning.

Description: CRF ANALOGS 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. Over 25 years ago, Guillemin, Rosenberg and Saffran and Schally independently demonstrated the presence of factors in hypothalamus which would increase the rate of ACTH secretion by the pituitary gland incubated in vitro or maintained in an organ culture. None of the secretagogs characterized met the criteria expected of a physiologic corticotropin releasing factor (CRF) until ovine CRF (oCRF) was characterized in 1981 and, as disclosed in U.S. Patent No. 4,415,558, was found to have the formula: 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-NH 2 . Sauvagine is a 40-residue, amidated generally similar peptide which was isolated from the skin of the South American frog Phyllomedusa sauvagei. It was characterized by Erspamer et al. and was described in Regulatory Peptides. Vol. 2 (1981), pp. 1-13. Sauvagine has the formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu- Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln- Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu- Asp-Thr-Ile-NH 2 . Sauvagine and oCRF have been reported to have biological activity in lowering blood pressure in mammals and in stimulating the secretion of ACTH and β-endorphin. Rat CRF(rCRF) has been isolated, purified and characterized as a hentetracontapeptide having the formul : 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-NH 2 • It may alternatively be referred to as rat Amunine. The formula of human CRF has now been determined to be the same as that of rCRF. Synthetic rCRF and oCRF stimulate ACTH and β-endorphin activities in vitro and in vivo and substantially lower blood pressure for an extended time period. SUMMARY OF THE INVENTION Analogs of these 41-residue CRF peptides of the following formula have at least substantially the same biological activity in the foregoing respects as the native peptides: Y-R-L-R^R- j -R^Rg-Ile-Ser-Rg-Rg- leu-R 11 -R 12 ~ R i3 "R 14 ""leu ~ Ar 5~ R 17~ R 18~ R 19~ R 20~ R 21~ R 22 " R 23~ R 24~ R 25~ R 26~ R 27~ R 28~ R 29~ Gln ~ a l - R 32~ R 33- Asn ~ Ar g~ R 36" R 37~ R 38~ R 39~ R 40~ R 41~ NH 2 wnere i n γ i s an acyl group having 7 or less carbon atoms or hydrogen; R is Ser, D-Ser or des R^; R 2 is Glu, Gin, pGlu, D-pGlu or des R 2 ; R 3 is Glu, Gly, D-Tyr or des R 3 ; R 4 is Pro, D-Pro or des R 4 ; 5 is Pro or desR 5 ; R 8 and R 19 are selected from the group consisting of leu, lie, ala, Gly, Val, Nle, Phe and Gin; R g is Asp or Glu; R 1:L is Thr or Ser; 12 is Phe, leu, ala, lie, Gly, Val, Nle or Gin; R 13 is His, Tyr or Glu; R 14 is leu or Met; R 17 is Glu or Lys; R 18 is Val, Nle or Met; R 20 is His or Glu; R 21 is Arg, Met, Nva, lie, ala, leu, Nle, Val, Phe or Gin; R 22 is ala, Thr, Asp or Glu; R 23 is Arg, Orn, Har or Lys; R 2 is Met, leu, lie, ala, Gly, ' Val, Nle, Phe and Gin; R 25 is Glu or Asp; R 26 is Gly, Gin, Asn or Lys; R 27 is leu, lie, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gin or Glu; R 28 is ala, Arg or Lys; R 9 is Gin or Glu; R 32 is Leu, His, Gly, Tyr or ala; R 33 is lie, Ser, Asn, leu, Thr or ala; R 36 is Asn, Lys, Orn, Arg, Har or Leu; R 37 is leu or Tyr; R 38 is Met or leu; R 39 is Glu or Asp; R 4Q is lie, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly, Asn or Gin; R 41 is lie, ala, Gly, Val, Leu, Nle, Phe, Gin or des R 41 , provided however that at least one of the following residues is present: R 14 is Met, R 20 is His, 21 is Arg, R 24 is Met, R 26 is Gly, R 3 is Leu, R 33 is lie, R 36 is Asn; or a nontoxic addition salt thereof. 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, β-endorphin, β-lipotropin, other products of the pro-opiomelanocortin gene and corti¬ costerone and/or for the lowering of blood pressure and/or for affecting mood, behavioral and gastro- intestinal 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 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, Nle = L-norleucine, Nva = norvaline, Har = homoarginine, Orn = ornithine etc. In addition the following abbreviations are used: leu = either L-leucine or C CH 3 -L-leucine (CML) and ala = either L-alanine or C CH 3 -L-alanine(CMA) . The invention provides analogs of- CRF of the following Formula (I) : Y-R 1 -R 2 -R 3 -R 4 -R 5 -Ile-Ser- R 8~ R 9~^ eu ~ R ll~ R 12" R 13~ R i4~l eu_Ar g~ R i7~ R 18~ R 19~ R 20~ R 21 -R 22 -R 23 -R 24 -R 25 -R 2 g-R 27 -R 28 -R 29 -Gln-ala-R 3 - R 33""Asn-Arg-R 3 --R 3 7 _R 38 - 39 - 4 Q~ R 41~ NH 2 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R- ^ is Ser, D-Ser or des R-^ j R 2 is Glu, Gin, pGlu, D-pGlu or des R 2 ; R 3 is Glu, Gly, D-Tyr or des R 3 ; R 4 is Pro, D-Pro or des R 4 ; R 5 is Pro or desR 5 ; R 8 and R 19 are selected from the group consisting of leu, lie, ala, Gly, Val, Nle, Phe and Gin; R g is Asp or Glu; .R-^ is Thr or Ser; R 12 is Phe, leu, ala, lie, Gly, Val, Nle or Gin; R 13 is His, Tyr or Glu; R 14 is leu or Met; R 17 is Glu or Lys; R 18 is Val, Nle or Met; R 20 is His or Glu; R 21 is Arg, Met, Nva, lie, ala, leu, Nle, Val, Phe or Gin; R 22 is ala, Thr, Asp or Glu; R 23 is Arg, Orn, Har or Lys; R 24 is Met, leu, lie, ala, Gly, Val, Nle, Phe and Gin; R 25 is Glu or Asp; R 26 is Gly, Gin, Asn or Lys; R 27 is leu, lie, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gin or Glu; R 28 is ala, Arg or Lys; R 29 is Gin or Glu; R 32 is Leu, His, Gly, Tyr or ala; R 33 is lie, Ser, Asn, leu, Thr or ala; R 36 is Asn, Lys, Orn, Arg, Har or Leu; R 37 is leu or Tyr; R 38 is Met or leu; R 39 is Glu or Asp; R 40 is lie, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly, Asn or Gin; R 41 is lie, ala, Gly, Val, Leu, Nle, Phe, Gin or des R 4 ^ r provided however that at least one of the following residues is present: 14 is Met, R 20 s His, R 21 is Arg, R 24 is Met, R 26 is Gly, R 32 is Leu, R 33 is lie, R 36 is Asn. Nontoxic addition salts of these peptides can be used as well. In this formula, at least one and preferably at least two of the following substituents are present: R 20 is His, R 21 ^- s Ar 9' R 26 ^- s G - L Y^ and/or R 36 is Asn. Most preferably, the residue His in the 20-position is one of these two substituents. These analogs are considered to be at least as potent as native CRF. These analogs may include residues having a high alpha-helical forming potential such as: R- ^ is Ser, R 2 is Gin or Glu, R 3 is Glu, R 4 and R 5 are Pro, R 8 is leu, R 1] is Thr, R 1 is Phe or leu, R 13 is His or Glu, R 17 is Glu, R 18 and R 21 are Met or Nle, R 19 and R 37 are leu, R 22 and R 41 are ala, R 23 is Lys, R 24 and R 28 are ala,' R 25 and R 39 are Glu, R 26 is Gin, R 27 is Glu or leu, R 29 is Glu, R 32 is His or ala, R 33 is Ser or leu, R 38 is Leu and R 40 is lie or Glu. One analog which has been found to be particularly potent is: H-Ser-Gln-Glu- Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Met-Leu-His-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala- Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 . It remains potent even if slightly shortened at the N-terminus, i.e., by a sequence of up to about 5 residues. Two other analogs which are particularly potent are those having the formulas: H-Ser-Gln-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu- His-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu- Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala-NH 2 and H-Ser-Gln-Glu- Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Met-Leu-His-Met-Ala-Lys-Ala-Glu-Gly-Glu-Ala-Glu-Gln-Ala- Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 . 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. Certain CRF analogs which do not include D-isomer residues or unnatural amino acid residues may also be synthesized by recently developed recombinant DNA techniques. Synthesis by the use of recombinant DNA techniques, for purposes of this application, should be understood to include the suitable employment of a structural gene coding for the desired form of CRF analog. Such a synthetic CRF peptide may be obtained by transforming a microorganism using an expression vector including a promoter and operator together with such structural gene and causing such transformed microorganism to express the CRF analog peptide. A non-human animal may also be used to produce the CRF analog peptide by gene-farming, the general techniques of which are known to those skilled in the art. For example, microinjection of embryos as described in W083/01783, published 26 May 1983, and W082/04443, published 23 December 1982, may be used. The synthetic CRF analog peptide is then suitably recovered from the animal by extraction from sera or the like. 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 (II) : X 1 -R 1 (X 2 )-R 2 (X 4 or X 5 )-R 3 (X 5 or X)-R 4 -R 5 -Ile-Ser(X 2 )- R 8 -R g (X 5 )-leu-R 1:L (X 2 )-R 12 (X 4 )-R 13 (X or X 5 )-R 14 -leu- Arg(X 3 )-R 17 (X 5 or X 6 )-R 18 -R 19 (X 4 )-R 20 (X 5 or X)- R 21 (X 3 )-R 22 CX 2 or X 5 )-R 23 (X 3 or X 6 )-R 24 (X 4 )-R 25 (X 5 )- R 26 (X or X 6 )-R 27 (X 4 or X 5 )-R 28 (X 3 or X 6 )-R 29 (X' or X 5 )-Gln(X 4 )-ala-R 32 (X)-R 33 (X 2 or X 4 )-Asn(X )-Arg(X 3 ) R 36 (X 6 or X 4 )-R 37 (X)-R 38 -R 39 (X 5 )-R 40 (X 2 or X 4 or X 5 )- R 41 (X 4 )-X 7 wherein: the R-groups are as hereinbefore defined. χ is either hydrogen or an -amino protecting group. The -amino protecting groups contemplated by X are those known to be useful in the art in the step-wise synthesis of polypeptides. Among the classes of -amino protecting groups covered by X 1 are (1) acyl-type protecting groups, such as for yl, 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 fluorenyl ethyloxycarbonyl(FMOC) , cyclopentyloxycarbonyl, adamantyloxycarbonyl,and cyclohexyloxycarbonyl; and (5) thiourethan-type protecting groups, such as phenylthiocarbonyl. The preferred -amino protecting group is BOC. X 2 is a protecting group for the hydroxyl group of Thr and Ser and is preferably selected from the class consisting of acetyl(Ac), benzoyl(Bz), tert-butyl, triphenylmethyl(trityl) , tetrahydropyranyl, benzyl ether(Bzl) and 2,6-dichlorobenzyl (DCB) . The most preferred protecting group is Bzl. X 2 can be hydrogen, which means there is no protecting group on the hydroxyl group. X 3 is a protecting group for the guanidino group of Arg or Har preferably selected from the class consisting of nitro, p-toluenesulfonyl(Tos) , Z, adamantyloxycarbonyl and BOC, or is hydrogen. Tos is most preferred. X 4 is hydrogen or a protecting group, preferably xanthyl(Xan) , for the amido group of Asn or Gin. X 5 is hydrogen or an ester-forming protecting group for the β- or -carboxyl group of Asp or Glu, preferably selected from the class consisting of benzyl, 2,6-dichlorobenzyl, methyl, ethyl and t-butyl ester. OBzl is most preferred. X is hydrogen or a protecting group for the. side chain amino substituent of Lys or Orn. 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. When His is present, X is hydrogen or a protecting group for the imidazole nitrogen such as Tos or 2,4-dinitrophenyl(DNP) , and when Tyr is present, X is hydrogen or a protecting group for the hydroxyl group such as DCB. 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 -amino groups during the synthesis. Hence, the -amino protecting group and the side chain amino protecting group cannot be the same. X is NH 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 a methyl-derivative of such a resin, a methyl-substituted amide can be created which is considered to be the equivalent of the unsubstituted amide. In the formula for -±føe iaatsaπmediate, at least one of X, X 1 , χ 2 , χ 3 , X 4 , X 5 and X 6 As a protecting group. The particular : ό iL o acid chosen for each the R—group determines-szhether -tcfaiere will also be a protecting group attached ss spe ied hereinbefore and as generally known in the cr . In .selecting a particular side chain protecting group ±ss ~ be uss≤l in the synthesis of the peptides, the .following ules are followed: (a) the protecting group should ±*e stabike to the reagent and under the reaction nonditiαss selectjefl for removing the -amino protecting group al each step of the synthesis, (b) the protecting -group should retain its protecting properties and not be. split raff under (coupling conditions and (c) the side chain prrrbjac gs?αnp must be removable, upon tluε completion of the .synthesis containing the desiaπad amiiES sc -d segraence, under reaction conditions that ill :uot alder ±he peptide chain. For the acyl group at "tfae Sf~*e.τminus represented by Y, acetyl, fomτy3. ; , acrjjφy! and toenzaEryl are preferred. For the 1 to 10 amiao acixS ipeptiiJSf ^ _Lch may be optionally included sexthout a verse!!^ affecting the potency, any aminα> acids joasy .be -usa ,, but the L- or D- forms of the naturally ΛCcttrrJ g aadaro acids would normally be used. a_tøreov.er>,.. as in&iLcated hereinbefore, the N-terminus can Ibe sligfe±lLy shortened without significantly affecting -bisilOgicjal αrtency. Thus, the present .iϋRrenticm is also considered to provide a process for ~ϊ3 manufacture of compounds defined by the Formula {!)) rjomprisiaag (a) forming a peptide having at ieast one protective group and having the Formula (II) wherein: 3£„ X 1 , X 2 „ X 3 , X 4 , X 5 and 6 are each either hydrogen or a protective group, *— and X is either a protective group or an anchoring bond to resin support or NH 2 and (b) splitting off the protective group or groups or anchoring bond from said peptide of the Formula (II) 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, _________ 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 usually commenced from the C-terminus end of the peptide by coupling a protected -amino acid to a suitable resin as generally set forth in U.S. Patent No. 4,244,946 issued Jan. 21, 1981 to Rivier et al., the disclosure of which is incorporated herein by reference. Such a starting material for rCRF analogs can be prepared by attaching -amino-protected lie to a BHA resin. lie protected by BOC is coupled to the BHA resin using methylene chloride and dimethylformamide (DMF) . Following the coupling of BOC-Ile to the resin support, the -amino protecting group is removed, as by using trifluoroacetic acid(TEA) 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 0C and room temperature. Other standard cleaving reagents and conditions for removal of speci ic -amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", 1 , pp 72-75 (Academic Press 1965) . After removal of the -amino protecting group of lie, the remaining -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(DCCI) 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 N,N'-diisopropyl carbodii ide 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 1-27 (1970). 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 2 C1 2 (1:1) or in DMF or CH 2 C1 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 -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, 1978, 17, pp.1927-1938. 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, X 2 , X 3 , X 4 , X 5 and X 6 and the -amino protecting group X 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 [His 20 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Va . l-Leu-His-Met-Ala-Arg-Ala-Glu- Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu- Ile-Ile-NH 2 is conducted in a stepwise manner on a MBHA hydrochloride resin, such as available from Bache , Inc. , having a substitution range of about 0.1 to 0.5 m oles/gm. resin. The synthesis is performed on an automatic Beckman 990B peptide synthesizer using a suitable program, preferably as follows: STEP REAGENTS AND OPERATIONS MIX TIMES MIN. 1 CH 2 C1 2 wash-80 ml. (2 times) 3 2 Methanol(MeOH) wash-30 ml. (2 times) 3 3 CH 2 C1 2 wash-80 ml. (3 times) 3 4 50 percent TFA plus 5 percent 1,2-ethane- dithiol in CH 2 Cl 2 -70 ml. (2 times) 12 5 Isopropanol wash-80 ml. (2 times) 3 6 TEA 12.5 percent in CH 2 Cl 2 -70 ml. (2 times) 5 7 MeOH wash-40 ml. (2 times) 2 8 CH 2 C1 2 wash-80 ml. (3 times) 3 9 Boc-amino acid (10 mmoles) in 30 ml. of either DMF or CH 2 C1 2 , depending upon the solubility of the particular protected amino acid, (1 time) plus DCCI (10 mmoles) in CH 2 C1 2 30-300 Coupling of BOC-Ile results in the substitution of about 0.35 mmol. lie 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 DCCI 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. P-nitrophenyl ester(ONp) is used to activate the carboxyl end of Asn or Gin, and for example, BOC-Asn(ONp) is coupled overnight using one equivalent of HOBt in a 50% mixture of DMF and methylene chloride. The amido group of Asn or Gin is protected by Xan when DCCI coupling is used instead of the active ester method. 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 OBzl. At the end of the synthesis, the following composition is obtained BOC-Ser(Bzl)-Glu(OBzl)- Glu(OBzl)-Pro-Pro-Ile-Ser(Bzl)-Leu-Asp(OBzl)-Leu-Thr(Bzl)- Phe-His(Tos)-Leu-Leu-Arg(Tos)-Glu(OBzl)-Val-Leu-His(TOS)- Met-Ala-Arg(Tos)-Ala-Glu(OBzl)-Gin(Xan)-Leu-Ala-Gin(Xan)- Gin(Xan)-Ala-His(Tos)-Ser(Bzl)-Asn(Xan)-Arg(Tos)-Lys (2-Cl-Z)-Leu-Met-Glu(0Bzl)-Ile-Ile-resin support. Xan may have been partially or totally removed by TFA treatment used to deblock the -amino protecting group. In order to cleave and deprotect the resulting protected peptide-resin, it is treated with 1.5 ml. anisole, 0.5 ml. of methylethylsulfide and 15 ml. hydrogen fluoride (HF) per gram of peptide-resin, first at -20C. for 20 min. and then at O.C. for one-half hour. After elimination of the HF under high vacuum, the resin-peptide is washed alternately with dry diethyl ether and chloroform, and the peptides are then extracted with de-gassed 2N aqueous acetic acid and separated from the resin by filtration. The peptide is purified by gel permeation followed by semi-preparative HPLC as described in Rivier et al. , Peptides: Structure and Biological Function (1979) pp. 125-128, and Rivier et al., J. Chromatographv (1983) . The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity were pooled. To check whether the precise sequence was achieved, the rCRF analog is hydrolyzed in sealed evacuated tubes containing constant boiling HC1, 3ul of thioglycol/ml. and 1 n ol of Nle (as an internal standard) for 9 hours at 140C. Amino acid analyses of the hydrolysates using a Beckman 121 MB amino acid analyzer show the following amino acid ratios: Asx(l.δ), Thr(0.9), Ser(3.1), Glx(7.9), Pro(2.2), Ala(3.9), Val(0.9), Met(1.8), Ile(2.8), Leu(6.9), Phe(O.δ), Lys(l.O), His(2.9) and Arg(2.9), which confirms that the 41-residue peptide structure is obtained. EXAMPLE II The synthetic peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Met-Leu-His-Met-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized using a procedure generally as set forth in Example I. The peptide is purified in the manner set forth in Example I. Amino acid analysis shows the expected amino acid ratios. EXAMPLE III The synthetic peptide from Example I and synthetic oCRF are examined for their effects on the secretion of ACTH and β-endorphin in vitro and also in vivo. The potency of synthetic oCRF to stimulate the secretion of ACTH and β-endorphin by cultured rat pituitary cells is measured using the procedure as generally set forth in Endocrinology. 91, 562 (1972) . Half-maximal responses are observed at concentrations of the peptide very substantially less than the synthetic oCRF concentrations of about 250 picomolar which are needed to achieve this response. In vivo testing is carried out using the general procedure set forth in C. Rivier et al. , Science, 218, 377 (1982), and biological potency is ascertained. EXAMPLE IV The peptide [des Ser-^-Glu 2 , lie 33 , Asn 36 ]-rCRF having the formula: H-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-Ile-Asn-Arg-Asn-Leu-Met-Glu-Glu-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE V The peptide [His 20 , Gly 26 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Met-Ala-Arg-Ala-Glu-Gly-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE VI The peptide [Acetyl-Gly 1 , He 33 , Glu 40 ]- rCRF having the formula: Ac-Gly-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-Ile-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE VII The peptide [Met 14 ' 24 , Arg 21 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Met- Leu-Arg-Glu-Val-Leu-Glu-Arg-Ala-Arg-Met-Glu-Gln-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE VIII The peptide [Acrylyl-Val 1 , Ser 2 , Arg 21 ]-rCRF having the formula: Acr-Val-Ser-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Val-Leu-Glu-Arg-Ala-Arg-Ala-Glu-Gln-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE IX The peptide [Glu 9 , His 20 , Nle 21 , Glu 29 , Ile 33 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Glu-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala- Glu-Gln-Ala-His-Ile-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF. EXAMPLE X The peptide [Benzoyl-Gly 1 , Nle 12 , His 20 , Arg 36 ]-rCRF having the formula: Bz-Gly-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Nle-His- Leu-Leu-Arg-Glu-Val-Leu-His-Met-Ala-Arg-Ala-Glu-Gln-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Met-Glu-Ile-Ile- NH is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XI The peptide [Nle 8 , Ser 11 , Gly 26 , Leu 32 , Glu 0 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Nle-Asp-Leu-Ser-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gly-Leu-Ala- Gln-Gln-Ala-Leu-Ser-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XII The peptide [Nle 21 , Har 23 , Asn 36 , Glu 40 , Nle 1 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Har-Ala-Glu-Gln-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Asn-Leu-Met-Glu-Glu-Nle-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XIII The peptide [Acetyl-Pro 4 , His 20 , Glu 29 ' 40 , Asn 36 ]-rCRF(4-41) having the formula: Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg- Glu-Val-Leu-His-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln- Ala-His-Ser-Asn-Arg-Asn-Leu-Met-Glu-Glu-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF. EXAMPLE XIV The peptide [Gin 2 , Orn 23 , Gly 26 , Glu 29 , Leu 38 ]-rCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Orn-Ala-Glu-Gly-Leu-Ala- Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Glu-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XV The peptide [His 20 , Nle 21 , Leu 32 , He 33 , Glu 40 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala- Gln-Gln-Ala-Leu-Ile-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF. EXAMPLE XVI The peptide [His 20 , Arg 21 , Leu 32 , Ile 33 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Arg-Ala-Arg-Ala-Glu-Gln-Leu-Ala- Gln-Gln-Ala-Leu-Ile-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF. EXAMPLE XVII The peptide [His 19 , Ala 20 , Arg 21 , Glu 28 , lie 39 ]-sauvagine having the formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu- Arg-Lys-Met-Ile-His-Ala-Arg-Lys-Gln-Glu-Lys-Glu-Lys-Glu- Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XVIII The synthetic peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Met-Leu-His-Arg-Ala-Lys-Ala-Glu-Gly-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized using a procedure generally as set forth in Example I. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XIX The peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Glu-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Nle-Leu-His-Arg-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XX The peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Nle-Leu-His-Nle-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXI The peptide having the formula: H-Ser-Gln-Glu-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Ile-Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXII The peptide having the formula: H-Ser-Gln-D-Tyr-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Nle-Leu-His-Nle-Ala-Lys-Ala-Gly-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXIII The peptide having the formula: H-Ser-Glu-Glu-Pro-Pro.-Ile-Ser-Leu-Asp-Leu-Thr-Leu-Glu- Met-Leu-Arg-Glu-Met-Leu-Glu-Met-Glu-Orn-Met-Glu-Lys-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 . Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXIV The synthetic peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Met-Leu-Arg-Glu-Met-Leu-His-Arg-Ala-Lys-Met-Glu-Gly-Glu- Ala-Glu-Gln-Ala-Leu-Ile-Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXV The peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-Glu- Leu-Leu-Arg-Glu-Met-Leu-Glu-Arg-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Asn-Tyr-Leu-Glu-Glu-Ala- NH 2 . Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXVI The peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-CML-Thr-Phe-His- CML-Leu-Arg-Glu-Met-CML-Glu-Met-Ala-Lys-Ala-Glu-Gln-CML- Ala-Glu-Gln-Ala-Ala-CML-Asn-Arg-Asn-Leu-CML-Glu-Glu-Ala- NH 2 . Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXVII The peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-CML-Thr-Phe-His- Leu-CML-Arg-Glu-Met-Leu-His-Met-CMA-Lys-Ala-Glu-Gln-CML- Ala-Glu-Gln-Ala-CMA-Leu-Asn-Arg-Leu-CML-Leu-Glu-Glu-CMA- NH 2 . Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXVIII The peptide having the formula: H-D-Tyr-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Glu-Nle-Leu-Glu-Arg-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu- Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI and also causes a very significant lowering of blood pressure. EXAMPLE XXIX The peptide having the formula: H-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg- Glu-Nle-Leu-His-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln- Ala-Ala-Ile-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI to an amount equal to about 160% of oCRF and also causes a very significant lowering of blood pressure. EXAMPLE XXX The peptide having the formula: H-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-His-Leu-Leu-Arg- Glu-Met-Leu-Glu-Met-Glu-Orn-Ala-Glu-Gln-Glu-Ala-Glu-Gln- Ala-Ala-Leu-Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala-NH 2 . Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXI The peptide [His 20 , Asn 36 ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Asn-Leu-Leu-Asp-Ile-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI and also causes a very significant lowering of blood pressure. EXAMPLE XXXII The peptide [His 20 , Nle 21 ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-His-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXIII The peptide [Nle 18,21 ,Asn 36 ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Nle-Leu-Glu-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Asn-Leu-Leu-Asp-Ile-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXIV The peptide [D-Pro 4 , His 20 ]-oCRF(4-41) having the formula: H-D-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Val-Leu-His-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala- His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXV The peptide [Met 14 ' 24 , CML 33 ]-oCRF(3-41) having the formula: H-Glu-Tyr-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Met-Leu- Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Met-Asp-Gln-Leu-Ala-Gln- Gln-Ala-His-CML-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXVI The peptide [Nle 18 ,Gly 26 ,Asn 36 ,Glu 40 ]-oCRF(2-41) having the formula: H-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Nle-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gly-Leu-Ala- Gln-Gln-Ala-His-Ser-Asn-Arg-Asn-Leu-Leu-Asp-Glu-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXVII The synthesis of [Arg 1 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Arg-Ala-Arg-Ala-Glu-Gln-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXVIII The synthesis of [Asn 36 ]-rCRF having the 0 formula: 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-Asn-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the 5 general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XXXIX The peptide [Gly 26 ]-rCRF having the formula: o 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-Gly-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it 5 likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XL The synthesis of [Leu 3 ]-rCRF having the formula: o 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-Leu-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it 5 likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLI The peptide [Ile 33 ]-rCRF having the formula: 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-Ile-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XVII The synthesis of [Met 4 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-Hiε-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Met-Glu-Gln-Leu- Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLIII The synthesis of [Met 14 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Met- 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- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLIV The peptide having the formula: H-D-Tyr-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Nle-Leu-His-Nle-Ala-Lys-Ala-Glu-Gly-Glu- Ala-Glu-Gln-Ala-Leu-Leu-Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLV The peptide having the formula: H-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu- Nle-Leu-His-Nle-Ala-Arg-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala- Leu-Leu-Asn-Arg-Asn-Leu-Leu-Glu-Glu-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLVI The synthetic peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Met-Leu-Arg-Glu-Met-Leu-His-Met-Ala-Lys-Met-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized using a procedure generally as set forth in Example I. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. EXAMPLE XLVII The synthetic peptide having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Met-Leu-His-Arg-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Ile-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala- NH 2 is synthesized using a procedure generally as set forth in Example I. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure. CRF analogs exhibit such lowering of blood pressure that they may be particularly valuable for the treatment of high blood pressure conditions and also for the treatment of patients who are to undergo certain types of surgery. CRF profoundly stimulates the pituitary- adrenalcortical axis, and CRF analogs should be useful to stimulate the functions of this axis in some types of patients with low endogenous glucocorticoid production. For example, CRF 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 β-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. Accordingly, CRF should also find application in modifying the mood, learning and behavior of normal and mentally disordered individuals. Because CRF analogs elevate the levels of ACTH, β-END, β-lipotropin, other pro-opiomelanocortin gene products and corticosterone, its administration can be used to induce their effects on the brain and its 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 analogs should also be of use for increasing blood flow to the gastrointestinal tract of mammals, particularly humans and other mammals. All CRF related peptides have been shown to dilate the mesenteric vascular bed. Also, oCRF inhibits gastric acid production, and CRF 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 % 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 gluco-corticoid 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. 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 °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. For example, substitutions and modifications at other positions in the CRF peptide chain can be made in accordance with present or future developments without detracting from the potency of the analogs. It appears important that the amino acid sequence from about positions 6 through 41 or an equivalent sequence of that length be present in the synthetic peptide, whereas the remainder of the N-terminus of the molecule does not appear as critical. In addition, instead of the simple amide at the C-terminus, a lower alkyl-substituted amide, e.g. methylamide, ethylamide, etc, may be incorporated, and such substituted amides are considered equivalent to the unsubstituted amide. Likewise from one to ten additional amino acid residues can be included at the N-terminus without significantly adversely affecting biological potency. Such peptides are considered as equivalents which fall within the scope of the invention. Various features of the invention are emphasized in the claims which follow.

Claims: WHAT IS CLAIMED IS: 1. A peptide having the formula: Y-R 1 -R 2 -R 3 -R 4 -R 5 -Ile-Ser-R 8 -R 9 -leu-R 11 - R 12~ R 13~ R 14~ leu ~ Arg-R 17~ R 18~ R 19 ""R 20~ R 21~ R 22~ R 23~ R 24~ R 25~ R 26~ R 27~ R 28~ R 29~ Gln ~ ala ~ R 3 2~ R 3 3~Asn-Arg-R 3 --R 37 -R 38 -R 3 - -R > Q -R _- H 2 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R_ is Ser, D-Ser or des R- ^ ; R is Glu, Gin, pGlu, D-pGlu or des R 2 ; R 3 is Glu, Gly, D-Tyr or des R 3 ; R 4 is Pro, D-Pro or des R 4 ; R 5 is Pro or desR 5 ; R g and R 19 are selected from the group consisting of leu, lie, ala, Gly, Val, Nle, Phe and Gin; R 9 is Asp or Glu; R_-. -- s Tnr or Ser; R 12 is Phe, leu, ala, lie, Gly, Val, Nle or Gin; R 13 is His, Tyr or Glu; R 14 is leu or Met; R 17 is Glu or Lys; R 18 is Val, Nle or Met; R 20 is His or Glu; R 21 is Arg, Met, Nva, lie, ala, leu, Nle, Val, Phe or Gin; R 22 is ala, Thr, Asp or Glu; R 23 is Arg, Orn, Har or Lys; R 24 is Met, leu, lie, ala, Gly, Val, Nle, Phe and Gin; R 25 is Glu or Asp; R 26 is Gly, Gin, Asn or Lys; R 27 is leu, lie, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gin or Glu; R 28 is ala, Arg or Lys; R 2g is Gin or Glu; R 32 is Leu, His, Gly, Tyr or ala; R 33 is lie, Ser, Asn, leu, Thr or ala; R 36 is Asn, Lys, Orn, Arg, Har or Leu; R 37 is leu or Tyr; R 38 is Met or leu; R 39 is Glu or Asp; R 40 is lie, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly, Asn or Gin; R 41 is lie, ala, Gly, Val, Leu, Nle, Phe, Gin or des R 41 , provided however that at least one of the following residues is present: R 14 is Met, R 20 is His, R 21 is Arg, R 24 is Met, R 26 is Gly, R 32 is Leu, R 33 is lie, R 36 is Asn; or a nontoxic addition salt thereof. 2. A peptide according to Claim 1 wherein R 20 is His. 3. A peptide according to Claim 1 wherein R 36 is Asn. A peptide according to Claim 1 wherein 21 is Arg. 5. A peptide according to Claim 1 wherein R 32 is Leu. A peptide according to Claim 1 wherein R 33 is lie. A peptide according to Claim 1 wherein R 26 is Gly. A peptide according to Claim 1 wherein R 1 is Met. A peptide according to Claim 1 wherein R 24 is Met. 10. A peptide according to Claim 1 wherein R 20 is His and R 26 is Gly. 11. A peptide according to Claim 1 wherein R 20 is His and R 36 is Asn. 12. A peptide according to Claim 1 having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Val-Leu-His-Met-Ala-Arg-Ala-Glu- Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu- Ile-Ile-NH 2 . 13. A peptide according to Claim 1 having the formula: 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-Asn-Leu-Met-Glu- Ile-Ile-NH 2 . 14. A peptide according to Claim 1 having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Arg-Ala-Arg-Ala-Glu- Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu- Ile-Ile-NH 2 . 15. A peptide according to Claim 1 having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Val-Leu-His-Met-Ala-Arg-Ala-Glu- Gly-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu- Ile-Ile-NH 2 . 16. A peptide according to Claim 1 having the formul : H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Val-Leu-His-Met-Thr-Lys-Ala-Asp- Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Asn-Leu-Leu-Asp- Ile-Ala-NH 2 . 17. A composition for lowering the blood pressure of mammals comprising an effective amount of a synthetic CRF peptide or a nontoxic addition salt thereof in accordance with Claim 1 and a pharmaceutically or veterinarily acceptable liquid or solid carrier therefor. 18. A method of modulating the secretion of ACTH and corticosteroids or the secretion of β-END-LI, and other pro-opiomelanocortin gene products which comprises administering an effective amount of the composition of Claim 17.

Inventors: RIVIER JEAN EDOUARD FREDERIC, VALE WYLIE WALKER JR, RIVIER, JEAN, EDOUARD, FREDERIC, VALE, WYLIE, WALKER, JR.
Applicants: SALK INST FOR BIOLOGICAL STUDI, THE SALK INSTITUTE FOR BIOLOGICAL STUDIES
Application year: 1989
Main classification: C07K7
IPC codes: A61K38, A61P9, C07K14