Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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 the hentetraeontapeptide CRF, to analogs of CRF, to pharmaceutical compositions containing CRF or such analogs and to methods of treatment of mammals using CRF or such analogs.
• hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells secretory functions. Factors in hypothalamus increase the rate of ACTH secretion by the pituitary gland.
• a physiologic corticotropin releasing factor (CRF), i.e., ovine CRF (oCRF) was characterized in 1981 and disclosed in U.S. Patent No. 4,415,558 to have the formula:
• Rat CRF(rCRF) has been characterized as 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-Lys-Leu-Met- Glu-Ile-Ile-NH 2 and may alternatively be referred to as rat Amunine.
• the formula of human CRF has apparently been determined to be the same as that of rCRF. Synthetic rCRF and oCRF stimulate ACTH and ß-endorphin activites in vitro and in vivo and substantially lower blood pressure for an extended time period.
• R 8 -R 9 or R 8 or R9 or desQ is Ser, D-Ser or des R 1 ;
• R 2 is Gln, pGlu, Glu, 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 , R 12 , R 19 and R 24 are selected from the group consisting of leu, lie, ala, Gly, Val, Nle, Phe and Gln;
• R 9 is Asp or Glu;
• R 11 is Thr or Ser;
• R 13 is His, Tyr or Glu;
• R 17 is Glu or Lys;
• R 18 is Val, Nle or Met;
• R 21 is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln;
• R 22 is ala, Thr, Asp or Glu;
• R 23 is Arg, Orn, Har or Lys;
• R 25 is Asp or Glu;
• R 26 is Gln, Asn or Lys;
• R 27 is leu, Ile, ala,
• 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.
• 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, ß-endorphin, ß-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 gastrointestinal functions and autonomic nervous system activities.
• CRF analogs may be used for the evaluation of the status of pituitary, cardiovascular, gastrointestinal or central nervous system functions.
• CML L-alanine or C ⁇ CH 3 -L-alanine (CMA) .
• the invention provides analogs of CRF having the following Formula (I) : Y-Q-leu-R 11 -R 12 -R 12 - leu-leu-Arg-R 17 -R 18 -R 19 -Glu-R 21 -R 22 -R 23 - R 24 -R 25 -R 26 -R 27 -R 28 -R 29 -Gln-ala-R 32 -R 33 - Asn-Arg-R 36 -R 37 -R 38 -R 39 -R 40 -R 41 -NH 2 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; Q is R 1 -R 2 -R 3 -R 4 -R 5 -Ile-Ser-R 8 - R 9 or -R 8 -R 9 or R 8 or R 9 or desQ; R 1 is Ser, D-Ser or des R 1 ; R 2 is Gln, pGlu, Glu, D-pGlu or des R 2 ;
• R 28 is ala, Arg or Lys
• R 29 is Gln or Glu
• R 32 is His, Gly, Tyr or ala
• R 33 is Ser, Asn, leu, Thr or ala
• R 36 is 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 Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln
• R 41 is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R 41 .
• R 3 is D-Tyr
• R 4 is D-Pro
• R 28 is Arg
• R 29 and/or R 4Q is Glu
• R 36 is either Arg or Har
• R 37 is Tyr.
• at least one of these two substituents is the residue Glu in the 29- or 40-position.
• CRF agonists have been synthesized which are at least as potent as and often more potent than native CRF.
• These analogs preferably include the following residues having a high alpha-helical forming potential: R 1 is Ser, R 2 is Gln or Glu, R 3 is Glu, R 4 and R 5 are Pro, R 8 is leu, R 11 is Thr, R 12 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 Gln, 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 Ile or Glu.
• R 40 is Ile or Glu.
• One analog which has been found to be particularly potent is:
• AHC for alpha-helical CRF
• 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.
• the 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 peptide.
• a non-human animal may also be used to produce the CRF peptide by gene-farming using such a structural gene and the general techniques set forth in U.S. Patent No. 4,276,282 issued June 30, 1981 or using microinjection of embryos as described in W083/01783 published 26 May 1983 and W082/04443 published 23 December 1982.
• the synthetic CRF peptide is then suitably recovered from the animal by extraction from sera or the like.
• suitable protecting groups which. will prevent a chemical reaction from occurring at that site until the group is ultimately removed.
• X 1 is either hydrogen or an ⁇ -amino protecting group.
• the ⁇ -amino protecting groups contemplated by X 1 are those known to be useful in the art in the step-wise synthesis of polypeptides.
• acyl-type protecting groups such as formyl, acrylyl(Acr), benzoyl(Bz) and acetyl(Ac) which are preferably used only at the N-terminal
• aromatic urethan-type protecting groups such as benzyloxycarbonyl(Z) and substituted Z, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl;
• aliphatic urethan protecting groups such as t-butyloxycarbonyl (BOC) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl;
• cycloalkyl urethan-type protecting groups such as fluorenylmethyloxycarbonyl(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 Gln.
• X 5 is hydrogen or an ester-formmg protecting group for the ß- 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 6 is hydrogen or a protecting group for the side chain amino substituent of Lys or Orn.
• 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.
• 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.
• Met the sulfur may be protected, if desired, with oxygen.
• X 7 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:
• At least one of X, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 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.
• the protecting group should be stable to the reagent and under the reaction conditions selected for removing the ⁇ -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.
• the acyl group at the N-terminal represented by Y acetyl, formyl, acrylyl and benzoyl are preferred.
• the present invention is also considered to provide a process for the manufacture of compounds defined by the Formula (I) comprising (a) forming a peptide having at least one protecting group and having the Formula (II) wherein: X, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each either hydrogen or a protecting group, and X 7 is either a protecting 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
• the peptides are prepared by chemical synthesis, they are preferably prepared using solid phase synthesis, such as that described by Merrifield,
• Such a starting material for rCRF analogs can be prepared by attaching ⁇ -amino-protected Ile to a BHA resin. Ile protected by BOC is coupled to the BHA resin using methylene chloride and dimethylformamide
• 1,2 ethanedithiol 1,2 ethanedithiol.
• the deprotection is carried out at a temperature between about 0°C and room temperature.
• other standard cleaving reagents and conditions for removal of specific ⁇ -amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", 1 pp 72-75 (Academic Press 1965).
• the remaining ⁇ -amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore.
• some of them may be coupled to one another prior to addition to the solid phase reactor.
• coupling reagent N,N'-dicyclohexyl carbodiimide (DCCI) and N,N'-diisopropyl carbodiimide (DICI) .
• DCCI N,N'-dicyclohexyl carbodiimide
• DICI N,N'-diisopropyl carbodiimide
• activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
• suitable activating reagents are carbodiimides, such as N,N'-diisopropyl carbodiimide 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 Cl 2 (1:1) or in DMF or CH 2 Cl 2 alone.
• DMF dimethylformamide
• 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).
• 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.
• 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 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.
• a reagent such as liquid hydrogen fluoride
• anisole or cresole and methylethyl sulfide are included in the reaction vessel as scavengers.
• 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 S-alkylation.
• TFA trifluoroacetic acid
• Example sets forth the preferred method for synthesizing CRF analogs by the solid-phase technique.
• Ile-Ile-NH 2 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: STEP REAGENTS AND OPERATIONS MIX TIMES MIN.
• 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.
• 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 Gln, 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 Gln 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 hhe guanidino group of Arg and the imidazole group of His, and the side chain carboxyl group of Glu or Asp is protected by OBzl.
• Xan may have been partially or totally removed by TFA treatment used to deblock the ⁇ -amino protecting group.
• TFA treatment used to deblock the ⁇ -amino protecting group.
• anisole 0.5 ml. of methylethylsulfide
• HF hydrogen fluoride
• 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. Chromatography (1983) .
• the chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity were pooled.
• the rCRF analog was hydrolyzed in sealed evacuated tubes containing constant boiling HCl, 3 ⁇ l of thioglycol/ml. and 1 nmol of Nle (as an internal standard) for 9 hours at 140oC.
• Amino acid analyses of the hydrolysates using a Beckman 121 MB amino acid analyzer showed the following amino acid ratios: Asx(1.9), Thr(0.8), Ser(3.1), Glx(9.0), Pro (2.1), Ala(3.8), Val (0.9), Met(1.9), Ile(2.6), Leu (7.0), Phe(0.9), Lys(1.0), His(2.0) and Arg(3.0), which confirmed that the 41-residue peptide structure had been obtained.
• H-Ser-Gln-Glu-Pro-Pro Ile-Ser-Leu-Asp-Leu-Thr-Phe-His- Leu-Leu-Arg-Glu-Met Leu-Glu-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.
• EXAMPLE III The synthetic peptide AHC and oCRF were examined for their effects on the secretion of ACTH and ß-endorphin in vitro and was also in vivo.
• the potency of synthetic oCRF to stimulate the secretion of ACTH and ß-endorphin by cultured rat pituitary cells was measured using the procedure as generally set forth in Endocrinology, 91, 562 (1972) .
• Half-maximal responses were observed at about 65 picomolar concentrations of the peptide AHC, while synthetic oCRF concentrations of about 250 picomolar were needed to achieve this response.
• the secretory response to maximal (1-5 nM) concentrations of AHC is at a plateau level.
• the peptide [des Ser 1 -Glu 2 -Glu 3 , Leu 33 , Glu 40 ] -rCRF having the formula:
• His-Leu-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 ß-END-LI and causes a very significant lowering of blood pressure.
• H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Arg-Ala-Glu-Gln-Leu-Ala- Glu-Gln-Ala-His-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 ß-END-LI and causes a very significant lowering of blood pressure.
• 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-Gln-Leu-Ala- Gln-Gln-Ala-His-Leu-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 ß-END-LI and causes a very significant lowering of blood pressure.
• H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala- Gln-Gln-Ala-Tyr-Leu-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 ß-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF.
• the peptide [des pGlu 1 -Gly 2 , Ala 21 , Glu 28 , Met 37 ]-sauvagine 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-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 ß-END-LI and causes a very significant lowering of blood pressure.
• 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-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 ß-END-LI and causes a very significant lowering of blood pressure.
• Example III Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and ß-END-LI and causes a very significant lowering of blood pressure.
• H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-Ala- Leu-Leu-Arg-Glu-Met-Leu-Glu-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. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and ß-END-LI and causes a very significant lowering of blood pressure.
• EXAMPLE XXV The peptide [Leu 12 , Glu 13 , Tyr 37 ] -AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-Glu- Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu- Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Tyr-Leu-Glu-Glu-Ala- NH 2 .
• EXAMPLE XXX The peptide [Glu 22 , Leu 12 , Orn 23 , Har 36 ] -AHC (4-41) 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-Har-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 ß-END-LI and causes a very significant lowering of blood pressure.
• H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Ala- Glu-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 ß-END-LI and causes a very significant lowering of blood pressure.
• 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-Arg- 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 ß-END-LI and causes a very significant lowering of blood pressure.
• EXAMPLE XLI The synthesis of the human CRF (9-41) having the formula: H-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 is conducted in a stepwise manner on a MBHA hydrochloride resin.
• EXAMPLE XLIII The synthetic CRF antagonists from Examples XLI and XLII are examined for their effects on the secretion of ACTH and ß-endorphin in vitro and the synthetic AHC peptide is also examined in vivo.
• the effectiveness of synthetic CRF antagonists to block the secretion of ACTH and ß-endorphin by cultured rat pituitary cells is measured using the procedure as generally set forth in Vale et al., Endocrinology, 91, 562 (1972). In vivo testing is carried out using the general procedure set forth in C. Rivier et al.. Science, 218, 377 (1982).
• the standard antagonist, AHC (9-41) blocks the secretion of ACTH due to 1 nMoCRF by 50%, at a concentration of 197 ⁇ 72 nM.
• the specificity of this inhibition is demonstrated by the finding of no effect of the standard antagonist on the GRF-stimulated secretion of GH, the GnRH-stimulated secretion of LH and FSH or the TRF-stimulated secretion of TSH and prolactin.
• the effects of the antagonist on a number of different concentrations of oCRF and the ability of several different concentrations of AHC (9-41) to inhibit ACTH secretion stimulated by a constant dose of oCRF (1 nM) are considered to demonstrate competitive inhibition.
• CRF antagonists The in vivo effect of CRF antagonists is tested on the spontaneous ACTH release by adrenalectomized rats.
• the iv injection of 3 mg/kg BW (2.7 nmole) causes a marked decrease in plasma ACTH levels (measured as described in Vale et al. Science, 213, 1394, 1981), which is statistically significant for 2 hours.
• the antagonist In the intact, non-anesthetized rats, the antagonist induces a dose-related inhibition of CRF-induced ACTH secretion, which is significant at the 0.09 pmole dose level.
• the antagonist AHC (9-41) also prevents most, but not all, of the ACTH rise due to ether-exposure.
• hCRF has been shown to be a powerful stimulator of ACTH and ß-END-LI secretion in vivo in several rat preparations.
• Plasma levels of ACTH and ß-END-LI are elevated for at least 5-20 minutes following the intraveneous administration of hCRF to nembutal-anesthesized male rats and to quiescent male or female rats with indwelling intravenous cannulae.
• hCRF is found to have a dramatic effect to lower blood pressure in rats and dogs.
• EXAMPLE XLIV The peptide hCRF(8-41) having the formula: H-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 is synthesized. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI. EXAMPLE XLV
• the peptide hCRF(10-41) having the formula: H-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 is synthesized. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE XLVII The peptide [Ala 19 , Thr 22 ] -hCRF (9-41) having the formula: H-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg- Glu-Val-Ala-Glu-Met-Thr-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE IL The peptide [Glu 13 , Val 21 ] -hCRF (9-41) having the formula: H-Asp-Leu-Thr-Phe-Glu-Leu-Leu-Arg- Glu-Val-Leu-Glu-Val-Ala-Arg-Ala-Glu-Gln-Leu-Ala-
• the peptide [Nle 8 , Ser 11 , Leu 33 ]-hCRF(8-41) having the formula: H-Nle-Asp-Leu-Ser-Phe-His-Leu-Leu- Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln- Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LIII The peptide [Acetyl-Asp 9 , Asp 39 ] -hCRF (9-41) having the formula: Ac-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-Asp-Ile-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example XlIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI. EXAMPLE LIV
• the peptide [Lys 23 , Leu 38 ] -hCRF (8-41) having the formula: H-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Glu-Val-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Leu-Ala- Gln-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI. EXAMPLE LV
• Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Leu-Lys-Gln- Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Met-Asp-Thr-Ile-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LIX The peptide [CML 10,15,27,37 , CMA 22,32,41 ]- AHC(9-41) having the formula: H-Asp-CML-Thr-Leu- Glu-CML-CML-Arg-Glu-Met-CML-Glu-Met-CMA-Lys-Ala-Glu-Gln- CML-Ala-Glu-Gln-Ala-CMA-CML-Asn-Arg-Leu-CML-Leu-Glu-Glu- CMA-NH 2 .
• Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LX The peptide [Nle 18,21 ] -AHC (9-41) having the formula: H-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu- Glu-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LXI The peptide [Nle 18,21 ] -AHC (9-41) having the formula: H-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu- Glu-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI. EXAMPLE LXII
• the peptide [Nle 18,21 ] -AHC (8-41) having the formula: H-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- Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 is synthesized. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LXIII The peptide [Glu 13,22 , Leu 12 , Lys 26 ] -AHC (8-41) having the formula: H-Leu-Asp-Leu-Thr-Leu-Glu-Leu-Leu- Arg-Glu-Met-Leu-Glu-Met-Glu-Lys-Ala-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LXIV The synthetic peptide [Ala 13 ] -AHC(9-41) having the formula; H-Asp-Leu-Thr-Phe-Ala-Leu-Leu-Arg- Glu-Met-Leu-Glu-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. Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI.
• EXAMPLE LXV The peptide [Leu 12 , Glu 13 ] -AHC(9-41) having the formula: H-Asp-Leu-Thr-Leu-Glu-Leu-Leu-Arg- Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-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 XLIII shows that it likewise inhibits the secretion of ACTH and ß-END-LI. EXAMPLE LXVI
• the peptide [CML 10,14,19,27,33,38 ]-AHC(9-41) having the formula: H-Asp-CML-Thr-Leu-Glu-CML-Leu-Arg- Glu-Met-CML-Glu-Met-Ala-Lys-Ala-Glu-Gln-CML-Ala-Glu-Gln- Ala-Ala-CML-Asn-Arg-Leu-CML-Leu-Glu-Glu-Ala-NH 2 .
• Testing in accordance with the general procedure set forth in Example XLIII shows that it likewise stimulates the secretion of ACTH and ß-END-LI and causes a very significant lowering of blood pressure.
• CRF agonists 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
• CRF analogs should be useful to stimulate the functions of this axis in some types of patients with low endogenous glucocorticoid production.
• CRF should be useful in restoring pituitary-adrenal function in patients having received exogenous glucocorticoid therapy whose pituitary-adrenalcortical functions remain supressed.
• 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 central nervous system and upon the gastrointestinal tract.
• CRF analogs delivered to the brain should also find application in modifying the mood, learning and behavior of normal and mentally disordered individuals. Because CRF agonists elevate the levels of ACTH, ß-END, ß-lipotropin, other pro-opiomelanocortin gene products and corticosterone, their 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, whereas CRF antagonists could be used to achieve opposite effects.
• CRF increases activity and improves learning performance in rats and thus CRF agonists may function as a natural stimulant.
• CRF antagonists in the brain could ameliorate stress-induced conditions to which endogenous CRF might contribute, including some types of hypertension, infertility, decreased libido, impotentcy and hyperglycemia, as well as to modulate the immune system, gastrointestinal tract and adrenalcortical growth and function.
• 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 dialate the mesenteric vascular bed.
• CRF antagonists may also be of use for decreasing blood flow to the gastrointestinal tract of mammals, particularly humans.
• 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.
• agonists 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.
• administration may be used as a diagnostic tool to evaluate the basis of 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).
• 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.
• 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.
• 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.
• a lower alkyl-substituted amide e.g. 1 to 4 carbons, i.e. methylamide, ethylamide, etc.
• a lower alkyl-substituted amide e.g. 1 to 4 carbons, i.e. methylamide, ethylamide, etc.
• additional amino acid residues can be included at the N-terminal without significantly adversely affecting biological potency.
• Such peptides are considered as equivalents which fall within the scope of the invention.

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