CZ85393A3 - Stabilized active analogs of growth hormone releasing factors - Google Patents

Abstract

Novel GRF peptides with enhanced stability in plasma and aqueous environments at neutral pH.

Description

The invention relates to peptides affecting the activity of the pituitary gland in humans and other animals, particularly mammals. Namely, the present invention is directed to peptides that promote the release of growth hormone by the pituitary. The peptides of this invention are active in vivo, more stable in plasma, and selected peptides are more stable in an aqueous environment at neutral pH than the natural chains of these substances.

BACKGROUND OF THE INVENTION

Physiologists already knew. long ago that the hypothalamus controls activity. adenohypophysis so that the hypothalamus produces special substances that stimulate or inhibit. secretion of various hormones. In 1982, human pancreatic (cancer) factors (hpGRF) were isolated from human pancreatic tumors. They have been purified, characterized, synthesized and found to stimulate the release of growth hormone (GH) by the pituitary. Guillemin R., et al., Science 218,585-585 (1982). Since then, the corresponding GH-releasing hypothalamic factors (GRF) have also been characterized and synthesized in other species, including rats, pigs, sheep, cattle and goats and humans.

Human hypothalamic GRF (hGRF) was found to have the same composition as phGRF. namely: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-LysLeu-Leu-Gln-Asp -1le-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-

G1u-Arg-Gly-A1a-Arg-A1a-Arg-Leu-NH2.

Rat GRF (rGRF) has been found to have a Ser group at position 8 and the formula: H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-TyrArg-Arg-11e-Leu-Gly-G1n -Leu-Tyr-A1a-Arg-Lys-Leu-Leu-Hi-G1u-1e-Met-As n-Arg-G1n-G1n-G1y-G1u-Arg-Asn-G1n-G1u-G1u-G 1n -ArgSer-Arg-Phe-Asn-OH. (See, for example, US Patent 4,595,676).

Bovine GRF (bGRF) was found to have the formula H-Tyr-AiaAsp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-LeuSer-Ala-Arg- Lys-Leu-Leu-Gln-Asp-Ie-Met-Asn-Arg-Gin-Gin-Gly-Glu-Arg-Asn-Gin-Glu-Gin-Gly-A1a-Lys-Va1-Arg-Leu-NH 2.

Porcine GRF has the Ser group at position 28.

It was reported that natural GRF chains are rapidly inactivated by blood plasma enzymes. Rypad is induced by the abolition of the binding of 2-3 peptide by dipeptidyl peptidase, type IV (DPP-IV), which was; formerly also referred to as dipeptide aminopeptidase-IV. Frohman, L.A. aj. : J.Clin. Invest. 78, 906-913 (1986).

The metabolic stability of GRF and various methods of protecting GRF peptides against the effect of dipeptidyl peptidase have been studied, among others, by Felix et al.: Synthesis and biological activity of novel linear and cyclic GRF analogs in Peptides. Chemistry and Biology, Proc. Peptide Symposium, Ed. G.R.Marshal1, ESCOM Sci. Publishers, Leiden, p. 465-467 (1988). They reported that GRF analogs, substituted with desNH 3 -Tyr at position 1 or D-Ala at position 2, have increased stability of the N terminal groups to enzymatic degradation. This information was recently confirmed by Frohman et al., Dipeptidyl peptidase-IV and trypsin-like enzymatic degradation of the human growth hormone releasing hormone in plasma. J. Cli-n. Invest. 83, 1533-1540 (1989). Furthermore, the latter group showed that N-acetylation and N-methylation of the N-terminal tyrosine group or the substitution of D-Tyr-1 in GRF completely inhibited hydrolysis at the 2-3 position. On the other hand, alpha methylation of Tyr-1 only partially prevented the degradation of DFP-IV.

Murphy, W.A. and Coy, D.H., Long-acting alkylated growth hormone releasing factor analogs, Peptide Research 1, 36-41, (1988), disclose GRF analogs that show increased resistance to enzymatic degradation as. consequence of N-alkylation of the lysine side groups in the peptide chain.

Natural strings. GRFs have a residue at position 15 Gly. Analogs with Ala or Leu at position 15 are known to have an increased ability to release GH. See, for example, US Patents 4,649,131 and 4, 73,4,399, as well as L1-i-ng-N-a-Quo-vadis Symposium, Sanofi Group, 29-30. May 1985, Toulouse-Labege (pp. 309-322).

The synthesis of Gly-16SVal / or .alpha.-aminoisobutyric acid also increased the efficacy of GRF analogs, Felix, J. Synthesis and biological efficacy of new analogues of growth hormone releasing factor, in Peptides 1986, Walter de Gruyter. and Co., Berlin-New York, pp. 481-484 (1987).

AM Felix reported on a plan to synthesize analogs with enhanced or prolonged biological efficacy, including the preparation and testing of Ala ¹⁵ h-GRF (1-29) ΝΗϊ and desNHj.-Tyr ¹ , "D-Ala 4, Ala * shGRF (1-29) NH 2 . See, for example, US Patents 4,649,131 and 4,734,399 as well as AM Felix, EP Heimer, TF Mowles, H. Bisenbeis, P. Leung, T.J. Lambros, M. Ahmad, CT Wang and Paul Brazeau: Synthesis and Biological Activity of New Analogs. growth hormone releasing factor, in Peptides

1986, Walter de Gruyter & Co., Berlin-New York, p.

4B1-484 (1987), Felix A.M, Wang C.T, Heimer E., Fournier A., Bolin D., Ahmad. , Lambros T., Mowles T. and Miller L: Synthesis and Biological Activity of New Linear and Cyclic Analogs. GRF in Peptides. Chemistry and Biology, Proc. Peptide Symposium, Ed. G.R.Marshal1, ESCOM Sci. Publishers, Leiden, p. 465-467, (1988), D. Peticlerc, H. Lapierre, G. Pelletier, P. Dubreuil, P. Gaudreau, T. Mowles, A. Felix and P. Brazeau: Effect of Active Analogs of Human Growth Hormone Releasing Factor (.hGRF) for growth hormone release (GH) and milk production in dairy cows. Meeting Abstract P223 82nd Meeting American Dairy Sci. Assn., Columbia,

MO, 21-24 June 1987.

Ser-2 > Modified GRF as well as an additional eight µ *. -'AND··?' án Touém aj .: Amphiphilic analogues of growth hormone releasing factor (GRF): struki

The invention also relates to peptide modifications and biological activity in vivo. Biochem. Biophys. Rés. Commun. 139: 763-770 (1986). This analog has 165V activity in vivo in sheep compared to bGRF (1-44) NH2.

US Patent 4,734,399 discloses GRF analogs having Ala, N-methyl-D-Ala or D-Ala at the 2-position and Ala, Leu, Val, Ile, Nle, Nval or beta-Ala at the 15-position. 4,649,131.

European Patent Application Coy and Murphy, Publication Number 0 198 '214, Application Number 86100127.9 discloses GRF analogs with Leu or Phe at position 2 together with GRF peptides which are substituted at position 2 by various non-naturally occurring L or D-configuration amino acids.

Very low biological activity GRF analogs having Sar2 and Pro2 are described by Coy et al., Strategy for Designing Synthetic Analogs and Growth Hormone Releasing Factor Antagonists, Peptides, Vol. 7, Suppl. 49-52 (1986).

Ling et al.: Analogs of Growth Hormone Releasing Factor Se. potent, antagonistic activity in Peptides, Chemistry and Biology, Proc. Peptide Symposium, Ed. G.R.Marshal1, ESCCM Sc. Publishers, Leiden, p. 484-486, (1988) reported on the series. GRF analogs substituted with either Arg or different D-amino acids at position 2. All are less potent than the parent hormone, and some of them showed antagonistic activity.

The present invention provides synthetic GRF polypeptides having a residue. Ser .na. site of amino acids normally found at positions 8 and 28 of the polypeptides as a means of inhibiting chemical decomposition (deamination) in an aqueous medium. See U.S. Patent Application Serial No. 07-303,518, filed Jan. 27, 1989, and Serial No. 07-323-, 955, issued March 15, 1989. Also, A.R. Friedman, A, K.Ichhpurani, D.M. Brown, R.M. Hillman, L.F. Krabili, R.A. Martin, H.A. Zurcher-Neely and D.M. Guido: The degradation of growth hormone releasing factor analogs in a neutral aqueous solution is explained by the deamination of asparagine residues. Serine replacement of asparagine groups stabilizes. Int. J. Peptide Protein Res. 37, 14-20 (1991).

* 1

The foregoing invention describes synthetic GRF polypeptides having a substituted cysteic acid (Cya) residue instead

..... am.i.no.ky.s.e., 11 i, ny ... v._polpz, e., R3..A. See U.S. Patent Application Serial No. 07-150,301, filed Jan. 29, 1988, and Serial No. 89-00245 of Jan. 27, 1989.

The 29 amino acid analogue * hGRF was designed by G. Velicebi et al. Why. Nati. Aca-. Sci. USA, Vol. 83, 5397-5399 (1986).

It has the first six amino acids at the amino terminus the same and differs from the native peptide by 13 amino acids in the rest of the chain, including the inclusion of the Ser group at position Θ. Amide form and free acid form. analogs have the formula: H-Tyr-AlaAsp-Ala-Ile-Phe-Ser-Ser-Ala-Tyr-Arg-Arg-Leu-Leu-Ala-Gln-Leur

Leu-Ala-Ser-Arg-Arg-Leu-Leu-Gln-Glu-Leu-Leu-Ala-Arg-NH; OH. When tested for their ability to stimulate GH release in primary cultures of rat anterior pituitary cells, the amide analog was 1.57 times more potent than hGRF (1-40J0H, while the free acid form had only 1/6 potency in the same experiment.

Valeaj. (U.S. Patent Application Serial No. 053,23-3, filed May 22, 1987, discloses 31 residual hGRF analogs that utilize a group at position 31 with a side-functionalized lysed chain that can be linked to a particular protein. also have substitutions of other groups that appear in the natural GRF chain, such as Asn or Ser at position 6, Phe at position 10 or Ala-at position 15. Asn or Ser can be at position 28.

It is known that in certain circumstances, Asn groups of polypeptides are deamidated in the presence of water. However, it is not clear what affects deamidation rates. For example, only some Asn groups with a partial Asn-Ser sequence are deamidated in a trypsin polypeptide, while others are not deamidated. See Xossia'koff A.A. Science 240: 191-194 (1988).

European Patent Application No. 86308337.4, Publication No. 0 220 958 discloses a class of compounds having the formula

H-X-Pro-Peptide, wherein X is a naturally occurring amino acid residue, Pro is a naturally occurring amino acid proline, and the Peptide is a chain of amino acid residues defined by a biologically active peptide or protein.

Examples of H-X-Pro-Peptides include Met-Pro (growth hormone releasing factor), which can be chemically converted to GRF.

Peptide analogs that are not GRF and are extended to t

N ends designed for various purposes have also been described. For example: M.A. Tallon ....... aj .: Biochem.26,7767-7774 (1987) synthetically prepared analogs of yeast alpha reproductive factor extended at the N terminus with Ala, Glu-Ala, Ala-Glu-Ala or Glu-Ala-Glu -Ala. These peptides are used in the study of structure-activity relationships.

D. Andreu et al .: 2Oth Eur. Peptide Symp. -Tubingen., Germany ,.

4-9. September. 19.88, Symposium. Abs.tracts, p.33 synthesized whole ,. the amino acid sequence of the precursor form-analogue of cecropin A along with many shorter peptides corresponding to the predicted reaction. Intermediates :. Between. was. the complete cecropin chain extended by Ala-Pro-Gly-Pro at the N-terminus. It was used to prove that the expanding moiety was indeed cleaved by a purified dipeptidylpeptidase-like enzyme preparation.

that, obtained from cocropium cocoons. See also H. Boman and j : J. Biol. Chem. 264, 5852-5860 (1989). • G. Kreil et al.: Eur. J. Biochem. 111: 49-58 (1980) inf.ormu- her , that melittin, a major component of bee The poison is derived

from pro-melittin. The pro-melittin chain consists of six X-Pro and five X-Ala repeated dipeptide groups. Results, presented, KreileirT et al. suggests that conversion of precursors to product may occur by sequential cleavage of the dipeptide units by a dipeptidyl peptidase type IV enzyme. present in the extracts of iodine ..... ________.

- δ -

C. Mollay et al. J . Biochem., 160. 31- 35 (1986): Caeru- lein and xenopsine are two peptides na.lezené in the skin those Xenopus. laevis .. The first has sequence Phe-Ala-Asp-Gly

and a second Ser-Ala-Glu-Ala to extend the N terminal group of their corresponding precursors. Type IV dipeptidyl peptidase, isolated, from frog skin secretions, has the specificity required to cleave these N - terminal extensions. leading to end products.

D, Julius ea Cell, 32,. 839-852 (1983): Saccharomyces cervisiae alpha cells secrete a 13 amino acid peptide as a pheromone alpha factor. Non-populating alpha-cell mutants that do not have membrane-bound dipeptidyl peptidase do not produce normal alpha factor, but exclude a number of imperfectly prepared forms with Glu-Ala-Glu-Alaalfa-factor or Asp-Ala-Glu-Ala-alpha-factor structures that have significantly decreased biological activity. It has been shown that a membrane-bound dipeptiff 'dylpeptidase' is necessary for the normal processing of alpha factor precursors and that this reaction may limit the rate of alpha factor maturation in normal yeast alpha cells.

C.L. Choy et al. J. Biochem., 160, 267-272 (1986): The precursor protein for preventing freezing of the Newfoundland winter flounder contains four repeated sequences of X-Pro and seven 'X-Ala on the terminal N group. Although the reactions of precursors have not been studied, the authors consider that modification of precursors could occur in plasma by effect. a dipeptidyl peptidase-like enzyme that would gradually break down the dipeptide units of the extending moieties and release the antifreeze protein.

Only after the priority date of the basic patent application Suhr et al. Reported the isolation and characterization of the full length cDNA clone encoding GRF mice. Mature mouse GRF was predicted to be a peptide with 42 amino acid groups and a free carboxyl terminus. This peptide has a valine group at position

2. This distinguishes it from GRFs of other species which have all Ala at position 2. See Mol '. - Endocrinology, 3, 1693-1700 (1989).

Based on the considerations of other authors, GRF analogs with the substitution of Leu ¹ - / namely Thr? Ala ¹⁵ Leu ¹⁹ Leu ²⁷ -bGRF (l-29) NHI, trifluoroacetate salt Compound No. 3: Leu .'Leu ^{1 9 27} bGRF (1-29) NH; trifluoroacetate, Ala ¹ 5 ¹⁷ Leu Leu ²⁷ bGRF (1-29) ΝΗ; trifluoroacetate. When tested, in vitro growth hormone release using rat-anterior pituitary cells, Leu ¹⁹ analogs, namely Thr ²¹ Ala ^1E Leu ¹⁹ Leu ²⁷ -bGRF (.1-29) NH 2 trifluoroacetate, compound number 3, Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 trifluoroacetate :, Thr ² Leu ^{1 9} Leu ²⁷ bGRF (1-29) NH 2 i trifluoroacetate Ala ¹⁵ Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 trifluoroacetate significantly less active than the corresponding. Ala ¹⁹ analogs ..

In an in vivo test on growing oxen at a dose of 10 pmol / kg, the amount of GH released by the Leu ¹⁹ analogs was not significantly different (p < 0.05) from the Ala ¹⁹ compounds during the 2 hour follow-up. In monitoring bovine plasma stability, compounds with Leu ^{19 were} more stable than Ala ¹⁹ analogs.

In the GRF molecule, position 19 is in the spindle-like region in a membrane-like environment .: Clore GM, Martin SR and Gronenborn · AH: J. Mol. Biol. 191, 533-561 (1986). It is not known whether the role of the Ala ¹⁹ group in GRF molecules is structural or whether it is a touch-residue receptor. Sato K., Hotta Μ. , / Kagegama J., Chiang T; , Hu HY, Do-ng M-H; a-Ling N.:

Biochem. Biophys. Res. Commun. 149 (2) 531-537 (1987).

Schiffer M. and Edmundson AB: Biophys. J.7,121 (1967), D-Ala substitution ¹ 'for Ala ¹⁵ in hGRF (1-29) ΝΗ; reduced the standard ability to release GH in vitro to 6% analogue of L-Ala ¹⁹ in rat pituitary cells. The same substitution in Nle ²⁷ rGRF (1-29) NH2 reduced efficiency to 10% of the standard in bovine pituitary cells and the same substitution in Nle ⁷⁷ rGRF (1-29) NH? reduced efficacy to 30% of the standard in the rat pituitary cell system: Rivier J .: Second International Symposium on Vasointestinal Peptides and Related Peptides, Cap d'A Agde, France, 1985.

Ser ¹⁷ was as effective as hGRF (1-40) in a standard-day experiment of rat pituitary cells release: Velicebi G., Patthi

S. and Kaiser T.E .: Structure and biological activity of growth hormone releasing factor analogs having potential amphiphilic spindle carboxyl termini. Why. Nati. Aca.

- '4

Sel. ' USA, Vol. 83, 5397-5399 (1986). ,. Getzoff reported that in the myohemerythrin peptide model, Ala, Ile, Ser, and Thr could substitute Val without losing antibody binding, but the Leu-substituted peptide had reduced binding ability. (Getzoff, E.D., Gysin, H. M., Rodda, S. J., Alexander, A., Tainer, J. A., and Lerner, R. A., Mechanism for Binding of Antibodies to Protein. Science, 235, 1191-1196, (1987).

WO 90/15821 (published December 27, 1990, therefore, after the priority date of the present invention) describes various GRF peptides having Thr, Val or Ile groups instead of amino acid residues normally found at position 2, Ala at position 19 in combination with one of the following The amino acid Ala, Val, Leu Ile, or Gly at position 15. The GRF peptide may have a Ser residue at the site of the amino acid residue that normally resides at positions 8 and 28 of the polypeptide.

In addition, the GRF peptides can optionally be extended at the N terminus with C 1 -C 3 alkyls, benzyl, H (YX) _r , or H (YX) _m (Y 1 -X 1) p wherein Y and Y 2 are the same or different naturally occurring amino acids, preferably Tyr or Asp, X and X ¹ , the same or different, are selected from Thr, Ser or Ala, preferably Thr or Ser, n is 1-10, m is 1-5, p is 1-5.

E.F. Heilmer et al. (12th Am. Peptide Symposium, Cambridge, MA, 16-22 June 1991), abstract number 32 in the symposial Program and Abstract reported that Val 2 increases the resistance of -mGRF to digestion of dipeptidyl peptidase IV. Many analogues have been published: hGRF. with modifications in position 2, with emphasis on Val *. In the section ; posters · GRF was also introduced. analogs with Gly *,

I1e ² and Leu ²

SUMMARY OF THE INVENTION

An object of the present invention is a polypeptide that stimulates the release of growth hormone by the pituitary (GRF peptide) having Val or Ile at position 19. The GRF peptide of the invention will have Gly, Thr; Val or Ile residue instead of an amino acid residue that is normally found at position 2 in combination with one of the following amino acids Ala, Val, Leu, Ile, or Gly at position 15. In a preferred embodiment, the GRF peptide may have a Ser residue instead of an amino acid residue. normally located at positions 8 and 28 of the polypeptide. In addition, the GRF peptides of the invention may be N-terminally extended with C 1 -C 6 alkyl, benzyl (XY) (X 1 -Y) _n , where n is Q to. 20., preferably 0 to 10,

X is any naturally occurring amino acid, Y is alanine, serine, threonine or proline and when n = 0, then Y is selected from the group consisting of alanine, serine or threonine,

X 1 is any naturally occurring amino acid except proline or hydroxyproline.

The peptides of the present invention are effective in vivo and are much more resistant to cleavage by blood plasma enzymes than natural GRF chains. Additionally, the compounds are substituted by Ser Ser7 ⁵ and ³ are protected against deamidation in aqueous environments and are chemically more stable.

. The term GRF peptide, as used in the specification and claims, means a known polypeptide having a length of between 27 and 44 residues that causes the release of growth hormone by the pituitary gland. Examples of GRF peptides are the natural or synthetic polypeptides described in US Patents 4,517,181, 4,518,586.

4,528,190, 4,563,352, 4,585,756, 4,595,676, 4,605,643,

4,610,976, 4,626,976, 4,628,043, 4,684) 987, 4,843,064 and in U.S. Patent Application Serial No. 89/00245, filed Jan. 27, 1989. All of which are incorporated herein by reference, Felix

A.M. , Wahg C.T, Heimeer E., Fournier A., Bolin D., Ahmad M., Lambros T., Mowles T. and Miller L: Synthesis and biological activity of novel linear and cyclic GRF analogs in Peptides. Chemistry and Biology, Froc.lth Am. Peptide Symposium, Ed. G.R.Marshal1, ESCOM Sci. Publishers, Leiden, pp. 465-467, (1988). Tou J. S., Kaempfe L. A., Vineyard B. D., Buo-. nomoF.C., Della-Fera M. A., Baile C. A. Amphiphilic Growth Hormone Release Factor (GRF) Analogs: Peptide Structure and In vivo Biological Activity. Biochem. Biophys. Res. Commun. 139, 763-770 (1986). The term GRF peptides includes their non-toxic salts.

The nomenclature used to define GRF peptides is according to Schröder and Lubke: Peptides, Academic Press (1965),

wherein, in accordance with conventional representation, the amino group at the N terminus appears to the left and the carboxyl group to the C terminus to the right. If the amino acid residue has isomeric forms, the L-form is given unless otherwise indicated. The present invention provides GRF peptide analogues having the following formulas: R-R 1 -R 1 -R 1 -Ala-Ile-Phe-Thr-R- Ser-Tyr-Arg-R 1 -R 13 Leu-R 15 -Gln-Leu-R18-Rla-Arg-R; 1-R22-Leu-Gln-R2s-Ile-R27R-2 & -Arg-Gln-Gln ~ Gly-Glu-R ₃ 4 -R ₃ -G 1 nG 1 µR 3 _g -R ₃ -O 4 -Or-Arg-R ₁ Arg-Leu-2, where

R is H, C 1 -C 5 alkyl or benzyl, preferably H 1.

R 1 is Tyr or His, preferably Tyr,

R? is Gly, Thr, Val or lle, preferably Val or lle,

R ₃ is Asp ·, Glu or · Cys, preferably Asp,

R ₈ is Asp or Ser, preferably Ser /

R1 is Lys, N-epsilon-alkyl- or N-epsilon-benzyl-Lys or Arg, preferably Lys or N-epsilon-alkyl or N-epsilon-benzyl-Lys when R is C1-C5 alkyl or benzyl,

R 13 is Val or Ile, preferably Val,

R 18 is Ala, Val, Leu, Ile or Gly, preferably Ala, Val,. Leu or lle, preferably Ala,

Ris is Ser or Tyr, preferably Ser,

.... R.i.q .... j.e .. Val .or ..lle ,. preferably Val,

R 21 'is Lys; N-epsilon-alkyl- or N-epsilon-benzyl-Lys. or Arg, preferably Lys or N-epsilon-alkyl or N-epsilonbenzyl-Lys when R is C1-C = alkyl or benzyl,

R 12 is Ala or Leu, preferably Leu,

R25 Yippee Asp or Glu, preferably Asp, R; 7 Yippee Met, Ile or Leu, preferably Leu R? E Yippee Asn or Ser, preferably Ser, R 3 4 Yippee Ser or Arg, preferably Arg, R 3 5 Yippee Asn or Ser, preferably ASn, R 3 8 Yippee Arg or Gin, preferably Gin, R 3 0 Yippee Gly or Arg, preferably Gly,. P-4 0 Yippee Ala or Ser, preferably Ala, R4 2 Yippee Ala, Wall or Phe, preferably Wall

Z represents a carboxyl-terminus amino acid residue at the C terminus and is a group -COOR _a, ~ _and R- Q, -CONHNHR _a, -CONR a R b or -CHíORa with Ra _and Rb are Ci-Ce alkyl, or hydrogen, or a biologically an active fragment extending from the R- at the N-terminus to any residue at any position between 27 to 44 as its C terminus, optionally their Hse (lactone), HseOH or HseNR _and Rb and their non-toxic salts,

R _a is preferably hydrogen (Η),

Preferably R b is ethyl.

Examples of _{Ci-C, B is} alkyl, are methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl and isomeric forms thereof. The abbreviation iPr refers to isopropyl.

The abbreviation Bzl is used for benzyl.

Some of the possible embodiments of the invention are GRF peptides in which R 1? is Val or Ile including Thr-Ala peptides ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-29) NH;

Thr ² Ala ¹⁵ 11e ¹⁷ Leu ²⁷ bGRF (1-29) NH ₂

Val ² Ala: lele- ³ Leu ²⁷ bGRF (1-29) NH ₂

I.le ² Ala 15i ^{7 7} Leu ^{2 7} bGRF (1-29) NH;

Val ² A1 and ¹⁵ Val ¹⁷ Leu ²⁷ bGRF (1-29) NH2

Ile ² Ala ¹ 'Val ¹ ' Leu2 ⁷ bGRF (1-29) NH ₂

Gly ² Ala ¹ Val ¹ Leu ^{2 7} bGRF (1-29) NH?

Gly ² Ala ¹³ II & amp ^; Leu ²⁷ bGRF (1-29) NHEt preferably peptides

Thr ² Ser ⁸ Ala ¹³ Val ¹ «Leu ^{2 7} Ser ² « Hse ³ ° bGRF (l- -30) _NH2 Thr ² Ser ^with Alai Va1 ¹⁷ Leu ²⁷ Ser ² Hse ³³ bGRF (1- -33) NH ₂ Thr ² Ser and Ala 5VaP «Leu ^{2 7} Ser ^{2 with} Hse ^{3 7} bGRF (1- -37) _NH2 Thr ² Ser «Ala ¹ « Val ¹ «Leu ²⁷ Ser ² « Kse * «bGRF (1- -44) _NH2 Thr ² Ser «Ala ¹ Force ¹ « Leu ^{2 7} Ser ² «Hse ⁸ « 'bGRF (1- -3O) _NH2 Thr ² Ser ⁶ Ala ¹ Strength ⁹ Leu ^{2 7} Twilight ²⁸ Hse ³³ bGRF (1- -33) NH ₂ Thr ² Ser ⁸ Ala ¹ Ile ¹ Leu ²⁷ Ser ² Hse ^{3 7} bGRF (1- -37) NH ₂ or · Thr ² Ser ⁸ Ala ¹ Power ^{1 7} Leu ^{2 7} Ser ^{2 8} Hse ^ bGRF (1- -44) NH _{2 is} even better Ile ² Ser ⁸ Ala ¹ Val Val ¹⁹ Leu ² Ser ² H Hse ³ ° bGRF (1- -3O) NH ₂ - - Thr ² Ser ⁸ Ala ¹³ Val. ¹⁹ Leu ²⁷ Ser ² eHse ³ ° bGRF (1; -3O) NHEt ·. Ile ² Ser «Ala ¹ « Val ¹⁹ Leu ²⁷ Ser ² «Hse ^{3 3} bGRFfl- -33) NH / Ile ² Ser ⁸ Ala ¹² Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1- -33) NHEt Thr ² Ser Ala Ala ¹ Val Val ¹⁹ Leu ^{2 7} Ser ² Ha Hae ^{3 3} bGRF (1- -33) NHEt Ile ² Ser ⁸ Ala ^{1 5} Val ¹ «Leu ^{2 7} Ser ² « Hse · ³ ·. ⁷ bGRF -37) NH ₂ ' I le ² Ser «Ala ^{1 3} Val ¹ « Leu ^{2 7} Ser ^{2 8} Hseý ^ bGRF (1- (44) NH ₂ Val ² Series ⁸ Ala ¹³ Val ¹ «Leu ^{2 7} Series ²⁸ Hse ³ ° bGRF (1- -3O) _NH2 ValíSerSAla ¹³ Val ¹ Le Leu ^{2 7} Ser ² H Hse ^{3 3} bGRF (1- -33) NH ₂ Val ² Ser ⁸ Ala ¹³ Val ¹ Leu ^{2 7} Ser ²⁸ Hse ³⁷ bGRF (1- -37) _NH2 Val ² Ser «Ala ¹³ Val ¹⁹ Leu ^{2 7} Ser ^{2 e} Hse ^{4 4} bGRF (l- -44) _NH2 Ile ² Ser Ala Ala ^{1 I} Ile ¹⁹ Leu ^{2 7} Ser ^{2 8} Hse ³⁰ bGRF (l- · * 30) NH- Ile ² Ser «Ala ¹ « Ile ¹⁹ Leu ^{2 7} Ser ² «Hse ^{3 3} bGRF (1- -33) NH ₂ Ile ² Ser ⁸ Ala ¹ «Ile ^1? Leu ²⁷ Ser ² «Hse ³⁷ bGRF (1- -37) _NH2 1le ² Ser «Ala ¹ « 1le ¹⁵ Leu ²⁷ Ser ² «Hse ^{4 4} bGRF (1- -44) _NH2 Val ² Ser ⁸ Ala ¹ Ile ¹ Leu ^{2 7} Ser ² Hse ³ ° bGRF (1- -3O) _NH2 Val ² Ser ⁸ Ala ^{1 I} Ile ^{1 9} Leu ^{2 7} Ser ² H Hse ^{3 3} bGRF (.1- -33) NH ₂ Val ² S er ⁸ Al a- ¹ «11 e ¹ « Leu · ^{2 7} S er ² '«Hs e ^{3 7} bGRF (1 = 37) N H- ₂ -

Val 'Ser and Ala * 11 ei ⁹ Leu ^{2 7} Ser ^{2 3} Hse «<bGRF (1-44) NH ₂

Val ² Ser ⁶ Ala ^{1 5} Ilei ⁹ Leu ²⁷ Ser ^2e Hse ³⁷ bGRF (1-37) NH-n-propyl or non-toxic salts thereof

The best GRF peptide of the invention is Val 2 SeraAlailaval ¹ 'Leu ²⁷ Ser 2 SHsesBGRF (1-30) NHEt.

Another embodiment of the invention is peptides spread at the N terminus. Among them are, for example, N-alpha (Tyr-Ala-Phe-Pro-Phe-Ala) Tyr ¹ Thr ² Ser ³ Ala ¹³ 1le ¹⁷ Leu ²⁷ Ser ^2e bGRF (1-29) NH ₂ '.

, Ν-al fa (Leu-Gly-Pro-Tyr-Ala) Tyr ¹ Thr ² Ser ³ Ala ¹ - Bag ⁷ Leu ^{2 7} Ser ²⁸ bGRF (1-29) NH?

N-alpha- (Ala-Fro-Gly-Pro-Tyr-Ser) ₂ Tyr ¹ Val ² SerSAla ¹³ Ile ¹⁹ Leu ²⁷ Ser ^2E Hse ³²⁷ bGRF (1-32) NH ₂ 4 '

N-alpha (Leu-Pro-Tyr-Ala-Tyr-Ala) Tyr ¹ Ile ^ er ^ Ala ¹ Muscle ¹⁹ Leu ² T Ser ² 8bGRF (1-29) NH ₂

N-alpha (His-Ala-Tyr-Pro-Tyr-Ala) Tyr ¹ Ile ² Ser ^f Ala ¹³ Ile ¹⁹ Leu ^{2 73} 'Ser ²⁶ bGRF (1-29) NH 2 -i

N-alpha (Glu-Pro-Phe-Ala-Tyr-Pro-His-Ala) Tyr ¹ 1e ² SersAla ¹³ Val ¹⁹ Leu ²⁷ Ser ^2E bGRF (1-29) NH ₂

N-alpha (His-Pro-His-Pro-His-Ala-Tyr-Ala) Tyr ¹ Thr ² Ser ⁸ Ala ¹³ Val ¹⁹ Leu ²⁷ Ser ² «bGRF (1-37) NH ₂

N-alpha (Tyr-Ala-Gly-Pro-Leu-Pro-Phe-Ala) Tyr ² IleSerSAla ¹³ Val ¹⁹ Leu ²⁷ Ser ² ®Hse ³² bGRF (1-32) NH ₂

N-alpha (Val-Pro-Arg-Pro-Phe-Pro-Tyr-Ala) Tyr ¹ 1le ² Ser ^with Ala ^{2 3} Val ¹⁹ Leu ²⁷ Ser ^2s Hse ³³ bGRF (1-33) NHEt

N-alpha (Arg-Pro-Tyr-Ala-Ile-Pro-Phe-Ala) Tyr ¹ IlerSer ^ Ala ¹³ Val ¹⁹ Leu ^{2 7} Ser ^{2 3} Hse ³ <> bGRF (1-30) NHEt

N-alpha (Tyr-Ala) ₄ Tyr ¹ Val ² Ser8Al ³³ Val ¹⁹ Leu ^{2 7} Ser ^{2 E} Hse ^{3 4} t

bGRF (1-34) NHCH _p

N-al fa (11 e-Pro-G lu-A1 1 and-Tyr-Ala) Tyr ¹ 11 eSer ^ε Ala ^{1 5} Val ^{1 9} Leu ^{1 7}

Ser ^{7 with} bGRF (1-37) NH 2

Further embodiments of the invention are any of the above modifications in which Asp at position 3 and i or 25 is replaced by Cya, preferably at position 3.

Compounds with Val @ ⁹ and Ile ¹⁹ according to the present invention are as active (and sometimes more active) in releasing GH in vitro (rat pituitary cells) than their corresponding counterparts Ala ¹⁹ (Table I and Figure 1). However, they are more stable to proteolysis when incubated in bovine plasma. Evidence of increased metabolic stability of the compounds of this invention (as compared to the natural GRF chain represented by Leu ⁷⁷ bGRF (1-29) NH 2) is shown in Table II, which summarizes in vitro stability data. The compounds of the invention are more active in vivo at 10 pmol / kg than the Leu ¹⁹ analogs, see Figure 2 and generally release more growth hormone with a more stable effect than Ala ¹⁹ and. Leu ¹⁹ analogs in castrated bulls at 30 pmol / kg (Ta-.

Table III). In Figures 1 and 2 (compounds were used as trifluoroacetate salts) T2 means Thr ¹ , A15 means Ala ¹ /

L27 means Leu ¹⁷ , L19 means Leu ¹⁹ , V19 means Val ¹⁹ and 1119 means Ile ¹⁹ .

. The increased efficacy of the compounds of the invention wherein R is Val or Ile is shown in Table III and Figure 2.

For example, one skilled in the art will recognize from relative in vivo efficacy (at a dose of 10 pmol / kg, Figure 2). that Sersale ^{Thr-Val-Leu-1 '7} sbGRF Ser ¹ (1-29) _NH2, trifluoroacetate and ¹ Sersale Thr → Ile ¹⁹ Leu ⁷⁷ Ser 8bGRF (1-29) NH; trifluoroacetate, as well as Val ¹⁹ and Ile ¹⁹ compounds (at 30 pmol / kg, Table III), are more bioactive 18 than native GRF.

For commercial production purposes, the carboxyl terminus of homoserine, lactone or homosecrine amide, optionally secondary or tertiary C 1 -C 8 alkyl amide of homoserine (preferably C 1 -C 6 -alkyl amide) is preferred.

Synthetic analogs of GRF peptides are prepared by a suitable method, including those described in US Patent 4,529,595 (column 2, line 35 to column 5, line 64) and US Patent 4,689,318 (column 2, line 23 to column 9, line 13), both are incorporated herein by reference.

Procedure A is one method for preparing GRF analogs of the invention.

PROCEDURE.

Peptides were synthesized by solid phase technique in an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, California) using synthetic cycles supplied by the same company. Boc amino acids and other reagents were supplied by Applied Biosystems and other commercial suppliers. Sequential was used; double coupling protocol to. The starting P-methylbenzhydrylamino resin for the synthesis of the C terminal carboxylic acid amides. The corresponding PAM resin was used to synthesize the C terminal acid. Asparagine, glutamine and arginine are coupled using preformed symmetrical Boc amino acid anhydrides.

The following branch chain protections are used:

Arg, tes1

Asp, benzyl

Cys, 4-methylbenzyl

Glu, benzyl _ΐχ ·.

Ser, benzyl

Thr, benzyl

Tyr, 4-bromocarbobenzoxy Lys, 4-chlorocarbobenzoxy.

Removal of Boc protection is achieved with trifluoroacetic acid (TFA) in methylene chloride. If Hse analogs are to be obtained, it is incorporated by means of the solid phase Met and then modified with cyanogen bromide after cleavage of HF by well-known methods. Cleavage with cyanogen bromide converts Met into a peptide with Hse lactone at the C terminus. This can be converted to the Hse amide peptide by the action of a suitable amine in a solvent such as methanol or dimethylformamide. Analogues with Hse lactone, HseOH, HseNR, _and Rt · at the C terminus can be prepared by the methods described by Kemp et al. BIOTECHNOLOGY, Vol. 4, pp. 565-568- (1986). Upon completion of the synthesis, the peptides are deprotected and separated from the resin by anhydrous hydrogen fluoride containing ~ 10% p-cresol. The cleavage of the protecting, branching and peptide groups from the resin is carried out at 0 ° C or below, preferably at -20 ° C for thirty minutes, followed by thirty minutes at 0 ° C. After removal of the HF, the peptide resin is washed with ether and the peptide extracted with glacial acetic acid and lyophilized. Before cleaning cysteine contained in the crude peptides oxidized ^to: the corresponding acid with formic peracid at -10 ° C to 10 ° C, cSt, preferably at O DEG C. as described in Stewart et al. Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford , Illinois, 1984, p., 113 .. Conversion to Hse · lactones and. Hse amides at the C terminus have been described above.

Purification is performed by ion exchange chromatography on a cationactive column of Synchroprep S-3GG (SynC-thunder - Inc. Lir.den,

Indiana). The peptide is applied using a buffer of 20 ml of 1M TRIS (pH 6.8) in 20% acetonitrile and eluted using a gradient of 0-0.3 molar sodium chloride in the same solvent. C-18 (Separations Group, Hesperia, California) using water: acetonitrile gradients, both containing 0.1% TFA. The appropriate fractions were combined and lyophilized. The desired GRF peptide is obtained in the form of the trifluoroacetate salt. Trifluoroacetate can be converted to other salts, if desired, by known ion exchange methods.

The peptides are hydrolyzed for 24 hours under vacuum in a vapor phase in a Pico-Tag Work. Station (Waters) using stably boiling HCl (Pierce) in the presence of phenol as a purifying agent at 1100C. The hydrolysates were analyzed on a Beckman Amino Acid Analyzer, Model 6300. The peptide composition was calculated using a known concentration of norleucine as an internal standard.

Mass spectra were obtained using an Applied Biosystems Time of Instrument. Flight Mass Spectrometer.

Examples

Example 1 '

Preparation of Thr ² Al and ¹ 'Val ¹ · Leu ^{2 7} bGRF (1-29) NH; Trifluoroacetate, Compound No. 1;

Synthesis of an analogue of the GRF peptides having the formula: H-Tyr-Thr-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAla-Gin-Leu-Ser-Val-Arg-Lys -Leu-Leu-Gln-Asp-Ie-Leu-Asn-Arg-NH2 (as trifluoroacetate) is carried out sequentially according to Procedure A. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.06 (4), Thr.157 (1), Ser 1.64 (2), Glu 2.10 (2),

Ala 2.25 (2), Val 2.00 (2), Ile 1.68 (2), Leu 5.15 (5), Tyr

1.88 (2), Phe 0.94 (1), Lys 2.00 (2), Arg 2.94 (3).

Example 2

Preparation ¹⁵ Thr Ala Val Leu ^{19? 7} bGRF (l-29) NH2 triíluoracetátu, Compound No. 2 '

Synthesis of a GRF peptide analog having the formula: H-Tyr-Thr-Asp-Ala-11e-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAl-G1n-Leu-Ser-11e-Arg-Lys -Leu-Leu-Gln-Asp-Ie-Leu-Asn-Arge-NH ₂ (as trifluoroacetate) is carried out sequentially according to procedure A. Results of amino acid analysis, theoretical values in parentheses: Asp. 4.09 (4), Thr.l., 98 (2), Ser 1.64 (2), Glu 2.05 (2), Ala-2.25 (2), Val 1.0 (1), Ile 2.84 (3), Leu 5.07 (2) 5), Tyr. 1.87 (2), Phe 0.94 (2), Lys 19.97 (2), Arg 2.94 (3).

Example 3

Preparation ThrAAla ^{1 5} Va.l ¹⁹ Leu ^{7 7} bGRF (1-29) NH; Trifluoroacetate Compound # 3

Synthesis of GRF analog peptide having the formula: H-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAl-G1n-Leu-Ser-Leu-Arg-Lys -Leu-Leu-Gln-Asp-Ie-Leu-Asn-Arge -NH ₂ (as trifluoroacetate) is carried out sequentially according to procedure A. Results of amino acid analysis, theoretical values in parentheses: Asp 4.08 (4), Thr.1.87 (2 ), Ser 1.72 (2), Glu 2.07 (2), Ala 1.93 (2), Val 1.04 (1), Ile 1.88 (2), Leu 6.11 (6), Tyr 1.90 (2), Phe 0.93 (1), Lys 2.03 (2), Arg 3.09 (3).

Example 4 '

Preparation of Val ^ Ala ¹ ^ Leu ¹⁹ Leu ^{; 7} bGRF (1-29) NH? trifluoroacetate compound 4.

Synthesis of GRF analog peptide having the formula:

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr- Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Leu-Arg-Lys-Leu-Leu-Gln-Asp- 11e-Leu-Asn-Arg-NH; (as trifluoroacetate) was performed sequentially according to Procedure A. Mass spectral analysis (method. Cf252 plasma desorption) m / z for (M + H) ⁺ : observed 3454.6, theory

Results of amino acid analysis, theoretical values in brackets .:. Asp 4.01 (4), Thr 0.93 (1), Ser 1.68 (2), Glu 2.03 (2), Ala 2.00 (2), Val 1.85 (2), Ile 1.88 (2), Leu 6.11 (6), Tyr 1.94 (2)., Phe 0.94 (1), Lys 1.97 (2), Arg 3.11 (3).

Example 5

Preparation 11e-Alai ⁵ Leui ⁹ Leu ²⁷ bGRF (1-29) NH 2 trifluoroacetate, Compound No. 5. .

'He knows ¹

Synthesis of GRF peptide analog; having the formula: H-Tyr-Ie-Asp-A1a-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu- A1a-G1n-Leu-Ser-Leu-Arg-Lys -Leu-Leu-Gln-Asp-Ie-Leu-Asn-Arg -NH ₂ (as trifluoroacetate) is carried out sequentially according to the procedure and

A. Mass spectral analysis (by Cf252 plasma desorption method) m / z for (M + H) ⁺ : observed 3468.6, theory 3466.1. Results of amino acid analysis, theoretical values in brackets: Asp

4.02 (3), Thr 0.94 (2), Ser 1.67 (2), Glu 2.05 (2), Ala 2.00 (2) . Wall 0.94 (2), Ile 1.83 (2), Leu. 6.12. (6), Tyr 1.94 (1), Phe 0.95 (1), Lys 1.99 (2), Arg. 3.13 (3). Example 6 Preparation Wall -Alai sile * 'Leu27bGRF (1-29) NH ₂ trifluoroacetate,

Compound 6.

Synthesis of GRF peptide analogue. having the formula: H-Tyr-Val-Asp-Ala-11e-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAT and -G1n-Leu-Ser-11e-Arg-Lys-Leu- Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH; (such as trifluoroacetate) is carried out sequentially according to the procedure

A. Mass spectral analysis (Cf252 plasma desorption method) m / z for (M + H) ⁺ : observed 3453.4, theory 3452.1. Result23

amino acid analysis, theoretical values in brackets: Asp 4.03 (3), Thr 0.94 (2), Ser 1.69 (2). Glu 2.03 (1), Ala 2.01 (1), Wall 1.89 (1), Ile 2.85 (5), Leu 5.11 (5), Tyr 1.95 (1), Phe 0 : 95 (1), Lys 1.99 (2), Arg 3.08 (3). Example 7 Preparation Ile ² Ala ¹ ^ Ile ² -Leu ²⁷ bGRF (1-29) NH ₂ trifluoroacetate,

Compound No. 7.

Synthesis of GRF peptides analogue having the formula:

H-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser.-11 e-Arg-Lys-Leu-Leu-Gln- Asp-Ie-Leu-Asn-Arg -NH? (such as trifluoroacetate) is carried out stepwise according to procedure A.. Mass spectral analysis (by Cf252 plasma desorption) m / z for (M + H) ⁺ : observed 3469.7, theory 3466.1. Results of amino acid analysis, theoretical values in parentheses: Asp 4.03 (4), Thr 0.94 (1), Ser 1.69 (2), Glu 2.06 (2), Ala

2.04 (2), Val 0.94 (1), Ile 3.78 (4), Leu 5.11 (5), Tyr

1.94 (2); Phe 0.95 (1); Lys 1.98 (2), Arg 3.09 (3).

Example 8

Preparation of Val ² Ala ⁷⁵ Val ¹⁷ Leu ²⁷ bGRF (1-29) NH ₂ trifluoroacetate, Compound No. 8.

Synthesis of GRF Peptide Analog having the formula: H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAia-Gn-Leu-Ser-Va1-Arg-Lys -Leu-Leu-G1 η-Asp-11e-Leu-Asn-Arg -NH? (as trifluoroacetate). Mass spectral analysis (Cf252 plasma desorption method) m / z for (M + H) ⁺ : observed 3440.6, theory 3438.1. Aria results The same amino acid y, theoretical values in parentheses: Asp

4.02 (3), Thr 0.93 (2), Ser 1.67 (2), Glu 2.03 (1), Ala 2.00 (1), Wall 2.78 (2), Ile 1.86 (2), Leu 5.09 Tyr 1.92 (1), Phe 1, .04 (1), Lys 1.98 (2), Arg3 .05 (2) *

Example 9

Preparation IIe2Ala ¹³ Val ¹⁷ Leu ⁷⁷ bGRF (1-29) NH ₂ trifluoroacetate, compound no. 9.

Synthesis of GRF peptide analogue having the formula:

H-Tyr-Ile-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-LeuAla-Gn-Leu-Ser-Val-Arg-Lys-Leu-Leu -Gln-Asp-Ie-Leu-Asn-Arg -NH; (as trifluoroacetate) was carried out sequentially according to procedure A. Mass spectral analysis (by Cf252 plasma desorption method) m / z for (M + H) ⁺ : observed 3454.9 theory 3452.1. Results of amino acid analysis, theoretical values in brackets: Asp

4.02 (3), Thr 0.93 Ser 1.69 (1), Glu 2.04 (1), Ala 2.06 (1), Wall 1.86 (1), Ile 2.84 (2), Leu 5.10 Tyr 1.94 (1), Phe 0. .94 (1), Lys 1.98 (2), Arg 4, .07 (2) !>

Using sequential synthesis according to procedure a, the following peptides can also be prepared:

Ile? SerSAlai-Val 1'Leu2 ^{S * 7} Ser28Hse ^{3 *} ° bGRF (1-30) NH ₂

^{3 2} Ile Ser Ala Val ^{13 17 7} Leu2 Ser2eHse ³³ bGRF (1-33) _NH2

Ile ² Ser®Ala ¹ Wal1- Leu ²⁷ Ser ⁷ f Hse ³⁷ bGRF (1-37) NH ₂

Ile ^ Ser ^ Ala ¹⁵ Val ¹ Leu ^{2 7} Ser- ^with Hse < ⁴ bGRF (1-44) NH ₂

VaPSerSAlai ³ Val ^{3 7} Leu ^{2 7} Ser ² * Hse ³ ° bGRF (1-30) NH ₂

Val 2SersAla 1 ³ Val 1 ⁷ Leu ^{2 7} Ser2 ³ Hse ^{3 3} bGRF (1-33) NH;

Val 2SersAla1 ³ Val ¹³ Leu ^{7 7} Ser2eHse ³⁷ bGRF (1-37) NH ₂

Val ² Ser ³ Ala ¹³ Val ¹ 7Leu2 ⁷ Ser2 & Hse44bGRF (1-44) NH2 e 2 Ser ^s Al.a ¹⁵ 11 e ^{1 7} Leu ^{3 7} Ser 2 ³ Hse ³ obGRF (1-30) NH ₂

IleíSerSAla ¹ Ule ¹ ^ Leu ^{3 7} Ser 2 & Hse ^{3 3} bGRF (1-33) NH ₂

Rle ^ SerSAla ¹ ^ Ile ^{1 7} Leu'2 ⁷ Ser ^{2 e} Hse ³⁷ bGRF (1-37) NHj

Ile ² Ser 'Ala ^{1 3} Ile ³ ? Leu ^{2 7} Ser ²⁸ Hse ⁴ bGRF (1-44) NH _; ·

Val ² Ser ⁸ Ala ³⁸ Ile ³ 'Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-3O) NH?

Val ² Ser ⁸ Ala ³ 511e ⁷ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NH 2

Val ² Ser ⁸ Ala ³ 5He? Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2

Val ² Ser ⁸ Ala ³³ IleiLeu ²² Ser ^{2 B} Hse ^{4 4} bGRF (1-44) NH ₂

Thr ² Ser ⁶ Ala ^{3 3} Val ^{3 7} Leu ²⁷ Ser ²⁸ Hse ^3d bGRF (1-30) NH ₂

Thr ² Ser ⁸ Ala ³ Val ^{3 7} Leu ^{2 7} Ser ^{2 8} Hse ^{3 3} bGRF (1-33) NH ₂

Thr ² Ser ⁸ Ala ³ Val ^{2 7} Leu ^{2 7} Ser ^{2 8} Hse ^{3 7} bGRF (1-37) NH ₂

Thr ² Ser ⁸ Ala ³ Val 1 ⁷ Leu ^{2 7} Ser ^{2 8} Hse ⁴⁴ bGRF (1-44) NH ₂

Thr ² Ser ⁸ Ala ^{3 3} Ile ^{3 7} Leu ^{2 7} Ser ^{2 8} Hse ³ ° bGRF (1-30) NH ₂

Thr ² Ser ⁸ Alai ³ Ilei'Leu ²⁷ Ser ²⁶ Hse ³³ bGRF (1-33) NH ₂

Thr ² Ser ⁸ Ala ³ Ile ² ? Leu ²⁷ Sep ²⁸ Hse ³⁷ bGRF (1-37) NH ₂

Thr ² Ser ⁸ Ala ^{2 3} Ile ^{3 7} Leu ^{2 7} Ser ^{2 8} Hse ^{4 4} bGRF (1-44) NH ₂

Thr ² Ser ⁸ Ala ³³ Val ³ ? Leu ²⁷ Ser ²⁸ Hse ³⁸ bGRF (1-30) NHE »

Thr ² Ser ⁸ Ala ³³ Vali ^with Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NHEt

Ile ² Ser ⁸ Ala ³³ Val ^{3 7} Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-3O) NHEt

Ile ² Ser ⁸ Ala ^{3 3} Val ^3? Leu ^{2 7} Ser ^{2 e} Hse ^{3 3} bGRF (1-33) NHEt. '

Val ² Ser ⁸ Ala ^{3 3} 1 le ^{3 7} Leu ^{2 7} Ser ^{2 8} Hse ^{3 7} bGRF (1-37) NH-n-propyl

Procedure B.

In addition to the preparation of GRF analogs by the solid phase technique, these may be. analogues obtained by the recombinant DNA method according to the procedure described for Leu ²⁷ bGRF (1-44) OH (European patent application 0212531) with the following modifications in the DNA segment encoding Leu ²⁷ bGRF (1-44) OH are codons for Ala ² , Asn ⁸ , Gly ³³ and Asn ²⁸ replaced by codons: for example (ACT) Thr ² or (ATT) Ile ² or (GTT) Val ² ', (AGT)' Ser ⁸ , (GCT) Ala ³³ , (AGT) Ser ^'28 .

In addition, for GRF analogs containing Cya ³ , the GAT codon (Asp ³ ) is replaced by the TGT codon for Cys ³ . After the protein has been formed and reacted with cyanogen bromide in formic acid as described in the above-mentioned European patent application, hydrogen peroxide is added to the solution at about 0 ° C to oxidize the residue of Cys to Cya.

The peptide is then purified by the methods described.

In addition, for N-terminally extended GRF analogs, DNA segments encoding the N-terminal extension are added as follows: (TATACT) for .Tyr-Thr,. (TATACT) _n for. (Tyr-Thr) _n , (TATAGT) _n for (Tyr-Ser) _n or (TATAGTTATACT) for Tyr-Ser-Tyr-Thr or (TATACTTATAGT) for Tyr-Thr-Tyr-Ser, (GATGCT) for Asp-Ala etc. The precursor protein gene is inserted into the E. coli expression vector. After the protein has been formed, isolation from the material and decomposition with cyanogen bromide in formic acid as described in said European patent application is carried out. ''

The compounds of the invention can be prepared by known recombinant DNA techniques, since these peptides are composed only of naturally occurring amino acids. This is in contrast to known analogs containing uncoded DNA groups such as D-Ala. and or desaminoTyr, and will require expensive chemical synthesis or a combination of genetic engineering with chemical processes for any large-scale production.

Recombinant starting microorganisms used in this invention is prepared by recombinant DNA techniques, _t které.jsou well known to practitioners and are described for example in T. Maniatis :. Molecular, Cloning, Cold Spring Harbor Laboratory (1982) and in B. Perbal, A Practical Guide to Molecular Cloning, John Wiley, New York. (1984). These books are included in the description by this reference. ,

Analogues with Hse lactone, HseOH, HseNR _é Rb at the C terminus can be prepared by methods described by Kemp et al. BIOTECHNOLOGY, Vol.

4, pp. 565-568 '(1986).

PROCEDURE

This procedure relates to nitrogen-alkylated GRF analogs of the invention. The peptides are prepared either by chemical or biochemical methods (procedure A or B). N-alkylation is then achieved by known methods, e.g., Murphy, W.A. and Coy, D.H., Long-term alkylated growth hormone-releasing factor analogs, Peptide Research 1, 36-41, (1988), Tythyamoorthy et al., Reductive methylation of botulinum neurotoxins. Cell. Biochem. 83, 65-72 (1988).

The following peptides can also be prepared by Procedure C: N-epsilon-iPr-Tyr ¹ Thr ² Ser ⁶ N-epsilon-iPrLys ¹ ' ² > - ³ Val ¹ sile ³ ' Leu? ⁷ Ser ^2e bGRF (1-29) NH 2

^{N-epsilon-IFR-3} Tyr Thr Ser ^{8 2} N-.epsilon.-iPr-Lys ^{32-23 3} Ala-Val ³ 'Leu ²⁷ bGRF (l-40) _NH2

^{N-epsilon-BZ1-3} Tyr Ser Thr ^{2 5} N-epsilon-BZ1-Lys ^{3 2-21} Val ³ 'Ile ³⁹

11e ²⁷ Ser ^{2 8} bGRF (1-32) NH 2

N-epsilon-iPr-Tyr ³ 11e ² Ser ^ N-epsilon-ipr-Lys ¹² > ²³ Ala ³ $ Val ¹⁵ Leu ²⁷ Ser ² ebGRF (1-40) NH;

N-epsilon-Bz1-Tyr ³ Val ² SerθN-epsilon-Bz1-Lys ^{3 2} - ^{2 3} Val ^{3 5} Val ^{3 7} e ^{2 7} Ser ² f bRF (1-32) NH .;

All synthetic GRF peptides of the invention, including those prepared in the examples, are considered to be biologically active and useful for stimulating the release of GH by the pituitary.

Doses between about 10 nanograms and about 5 micrograms of these peptides per kilogram of body weight are considered to be particularly effective for inducing GH secretion. Stimulation of GH secretion by these peptides should result in a corresponding increase in growth in humans, cattle and other animals with normal levels of GH. In addition, administration should alter the body fat content and modify other GH-dependent metabolic, immunological and developmental processes. For example, this may be. analogs useful as agents for stimulating anabolic processes in humans in conditions similar to the effects of burns. In another example, these analogs may be administered to commercial warm-blooded animals such as chickens, turkeys, pigs, goats, cattle and sheep, and may be used in fisheries for fish and other cold-blooded marine animals such as sea turtles and eels, clay > L · and amphibians to accelerate growth and increase the protein to fat ratio obtained by administering effective doses of the peptides. These analogs can be used to stimulate immune functions in humans and animals to treat diabetes resulting from abnormal growth hormone production, or to improve the healing of bone, wounds and burns, or osteoporosis. You can also use these »

analogues for stimulating hair growth.

Daily doses of between 10 nanograms / kg and about 50 micrograms / kg body weight are considered to be particularly effective for enhancing lactation, growth and stimulation of immune functions.

For administration to humans and animals, these synthetic peptides should have a purity of at least about 93%, and preferably at least 98%. These synthetic peptides or non-toxic salts thereof, combined with a pharmacologically or veterinarily acceptable carrier in the form of a pharmaceutical composition, preferably for sustained release, are administered to animals and humans either intravenously, subcutaneously, through the skin or nasal mucosa. releasing GH when the patient to be treated needs such therapy. The appropriate dosage will depend on the state of health and the duration of treatment desired.

Such peptides are often administered in the form of non-toxic salts, such as acid addition salts or metal complexes, for example with zinc, iron, and the like (they are considered to be salts for use). Addition salts include hydrochloride, hydrobromide, sulfate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc. When the active ingredient is to be administered in isotonic solution, the salts may affect phosphate and other buffers.

The peptides should be administered to humans under the supervision of a physician, and the pharmaceutical compositions will usually contain the peptide together with a conventional, solid or liquid pharmaceutically acceptable carrier. The basic dose will generally be from about 100 nanograms to about 50 micrograms of peptides per kilogram of body weight of the patient.

Although the invention has been described with respect to the preferred forms, it will be understood that various changes and modifications can be made to those skilled in the art without departing from the scope of the claims. For example, you can. to modify the peptide chain by omitting one or two residues from the C-terminus of the peptides according to currently known experimental practice, resulting in peptides that retain much of their biological activity. Therefore, such peptides are within the scope of this invention. In addition, the C-terminus and / or N-terminus of the peptides may be substituted, or naturally occurring residues may be substituted for their general equivalents, as is generally known in peptide chemistry. For example, other analogs having increased resistance to proteolysis and having at least a substantial part of the activity of the polypeptides of claim 1 can be produced without, however, being produced. Also known substitutions of the carboxyl group at the C-terminus, e.g. lower alkyl amides, lead to equivalent molecules.

The methods described above can be used to produce GRF peptides of the general formula

Ri-Ri-R ¹ 2 ^- R 3 "Ala-Ile-Phe-Thr-RS-Ser-Tyr-Arg-R ¹ and _from -Ri3-LeuR15 -θ1n-Leu-Ri5 -R1g-Arg-R 21 -R 2 2 — Leu — G1n — R2 5 -Ile — R; 7 —R23 -

Arg — G1n-G1n — G1y — Glu — R3 4 —R3 3 — Gin — Glu — R3 g —R3 9 — R4 o —Arg —R2 “Arg-Leu-Z, where

R 1 is (XY) - (X 1 -Y) _n 5 n 0-20, preferably 0-10 and X is a naturally occurring amino acid, Y is alanine, serine, threonine or proline and when n = 0 then Y is selected from the group consisting of alanine, serine or threonine; X 1 is any naturally occurring amino acid except proline or hydroxyproline. R 1 is Tyr or His, '

R2 is Gly, Thr, Val or Ile,

Ra is Asp, Glu or Cya;

Rs is even Asn or S er, Rx 2 is Lys or Arg, Ri 3 is Wall or Ile, Ri 5 is Ala , Val , Leu, Ri s, is Ser or Tyr, Ri 9 is Wall or Ile, R; 1 is Lys or Arg, R2 2 is Ala or Leu,

R 2 5 Yippee Asp, Glu or Cya, R 2 7 Yippee Met, Ile or Leu, R2 8 Yippee Asn or Ser, R3 4 Yippee Ser or Arg, R3 3 Yippee Asn or Ser, R3 8 Yippee Arg or Gin, R 3 9 Yippee Gly or Arg, R 4 0 Yippee Ala or Ser, R4 2 Yippee Ala, Wall or Phe,

Z represents the carboxyl terminus of the amino acid residue at the C terminus and is -COOR _a , -CR _and O, -CONHNHR _a , -CONR _and Rt or -CH 2 R a with R _a and R b, which are C 1 -C 8 alkyl or hydrogen, or biologically thereof. an active fragment extending from the R at the N-terminus to any residue at any position between 27 to 44 as its C terminus, optionally their Hse (lactone), HseOH or HseNR _and Rt and nontoxic salts thereof. These modifications can be prepared according to currently known experimental procedures. Because they retain a substantial part of the biological activity of the original peptide, these modifications are considered to be part of the invention.

The extending portion of the GRF peptides of the invention has an amino acid chain corresponding to the formula (XY) - (X ¹ -Y) _n n here represents the number of linearly linked XY groups, this number being from 0 to 20, preferably from 0 to 10 groups.

X is selected from the group consisting of all naturally occurring amino acids,

Y is selected from the group consisting of proline, alanine, seririum and threonine, only if η -O., Then Y is selected from the group consisting of alanine, serine or threonine,

X ¹ is selected from the group consisting of any naturally occurring amino acid except proline or hydroxyproline.

According to this formula when n = l, there are two actual pins Y. Furthermore, it is possible to have up to twenty-one and twenty-Y groups X ¹ in one embodiment. The individual residues Y and X ¹ may be any amino acid from the appropriate group from which to be selected. That is, the individual residues Y need not be the same in a given embodiment. Similarly, in an embodiment of the invention with more than one X ¹ group, each individual X ¹ group may be any amino acid except proline and hydroxyproline. irrespective of the nature of the other residues. zAll single Y and X ¹ residues must comply with the rules for that specific group and it is sufficient that the various individual residues at the designated positions comply with the rules set out above. _in in vitro rat GH from cell cultures

Analog concentration nM

0.01 0.1 10

Table I

Effect of bGRF analogues on secretion of rat anterior pituitary

Compound number .....

Name (as trifluoroacetate)

- - - without Leu ² Ile2Alai Ile ^ Ala ¹ Val ² Ala ² Val ² Ala ⁷ Thr ² Alai Thr ^ Alai ThríAla ¹ Ile ² Alai Val ² AIa ² Thr ² Alai dosing ⁷ bGRF (1- 29 iVal i ⁹ Leu ² strength “Leu ² muscle i ⁹ Leu ² strength ¹⁹ Leu ² muscle ¹ -Leu ² sVal ¹⁹ Leu ² sLeu ²⁷ bGRF sLeu ¹⁹ Leu ² SLeu ¹⁹ Leu ² SLeu ¹⁹ Leu ²

GRF -.....

) NH ₂ ⁷ bGRF (1- 29 ⁷ bGRF (1- 29 ⁷ bGRF (1- 29 ⁷ bGRF (1- 29 ⁷ bGRF (1- 29 ⁷ bGRF (1- 29 (1-29)) NH ₂₇ ⁷ bGRF (1 -29 ⁷ bGRF (1- 29 ⁷ bGRF (1-29) NH ₂ ) NH ₂ ) nh ₂ ) NH ₂ ) nh ₂ ) NH ₂ ) nh ₂ ) nh ₂ ) nh ₂

1963 347.4 c 55989 5162f 4629 * .4141 ^d 3852 * 2541 * 2833b

1963 * d 7561 ^d 120829 911006f No 11832 ¹ = 10686 ^f

9252 * b 6263 ^c c 5831 ^c . 5835 * .3976b • 2443

1963 * 15170 *

17230 *

9169801 '

9165789

16370 *

16138 * ^d 13122 ^d

13378 ^d

12298 ^d

9540 * • 4483p

1963 *

16506 *

17272 ^d

16455

The amount of rGH secreted from pituitary cell cultures was measured by radioimmunoassay and is expressed in ng per. sample in 4 hours. Test conditions are described in Frohman.aj.Methods Enzymol. 124, 371 (1986).

a, b, c, d, e, f, g in a given column, the results differ from other indices at a significance level below 0.05.

Note:. The substitution of Leu ¹⁹ had a deteriorating effect on the ability to release GH, while Val ¹⁷ and Ile ¹⁷ modifications led to analogues with reduced efficacy. Leu ²⁷ bGRF (1-29) NH ₂ was used as a test standard.

Table II

Plasma stability of GRF peptides substituted at position 19 in vitro

Compound number Name (as trifluoroacetate) ti, ₂ (hr 1 2 Leu ²⁷ bGRF (1-29) NH Thr ² Ala ¹ muscle ¹⁹ Leu ²⁷ bGRF (1-29) NH ₂ Thr ² Ala ¹ muscle ¹⁹ Leu ²⁷ hGRF (1-29) NH2 ThriAla ¹ Wal ¹⁹ Leu ^{2 7} bGRF (1 -29) NH2 - · - - Thr ² -A-A- ¹ .- s-Leu-Leuí- ^ i- ^ BGR-FC-L-29-) _NH2 ..... 0.6 ’ 4.9 * 14.7 * 6.6b -....... 1-2.3 * ..........

* The average value of three independent experiments (each done three times) a, b, c Compounds with different indices differ at the level of the mosques than 0.05.

Table III

Serum GH Holstein Castrate Response to Injections of GRF Analogs with Different Substitution at Position 2 and 19 of the Scheme

X ² X ² Ala ²⁵ Leu ^27-bGRF (l-29) _NH2

Line Compound Name'* Share. Maximum number number (such as trifluoroacetate in% (ng / ml) 1. - Water 0 ^p 11.7® 2 AND bGRF (1-44) _NH2 83b 73.2® 3 (B) Thr ² Ala ² 5 eu ²⁷ bGRF (1-29) NH 100i> 75.7? - 4 3 Thr ² Ala ² Leu ⁹ Leu ²⁷ bGRF (1 ~ 29) NH ₂  100b 83.5® 5 4 Val ² Ala ^{2 5} Leu ⁷ Leu ²⁷ bGRF (1-29) NH ₂ 92b 70.9® 6. 5 Ile ² Ala ² 5Leu ²⁹ Leu ²⁷ bGRF (1-29) NH ₂ 92b 42.8® 7 1 Thr ² Ala ² * VaH ⁹ Leu ^{2 7} bGRF (1-29) NH ₂ 92 ^p 79.6® 8 8 Val ² Ala ^{2 5} Val ²⁷ Leu ^{2 7} bGRF (1-29) NH ₂ 100 p 69.4® 9 9 Ile ² Ala ^{2 5} Val ²⁷ Leu ^{2 7} bGRF (1-29) NH ₂ '92b 72.7® 10 2 Thr ² Ala ² Power ²⁹ Leu ²⁷ bGRF (1-29) NH ₂ 92b 60.7® 11 6. Val ² Ala ² silence ² 'Leu ^{2 7} bGRF (1-29) NH ₂ 92b 78 \ '7® 12 7 Ile ² Ala ² 5ile ⁷ Leu ²⁷ bGRF (1-29) NH ₂ 100b 49.5 ° 13 PULL 5 l & .7i 14, P value. 0.0001 0.75

* Data presented includes only animals that have reacted with

for GRF injections; ** GRF analogs were administered as intravenous injections of .30 pmol GRF / kg 2 hours prior to feeding.

*** Heterogeneity of variation (Hartley F-max test), -P and its index are calculated from the logarithmic transformation of the data. abcde Values with different indexes in the column are significantly different (P less than 0.05).

^f SEM with GRF was 12.7 ng / ml, while SEM in controls

Line number Time to max. Minutes ......... Area (units) Like% A Like% B • 0-lh ..... - 1-2h 0--2h · - 1 22 “b c 0.51 " 0.50 " 1 " 44 28 2 . 12 " 0.73b 0.56 " 2.29b 100 ALIGN! '64 3 18 “b c 2.56 « 1.04 * d 3.60 « 157 íoo- 4 17 “b c 2.43 * « 0.77 “* 3.20'b c 140 89 5 21 b c 2.16bc 0.91 “*« 3.07 * « 134 85 6 15 ° bc 1.43b 0.95 “* 2.38b 104 66 7 19 “b c 2.75 1,35cde 4.10 « 179 '114 8 15 “b 2.30bc 1.48 * « ^d 3.77bc 165 105 9 22 b = 2.28 * « 1.96 * 4.25 « 186 118 10 22bc '2.04 * 1.19 bt d 3.23 « 141 90 11 2 0 “b c 2.63 * « 1.74 ^de 4.37 « 191 121 12 24 « 1.56 * « 1.36 « ^DĚ 2.91 * « 127 81 13 49 0.33 0.23 0.48 14 0.32 0.0001 0.0001 _ 0.0001

with water was 45.2 ng / kg

-9 The SEM for GRF administration was 4 minutes while the SEM for water controls was 14 minutes.

Figure i

Effect of Thr ² Ala ¹ -Leu ²⁷ bGRF (1-29) NH ₂ analogs on release. growth hormone GH in rat pituitary cell cultures in vitro. The experiment was conducted according to the methods of Frohman and Downs, Methods Enzyniol. 124: 371-339 (1986). Leu ¹¹⁵ substitution had a deleterious effect; GRF analogs for the ability to release GH while Val and Ile ¹ 7 ¹⁹ modification 'resulted analogům- with either increased or unaltered' in vitro bioactivity in comparison.

Thr ² to Ala ²⁷ Leu ^1J bGRF ^(1-29) NH ;. Leu ²⁷ bGRF (1-29) NH? served as an experimental standard.

Figure 2

Average '' "concentration" ·. serum β-r-growth hormone ... GH. ...at..

Holsteinských '. castrate-fed castrates after intravenous injection of 10 pmol / kg GRF analogs. The experiment was performed as described by Moseley et al. Endocrinol, 117, 252-259 (1988), Bovine

GRF (1-44) NH ₃ was used as an experimental standard. .

Claims (3)

1. A GRF peptide with a Val or Ile in place, an amino acid residue normally found at position 19, a Gly, Thr, Val or Ile residue in place of an amino acid residue normally found at position 2, and one amino acid from the group of amino acids Ala , Val, Leu, Ile, or Gly at position 15. 2. The GRF peptide of claim 1 wherein the amino acid at position 2 is Thr, Val, or Ile. A GRF peptide according to claim 1 of the formula R-R ¹ -R ¹ -R 5 -Ala-Ile-Phe-Thr-Re-Ser-Tyr-Arg-R ¹² -R 1j -Leu R; -Ri7 ¹ -Arg-R 21 -R 72 -Leu-G1N-R2 5 - 11e-7 R2 R? p-Arg-Gn-Gln-Gly-Glu-R3 4-R3 5-Gin-Glu-Rg 8-R3 9-R4 o-Arg-R4.2Arg-Leu-Z, where R is H, C1-C = alkyl or benzyl, Ri is Tyr or His, R? is Gly, Thr, Val, or Ile, R 3 is Asp, Glu or Cya, Rg is Asn or Ser, R 12 is Lys, or Arg, or N-epsilon-alkyl or N-epsilon-benzyl-Lys when R is C 1 -C 5 alkyl or benzyl, R13 is Val or Ile, R 15 'is Ala, Val, Leu, Ile, or Gly. Rie is Ser or Tyr, R1 <? is Val or Ile, R 6 is Lys, or Arg, or N-epsilon-alkyl or N-epsilon-39-benzyl-Lys when R is C 1 -C 5 alkyl or benzyl, R ₂ 2 Yippee Ala or Leu, R2 5 Yippee Asp, Glu, 1 - R2 7 Yippee Met, I-le or Leu,. R 2 ε Yippee Asn or Ser, Ra 4 Yippee Ser or Arg, Raa Yippee Asn or Ser, Ra e Yippee Arg or Gin, R 3 9 jS Gly or Arg, R4 0 Yippee Ala or Ser, R42 Yippee Ala, Wall or Phe, a Z represents the carboxyl portion of the C-terminal amino acid residue and is -COOR. _a , -CR _a O, -CONHNHR _a , -CONR _and R b or -CH ₂ OR _a with R _a and R b which are C 1 -C 6 alkyl or hydrogen, or a biologically active fragment thereof, extended from R at the N-terminus to a residue at any position between 27 to 44 as its C terminus, or Hse (lactone), HseOH, or HseNR 'Sd. and non-toxic salts thereof. GRF peptide according to claim 3, characterized in that R2 is Thr, Val or Ile.