SK49093A3 - Stabilized active analogues of factors releasing of growth hormone - Google Patents

Abstract

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

Description

The present invention relates to peptides affecting the activity of the under-gland 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 the invention are active in vivo, more stable in plasma, and selected peptides are more stable even in an aqueous environment at neutral pH than the natural chains of these substances.

BACKGROUND OF THE INVENTION

Physiologists have long known that the hypothalamus controls the activity of adenohypophysis by producing peculiar agents that encourage but inhibit the secretion of various hormones. In 1982, human pancreatic (cancer) factors (hpGRF) were isolated from human tumors of the pancreas. They were purified, characterized, synthesized and found to promote the release of growth hormone (GH) by the pituitary. [Guillemin R., et al., Science 218, 585-585 (1982)]. Since then, corresponding GH-releasing factors (GRF) have been characterized and synthesized from other species, including rats, pigs, sheep, cattle and goats and humans.

Human hypothalainic 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-Lys-Leu-Leu-Gln-Asp-Ile-Met-S-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg Gly-Ala-Arg-Ala-Arg-Leu-NH ?.

The rat GRF (rGRF) has been found to have a Ser group at the 8-position and the formula: H-H 1 s -A 1 -Ap -A 1 a-11e-Phe-Thr-Ser-Tyr-A rg-Ar g - 1.1 e - L e uG ly -G 1 n- L. eu -Ty r - A 1 a - A rg - Ly s - L eu - L eu - H is - G 1 u -Ie-Met-Asn-Arg- Gln-Gln-Glu-Arg-Asn-Gln-Glu-Glu-Glu-Arg-Ser-Arg-Phe-Asn-OH (see, for example, US Patent 595,676).

Bovine GRF (bGR1) was found to have the formula Il-Tyr-Ala2

As-1-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu-Gly-G1n-LeuSer-A 1a-Arg-Lys-Leu-Leu-Gin-Asp-Ile- Met-Asn-Arg-Gln-Gln-GlyGl-Arg-Asn-Gln-Gln-Gln-Gly-Ala-Lys-Va-Arg-I.eu-NH2.

Pork GRF has the Ser group at position 28.

Natural GRF chains have been reported to be rapidly inactivated by blood plasma enzymes. Rapid disintegration is induced by disruption of the binding of 2-3 peptides to ipeptides by 1-peptidase, type IV (DRP-IV), which was formerly also referred to as dipeptidylaminopeptidase-IV. [Frohman, L.A. et al., 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. Marshall, ESCOM Sci. Publishers, Leiden, p. 465-467 (1988), who reported that GRF analogs, substituted with desNH 2 -Tyr at position 1 and / or D-Ala at position 2, have increased stability of N-terminal groups to enzymatic degradation. This information was recently confirmed by Frohman et al .: Dipeptidyl peptidase-IV and trypsin-like enzymatic degradation of human growth hormone releasing hormone in plasma. J. Clin. Invest. 83, 1533-1540 (1989). In addition, 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, α 1 -methylation and Tyr-1 only partially prevented the degradation of DPP-IV.

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

Natural GRF chains have a Gly residue at position 15. Analogs with Ala or Leu at position 1.5 are known to have an increased ability to release GH [see, for example, US Patents 4,649,131 and 4,734,399 as well as I.ing N. et al., Quo Matters Symposium, Sanofi Group, pp. 29-30. May 1985, Toulouse-Labege (pp. 309-322)]. Sub3 substitutions of Gly-15 Val or α 1 α-aminoisobutyric acid also enhanced the efficacy of GRF analogs [Felix et al .: Synthesis and biological efficacy of new analogs of growth hormone releasing factor, in Peptides 1986, Walter de Gruyter and Co., Berlin-New York , p. 481-484 (1987)].

AM Felix reported on a plan to synthesize analogues with increased and / or prolonged biological activity, including preparation and testing of A1a ¹⁵ h-GRF (1-29) NH2 and desNH2-Tyr ¹ , D-Ala ² , A1 and ¹⁵ hGRF (1- 29) NH2 [see, for example, US Patents A, 649,131 and 4,734,399 as well as AM Felix, EP Heimer, TF Mowles, H. Bisenbeis, P. Leung, TJ Lambros, M. Ahmad, CT Wang and Paul Brazeau: Synthesis and Biological Efficacy of New growth hormone releasing factor analogs, in Peptides 1986; Walter de Gruyter and Co., Berlin-New Yokr, p. 481-484 (1987); Felix AM, Wang CT, Heimer 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, Proc. Peptide Symposium, Ed. GR Marshall, ESCOM Sci. Publishers, Leiden, p. 465-467 (1988); Peticlerc D., Lapierre H., Pelletier G., Dubreuil P., Graudreau P., Mowles T., Felix A. and Brazeau P.:

à

Effect of Active Human Growth Hormone Release Factor (hGRF) Analogs on Growth Hormone (GH) Release and Milk Production in Dairy Cows; Meeting Abstract P223 82nd Meeting American Dairy Sci. Assn., Columbia, MO, June 21-24 (1987)].

The Ser ² modified GRF analog, as well as eight other modifications of the same molecule, is described by Tou et al .: Amphiphilic growth hormone releasing factor (GRF) analogs: peptide structure and biological activity in vivo. Biochem. Biophys. Res. Commun. 139, 763-770 (1986). This analog has 165Y, in vivo activity in sheep compared to hGRF (1-4-4) NH2.

U.S. 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, No, N val, or β-Ala in Appendix 15 (see also U.S. Pat. 4,649,131).

The European patent application (Coy and Murphv, Publication Number 0 180 214, Application Number 86100127.9) discloses GRF halogens with Leu or Phe at position 2 together with GRF peptides which are substituted at position 2 with various non-naturally occurring amino acids with L or D-cores i gurác iou.

Very low biological activity GRF analogs having Sar ² or Pro ² are described by Coy et al.: Strategy for Designing Synthetic Analogs and Antagonists of Growth Hormone Release Factor, Peptides, Vol. 7, Suppl. 49-52 (1986).

Ling et al .: Analogues of Growth Hormone Releasing Factor with Strong Antagonist Activity, in Peptides, Chemistry and Biology, Proc. Peptide Symposium; Ed. G. R. Marshall, ESCOM Sci. Publishers, Leideri, p. 484-486 (1988) reported a series of GRF analogs

Arg or various

(see US pat. registration January 1989 and serial In the same way A.R. Fri edman, Hi11man. L. F. Krabi11, R

substituted with either D-amino acids at the 2-position. All are less potent than the parent hormone, and some of them showed antagonistic activity.

The present invention provides synthetic GRF polypeptides having a Ser residue at the amino acid site normally found at positions 8 and 28 of the polypeptides as an agent for inhibiting chemical decomposition (deamination) in an aqueous environment, application Serial No. 07-303,518, filed 27.

No. 07-323,955 of March 15, 1989). IF. Ichhpurani, D.M. Brown, R.M. i. Martin, H.A. Zurcher-Neely and D.M. Guido: The degradation of growth hormone releasing factor analogs in neutral aqueous solution is explained by deamination of asparagine residues. Serine replacement of asparagine groups stabilizes. Int. J. Peptide Protein Res. 37, 14-20 (1991).

The foregoing invention discloses synthetic GRF polypeptides having a substituted residue in place of the amino acid at the R3 position of cysteic acid (Cya) and / or R 25 (see US Pat.

Serial No. 07-150,301, filed Jan. 29, 1988 and Serial No. 89-00245 of Jan. 27, 1989).

29-amino acid hGRF hol analog designed by G. Velicebi et al., Proc. Natl. Aca. Sci. USA, Vol. 83, 5397-5399 (1986), which has the first six amino acids at. and the minus end of the same and differ from the native peptide by 13 amino acids in the rest of the chain including the incorporation of the Ser group at position 8. The amide form and the free acid analog form have the formula: H-Tyr-Ala-Asp-Ala-Ile-Phe-Ser- Ser-Ala-Tyr-Arg-Arg-Leu-Leu-Ala-G1n-Leu-A1a-Ser-Arg-Arg-Leu-Leu-G1n-G1u-Leu-Leu-A1a-Arg-NH2 / / 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 (1-40) OH, while the free acid form had. only 1/6 of efficacy in the same experiment.

Vale et al. (U.S. Pat. No. 053,233, filed May 22, 1907) discloses 31-residue hGRF analogs that utilize a group at position 31 with a side-functionalized chain that can be linked to a particular protein. The 31-residue hGRF analogs may also have substitutions. other groups that appear in the natural GRF chain, such as Asn or Ser at position 8, Phe at position 10, or Ala at position 15. Asn or Ser may be at position 28.

It is known that, in certain circumstances, the Asn groups of the polypeptides are deamidated in the presence of water. However, it is not clear what affects the 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 Kossiakoff A.A .: Science 240, 191-194 (1988)].

European Patent Application No. 86308337.4, Publication Number 0 220 958, describes a class of compounds having the formula HX-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 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.

Non-GRF peptide analogues that are extended to the N-terminus for various purposes have also been described. For example, M.A. Tallon et al. [Biochem. 26,777-7774 (1987)] synthetically prepared, yeast alpha-reproductive factor analogues extended at the N terminus with Ala, Glu-Ala, Ala-Glu-Ala or Glu-Ala-Glu-Ala. These peptides were used in the study of the relationships between me. t r r a a a a a a a t o u.

D. Andreu et al. [20 th Eur. Peptide Symp. Tubingen, Germany, 4-9. (1980), Symposium Abstracts, p. 33] synthesized the entire sequence of 64 amino acids of the cecropin A analog precursor form together with many shorter peptides corresponding to the putative reaction intermediate. Among them, the complete cecropin chain was extended by the A1a-Pro-Gly-Pro at the N-terminus. It has been used to demonstrate that the extension portion is indeed cleaved by a purified dipeptidyl peptidase-like enzyme preparation obtained from cecropylaid cocci [see also H. Boman et al., J. Biol. Chem. 264, 5832-5860 (1989)].

G. Kreil et al. [Eur. J. Biochem., 111, 49-58 (1980)] disclose that melittin, a major constituent of bee venom, is derived from progeny. í nu. The pro-melamine chain consists of six X-Pro and five X-Ala repeated dipeptide groups. The results presented by Kreil et al.

the prodrug to the product may be by an enzyme of the type suggest that the conversion by sequential cleavage of didipeptidyl peptidase IV, the peptide units present in the extracts of the venom glands.

C. Mollay et al. [Eur. J. Biochem., 160, 31-35 (1986)]: Caerulein and xenopsin are two peptides found in the skin secretions of Xenopus laevis. The first has the sequence Phe-A1a-Asp-Gly and the second Ser-Ala-Glu-Ala to extend the N-terminal group of their respective precursors. Dipeptides Type IV peptide, isolated from frog skin secretions, has the specificity required to cleave these N-terminal extensions, resulting in end products.

D. Julius et al. [Cell, 32, 839-852 (1983)]: Saccharomyces cerevisiae alpha cells secrete an alpha-factor peptide of 13 amino acids as a pheromone. Do not kick 1 mutants i

alpha cells that do not have membrane bound dipeptidyl peptidase do not produce normal alpha-factor, but exclude a number of imperfectly prepared forms with the structures G 1 µA 1a-G 1 µ-A1 and -alpha-factor or Asp-Ala-Glu-Ala-alpha factors having significantly reduced biological activity. It has been shown that membrane-bound dipeptidyl peptidase is essential for the normal processing of the precursor a 1 f a-f and kt o r and that this reaction may limit the reaction time.

maturation of alpha-factor in normal alpha-cells

C. L. Choy et al. [Eur. J. Biochem., 160, 267-272 (1986)]: The precursor protein preventing the freezing of the Newfoundland winter plateau contains four repeated sequences of X-Pro and seven X-Ala on the terminal N-group. Although the reactions of the precursor have not been studied, the authors contemplate that the modification of the precursor could occur in plasma by the action of a dipeptide 1 -peptidase-like enzyme, which in turn degrades the dipeptide units of the elongation moiety and releases the protein to prevent m.

Only after the priority date of basic Suhr et al. reported the isolation and length of the cDNA clone encoding mouse GRF.

mature mouse GRF is 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 GRF of other species having all Ala at position 2 [see Mol. Endocrinoogy, 3, 1693-1700 (1969)].

Based on the considerations of other authors, GRF preparation analogues with Leu ¹⁹ substitution (namely Thr ² A1 and ¹⁵ Leu ¹⁹ Leu ²⁷ -bGRF- (1-29) NH 2, tri-fluoroacetate salt, compound 3; Leu ¹⁹ Leu ²⁷ hGRF - (1-29) NH2 trifluoroacetate salt; Thr ² Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH2 testing the rat anterior pituitary growth cell release patent specification characterizing the whole (namely the trifluoroacetate salt). In vitro hormone using Leu were ¹⁹ analogs

Thr ² A1.a ^{1 5} Leu ^{1 9} Leu ^{2 7} - bGRF (1-29) NH 2, fluoroacetate compound. Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH2,

Thr ² Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 Leu ²⁷ bGRF (1-29) NH 2] significantly Ala ¹⁹ analogs. When testing.

three fluoroacetate # 3, three trifluoroacetate and three fluoroacetate. Ala ¹⁵ Leu ¹⁹ less active than the corresponding in vivo on growing humps at a dose of 10 pmol / kg, the amount of GH released by Leu ¹⁹ analogs was not observed for 2 h from the novel and was significantly different (p> 0.05) from Ala ¹⁹ . In monitoring bovine plasma stability, compounds with Leu ^{19 were} more stable than Ala ¹⁹ analogs.

position .19 in the spindle measuring zone

Mol. Biol.

In the GRF molecule, it is similar to G r a n b n r o A. H : J.

[C 1 O r G.M. , Martin S.R. .191. 553-561 (1986)]. It is not possible for GRF to be a cluster or whether it is a touch residue receptor [Sato K., Hotta M.,

Kagegama, J., Cziang, TC, Hu, HY, Biochein. Biophys. Res. Commun. 149 Schiffer M. and Edmundson AB: Biophys. J stitution of D-A 1 and ¹⁹ for Ala's ability to release GH experiments with rat pituitary cells.

in N1 e ²⁷ rGRF (1-29) NH2 reduced efficacy in bovine pituitary cells and in Nle ²⁷ rGRF (1-29) N112 reduced efficacy in rat pituitary cells [Rivier J.

Dong M.H. and Ling N .: (2) 531-537 (1987);

7, 121 (1967)]. Sub ¹⁹ in hGRF (1-29) NH2 reduced standard in vitro to 6% analog with L-Ala ¹⁹ at the same substitution of 10% standard in parallel substitution of 30% standard in Second International

Symposium on Vasointestinal Peptides and Related Peptides, Cap

In contrast, the GR ¹⁹ analogue with Ser ¹⁹ was in a standard release experiment in rat pituitary cells [Velicebi G., Patthi S. and Kaiser TE: Structure and biological activity of growth hormone releasing factor analogs having potent non-amphiphilic spindle carboxyl termini. Proc. At 1.1. Aca. Sci d'Agde, France (1985)]. as effective as hGRF (1-AO)

USA, Vol. 83.

5397-5399 (1986)].

? Getzoff reported that in the myohemerythrin peptide model, Ala, ile, Ser, and Thr can substitute for Val without losing antibody binding, but the peptide substituted by Leu ability [Getzoff E.D., Gysin H.M., Rodda Tainer J.A. and Lerner R.A .: Mechanism of Antibody Binding to Protein. Science, 235, 1191-1196 (1987)].

WO 90/15821 (published December 27, 1990, i.e. after the priority date of the present invention) discloses various GRF peptides having Thr, Val or ile groups instead of amino acid residues normally existing at position 2, Ala at position 19 in combination with one of The GRF peptide may optionally have a Ser residue at an amino acid residue site that normally resides at positions 8 and 28 of the polypeptide.

In addition, optionally, the GRF peptides may be extended to the N-terminus with C1-C5 alkyl, benzyl, H (YX) _n or H (YX) _m - (Y'-X ') _p , wherein Y and Y ¹ are the same or different naturally occurring amino acids, preferably Tyr or A sp, X and X ¹ , the same or having reduced SJ binding, Alexander H., different, are selected from Thr, Se.r or Ala, preferably Thr or S er, n is 1- 10, m is 1-5, p is 1-5.

EP Heilmer. et al. (121.h Am. Peptide Symposium, Cambridge, MA, 16-22 June 1991), Abstract No. 32 in the Symposium Program and Abst.ract reported that Val ² increases the resistance of mGRF to digestion of dipeptidyl peptidase IV. A series of hGRF analogs with modifications at the '2 position, with an emphasis on Val ^{2, has} been disclosed. Under the posters are tiež.uvedené GRF analogs of G1 ^y2, Ile, and Leu ^{2 second}

SUMMARY OF THE INVENTION

The present invention provides a pituitary growth hormone releasing polypeptide (GRF peptide) having a Val or clay at position 19. The GRF peptide of the invention will have a Gly, Thr, Val or clay residue in place of the amino acid residue normally found at position 2 in in combination with one of the following amino acids: Ala, Val, Leu, clay or Gly at position 15. In a preferred alternative, the GRF peptide may have a Ser residue instead of an amino acid residue normally found at positions 8 and 28 of the polypeptide. In addition, the GRF peptides of the invention may be N-terminally extended. C1-C5 alkyl, benzyl (XY) - (X - Y) n, wherein n is 0 to 20, preferably 0 to 10, X is any naturally occurring amino acid, Y is alanine, serine, threonine or proline, and if n = 0, then Y is selected from the group consisting of alanine, serine or threonine, X > 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 chains of natural GRF. Moreover, the compounds substituted with Ser, Ser ⁸ and ²⁸ protected e and d as iri can CII in an aqueous environment and are much more stable chemically.

The term GRF peptide, as used in the specification and claims, means a known polypeptide having a length of 27 to 6 residues and which causes the release of growth hormone by the pituitary. Examples of GRF peptides are the natural or synthetic polypeptides described in the IJ 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 US Pat. Application Serial No. 89/00245, filed Jan. 27, 1989. All of which are incorporated herein by reference. Felix A.M., Wang C.T., Heimer E., Fourier A., Bol in D., Ahmad M., Lambros T., Mowles, and biological activity of novel linear GRFs in Peptides. Chemistry and Biology

T. and ^- M 1 1 er L: Synthesis of cyclic ana ions

Proc. 10th Am. Leptide Syrnposium, Ed. G. R. Marshall, FSCOM (1988); By B.I., Buonomo F.C., Della-Fera M.A., analogues of the factor releasing growth and biological activity in viSci, Publishers, Leiden, p. 465-467, Kaempfe h.A., Vineyard

Bai C. e. : hormone (GRF) group C-conc

amphiphilic

Structure of peptides in. Biochern. Biophys. Res. Commun. 19, 763-770 (1986). The term GRF peptides includes non-toxic salts thereof.

The nomenclature used to define GRF peptides is according to the book by Schroder and Lubke: Peptides, Academic Press (1965), in which, in accordance with conventional representation, the amino appears to the left and the carboxyl group has the amino acid residue of the isomeric form. otherwise. The present invention provides GRF peptide analogs having the following formulas: R-R 1 -R 2 -R 3 -Ala-Ile-Phe-Thr-R-Ser-Tyr-Arg-R 12 -R 13 -Leu-R 15 -Gln-Leu-R 18- R19-Arg-R21-R22-Leu-Gln-R25-Ile-R27-R28-Arg at the N-terminus to the right. if

-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R38-R39-R40-Arg-R42-Arg-Leu-Z wherein R is H, C1-C5 alkyl or henzyl, preferably H,

R1 is Tyr or His, preferably Tyr,

R 2 is Gly, Thr, Val or clays, preferably Val or clays,

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

R 8 is Asp or Ser, preferably Ser,

R12 is Lys, N-e-alkyl- or N-e-benzyl-lys or Arg, preferably Lys; or N-e-alkyl- or N-e-benzyl-Lys when R is C 1 -C 5 alkyl or b e; n z y 1,

R 13 is Val or Ile, preferably Val,

K’is is Ala, V or l, Leu, I 1 t; or Gly, preferably Ala, Val, Leu or clays, most preferably Ala,

Plb is Ser or Tyr, preferably Ser,

R 19 is Val or clays, preferably Val,

R 21 is Lys, N-e-alkyl- or N-e-benzyl-Lys or Arg, preferably Lys or N-e-alkyl or N-e-benzyl-Lys if R is

alkyl or benzyl, R 2 2 i ^e A 1 a a 1 e b 0 Leu, with preferably Leu, R 2 5 is a Asp or Gl.u. with advantage, Asp, R 2 7 is a Met, Ile a 1 e bo ' Leu, preferably 1 R 2 8 is a same time or Ser. with preferably Ser, · R 3 4 is a Ser or Arg, with preferably Arg, R 3 5 is a same time or Ser, with preferably Asn, R 3 8 is a Arg or Gin, with preferably Gin, R 3 9 is a Gly or Arg, with preferably Gly, Rá 0 is a A 1 a or Ser, with preferably A1 a, R4 2 is a A1 a, wall or Phe, preferably 1

Z represents the carboxyl terminus of the amino acid residue at the C-terminus and is -COORa, -CRaO, -CONHNHRa, -CONRaRb or -CH 2 OR _and with Ε »and Rb which represent C 1 -C 6 alkyl or hydrogen or a biologically active fragment thereof extended 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 HseNRaRb and their non-toxic salts,

Ra is preferably hydrogen (H); Rb is preferably ethyl.

Examples of C 1 -C 6 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 19 is Val or clays, including Thr ² Ala ' ⁹ peptides Val ¹⁹ Leu ²⁷ bGRF (1- 29) NH 2

Thr ² Ala ¹ 'e 19 Leu ²⁷ bGRF (1- 29) NH 2

Val ² Ala ¹ 5 l le ⁹ l.eu ^{2 7} bGRF (1-29) NH 2

1 e ² A 1 and ^{1 5} 11 e ^{1 9} Leu ^{2 7} bGRF (1-29) NH 2

Va 1 ² Λ 1 and ¹ 'Va 1 ¹⁹ Leu ^{2 7} bGRF (1-29) NH2

T 1 e ² A 1 a ^{1 5} V 1 ^{1 9} Leu ^{2 7} bGRF (1-29) NH 2

G 1 y ² A 1 and ^{1 5} V and 1 ^{1 9} Leu ^{2 7} bG RF (1 - 2 9) NH 2

GI y ² Λ 1 and ^{1 11} e ¹⁹ Leu ²⁷ bGRF (1-29) NH 2, preferably peptides

Thr ² Se r ⁸ A 1 a ¹⁵ Va ^{1 19} Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NH2

Thr ² S er ⁸ A 1 and ¹⁵ Va 1 ^{1 9} Leu ^{2 7} S er ^{2 8} H with ^{3 3} bGRF (1 - 3 3) NH 2:

Thr ² Ser ⁸ A 1 a ¹⁵ Va 1 ¹⁹ Leu ²⁷ Ser ²⁸ Hs e ³⁷ bGRF (1-37) NH2

Thr ² Ser ⁸ Ala ^{1 5} Val ^{1 9} Leu ^{2 7} Ser ^{2 8} Lls e ^{4 4} bGRF (1-AA) NH 2:

Thr ² Ser ⁸ Ala ¹⁵ 11 e ^{1 9} Ie u ^{2 7} Se r ^{2 8} Hs e ^{3 o} bGRF (1-30) NH 2:

Thr ² Ser ⁸ A1 and ^{1 5} 11 e ^{1 9} Leu ^{2 7} Ser ^{2 8} H with ^{3 3} bGRF (1-33) NH 2:

Thr ² Ser ⁸ Ala ¹⁵ 11 e ¹⁹ Leu ^{2 7} S er ^{2 8} Hs e ^{3 7} bGRF (1-37) NH2: or

Thr ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-AA) NH 2 ;, more preferably Ile ² Ser ⁸ Ala ^{1 8} Va 1 ^{1 9} 1. eu ^{2 7} S er ^{2 8} H se ^{3 (,} bGRF (1-3O) NH2 Thr ² S er ⁸ A1 a ^{1 5} Va 1 ^{1 9} Leu ^{2 7} S er ^{2 8} H with ³ θ bGRF (1 -30) NIIEt II e ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ S er ²⁸ Hse ³³ bGRF (1-3 3) NH 2 Ile ² Ser ⁸ Alai ⁸ Val> ⁹ Leu ^{2 7} Se ^{2 2} Hs e ^{3 3} bGRF (1-3 -3) NHL t Thr ² Ser ⁸ AIa ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-3 3) NHEt Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH2 Ile ² Ser ⁸ A.la ¹⁵ Val ^{1 9} Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-AA) NH2 Val ² Ser ⁸ A1 and ¹⁵ Va1 ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NH2 Val ² Ser ⁸ Ala ¹⁵ Val ^{1 9} Leu ²⁷ S er ²⁸ Hse ³³ bGRF (1-33) NH2 Val ² Ser ⁸ Alal Muscle 1 ⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH2 Val ² Ser ⁸ ATat ⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-AA) NH2 Ile ² Ser ⁸ Ala ¹⁵ Ile ^{1 9} Leu ^{2 7} Se ^{2 2} Ilse ³ ° bGRF (1-30) NH2 Ile ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ^{3 3} bGRF (1-3 3) NH2 Ile ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2 11 e ² Ser ⁸ A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ Se r ²⁸ Hse ⁴⁴ bGRF (1-AA) NH2 Val ² Ser ⁸ Ala ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ^3O bGRF (1-30) NH2 Va 1 ² S er ⁸ A 1 and ¹⁵ 11 e ^{1 9} Leu ^{2 7} S er ^{2 8} H with ^{3 3} bGRF (1-3 3) NH 2 Val ² Ser ⁸ Ala ^I5 Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1 -37) NH2 Va 1 ² Ser ⁸ A 1 and ¹⁵ 11 e ^{1 9} Leu ²⁷ Ser ^{2 8} Hse ^{4 4} bGRF (1-AA) NH2 Val ² Ser ⁸ Ala ¹⁵ Ile ' ⁹ Leu ^{2 7} Ser ^{2 8} Hse ^{3 7} bGRF (1-37) NHnPr or non-toxic salts.

The best GRF of the present invention is Val ² Ser ⁸ Ala ' ⁵ Val ¹⁹ Leu ^{2 7} S er ²⁸ II with ³ ° bGRF (1-30) NHEt.

Another embodiment of the invention is peptides spread at the N-terminus. Among them, for example, N - a - (T yr - A ia - P11 e - P ro - P he - A 1 a) T yr ¹ T hr ² S er ⁸ A 1 a ^{1 5} ¾ 1 e ^{1 9} L eu ^{2 7} S er ^{2 8} -bGRF (1-29) NH 2

N-rt- (Leu - Pro-G 1 y - P r o-Ty rA 1 a) Ty r ¹ Thr- ² S er ⁸ A 1 a ^{1 3} V a 1 ^{1 9} Leu ^{2 7} Ser ^{2 8} -bGRF ( 1-29) NH2

Ν-α- (S-1 and Pro-Gly-Pro-Tyr-Ser) 2 four ¹ W 1 and S ² E R ⁸ Ala ^{1 5} 11 ^{9 1} e Leu Ser ^{2 7 28 3 2} -list bGRF ( 1-32) NH2

N a (Leu-Pro-Tyr-Ala-Tyr-Ala) ^{2 Tyr} Ile Ala Ser ^{8 15 19} Val Leu Ser ^{27 28} -bGRF (l-29) NH2

Na- (His-Ala-Tyr-Pro-Tyr-Ala) Tyr ¹ Ile ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ -bGRF (1-29) NH2

Ν-α - (G1 u-Pro-Phe-A1 1 a-Tyr-Pro-Hi s-A1 a) Tyr ¹ 11 e ² Ser ⁸ A1 a ' ^{1 5} Va 1 ^{1 9} /

-Leu ²⁷ Ser ²⁸ bGRF (1-29) NH 2

Na- (His-Pro-His-Pro-His-Ala-Tyr-Ala) Tyr ¹ Thr ² Ser ⁸ Ala ^15. Val ¹⁹ -Leu ²⁷ Ser ²⁸ bGRF (1-37) NH2

N a ^{(Tyr-Ala-Gly-Pro-Leu-I,} ro-Phe-Ala) ^{2 Tyr} Ile Ala Ser ^{8 15 19} Val - L eu ^{2 2} ER ^{7 8 3 2} H, the RF bG (1-3 2) NH 2

Na (Val-Pro-Arg-Pro-Phe-Pr'o-Tyr-Ala) Tyr ¹ Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ -Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NHEt

Ν-α - (Arg-Pro-Tyr-Al and -Ile-Pro-Phe-Ala) Tyr ¹ Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ -Leu ^{2 7} Ser ^{2 8} Hse ³ ° -bGRF (1-30) NHEt

Na-TTyr-AlajATyr'Val ² Ser ⁸ Ala ¹⁵ Va 11 ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁴ bGRF (1-3A) -NHCII3

Na- (Ile-Pro-Glu-Ala-Tyr-Ala) Tyr ² Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ -bGRF (1-37) NH2

Another embodiment of the present invention are any of the above modifications wherein Asp at position 3 and / or 25 is replaced by Cya, preferably at position 3.

The Val ¹⁹ and Il ¹⁹ compounds of the invention are as effective (and sometimes more effective) in GH release in vitro (rat pituitary cells) than their corresponding Ala ¹⁹ counterparts (Table I and Figure 1). However, they are more stable to proteolysis when incubated in bovine plasma. Evidence of increased metaboic stability of the compounds of this invention [as compared to the natural GRF chain represented by Leu ²⁷ bGRF (1-29) N112] is provided in Table II, where in vitro stability data are summarized. The compounds of the invention are more active in vivo at 10 pmol / kg than 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 pinol / kg. (Table III). In Figures 1 and 2 (the compounds were used in the form of three fluoroacetyl 14 to 2 (nitrite salts) T2 stands for Thr. ² , Ala1 stands for Ala ¹⁵ , 1.27 stands for Leu ²⁷ , Ĺ19 stands for Leu ' ^9. , V19 means Val ¹⁹ and 1.119 means Clay ¹⁹ .

The enhanced efficacy of the present invention in which R is Val or Ile is exemplified in Table III and Figure. For example, one skilled in the art will recognize from a relative in vivo efficacy dose of 10 pmol / kg, Figure 2) that three f 1uoroacetate

Thr ² Ser ⁸ A1 and ¹⁵ Va ^{19 19} Leu ²⁷ Se r ²⁸ bGRF (1-29) NH 2. and Trifluoroacetate Thr ² Ser ⁸ A1a ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ bGRF (1-29) NH 2 as well as Val ¹⁹ and Ile ^{19 of the} compound (dose: 30 pmol / kg, Table III) are more bioactive than natural GRF.

For commercial production purposes, the carboxyl terminus of homoserine, lactone, or homoserine amide, or a secondary or tertiary C 1 -C 6 (preferably C 1 -C 4) alkyoseride of homoserine 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 of which are incorporated herein by reference. incorporated by reference.

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

PROCEDURE

Peptides are 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. Boe amino acids and other reagents were supplied by Applied Biosystems and other commercial suppliers. A sequential double coupling protocol to the starting p-methylbenzhydrylamino resin was used to synthesize the C-terminal carboxylic acid amides. For the synthesis of users 1 and 2: P a M f a n i n e.

The nines are coupled using preformed symmetrical anhydrides of Boe amino acids.

Usage in 1 s: d u j e e e s h e s h e s h e s:

G r g, it s y 1

A s p, h e: n z y 1

The C-terminal acids are poAsparagine, glutamine and argi15

Cys, 4-methylbenzyl

G 1 u, b e n z y 1

Ser, benzyl.

Thr, benzyl

Tyr, 4-bromocarbobenzoxy

Lys, 4-chlorocarbobenzoxy.

The deprotection of Boe is achieved with three fluoroacetic 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. Digestion with cyanogen bromide converts Met into a Hse lactone peptide 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, IseOH, HseNRaRb at the C-terminus can be prepared by the methods described by Kemp et al. [BIOTECHNOLOGY, Vol. 4, p. 565-568 (1986)]. Upon completion of the synthesis, the peptides are deprotected and separated from the resin with anhydrous 10X β-cresol-containing hydrogen fluoride. The cleavage of the side protecting groups and the peptide from the resin is carried out at 0 ° C or below, preferably at -20 ° C for 30 minutes, followed by 30 minutes at 0 ° C. After removal of HF, the resin resin is washed with ether and the peptide is extracted with glacial acetic acid and lyophilized. Prior to purification, the cysteine contained in the crude peptides is oxidized to the corresponding acid with formic acid at -10 to 10 ° C, preferably at 0 ° C as described in Synthesis, Pierce (1984), p. 113].

S t e w a r d

Chemical Wheel [Solid Phase

Cumpany, Rockford,

Peptide

Illinois

Conversion to Hse lactones and Hse amides to

The C-terminus has been described above.

Purification is performed by ion exchange chromatography on a Synchroprep S-300 cationic column (SynChrorn Inc. Linden, Indiana). The peptide is loaded using buffer, 20 millimolar TRIS (pH 6.8) in 20X acetonitrile and eluted using a gradient of 0-0.3 molar sodium chloride in the same solvent. The compounds are furthermore freed from the salts by reverse phase liquid chromatography on a Vydac L-18 column (Separate Group, H e p i i i, K i j i f i i n i) using m g i d i e n t o

6 iý>

t;. · ..

water: acetonitrile, both phases containing 0.1% TFA. The desired fractions were pooled and lyophilized. The desired GRF peptide is obtained in the form of trifluoroacetate salt. The trifluoroacetate can, if desired, be converted to other salts by ion exchange methods.

The peptides are hydrolyzed under vacuum in a gas phase in a Pico-Tag Work St.ation (Waters) for 24 hours under constant boiling HCl (Pierce) in the presence of phenol as a purifying agent at 110 ° C. Hydrolysates are analyzed on a Beckman Amino Acid Analyzer, Model 6300. Peptide composition is calculated using a known concentration of norleucine as an internal standard.

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

DETAILED DESCRIPTION OF THE INVENTION

· IY ;. J / 2 · · · · ·

Example 1

Preparation of three FluoroC ethers Thr ² A1 and ¹⁵ Va1 ¹⁹ Leu ²⁷ bGRF (1-29) NH 2, Compound no. 1.

Synthesis of GRF Analog Peptide of Formula H-Tyr-Thr-Asp-Ala-Ile -Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Val-Arg -Lys-Leu -Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH2 (as three fluoroacetate) is carried out sequentially according to Procedure A. Results of amino acid analysis, theoretical values in parentheses: Asp 4.06 (4), Thr

1.57 (2), Ser 1.64 (2), Glu 2.10 (2), Ala 2.25 (2), Val 2.00 (2), Clay 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 of three fluoroacetate in Thr ² A and ¹⁵ 11 e ¹⁹ Leu ^{2 7} bGRF (1-29) NH 2, Compound # 2.

b ' ^{ ) ·

f.

Synthesis of GRF Analog Peptide of Formula H-Tyr-Thr-Asp-Ala-Ile -Phe-Thr-Asn-Ser-Tyr-Arg-Lvs-Va1-Leu-A1 and G 1 n-Leu-Ser-11e-Arg-1 ' ys-L eu-L eu-G 1 n-A sp-1 1 e -1. eu - A sn - A rge - NII2 (as trifl u 6-racetate) is carried out just after the time of pu A. The results of no-acids, theoretical 1.98 (2), Ser 1.64 (2 ) ho d 11 o o s samples: A sp

Glu 2.05 (2), Ala 2.25 (1),

Iu e 2.84 (3), Leu 5.07 (5), Tyr 1.87 (2),

Phe a n i z y y m 4.09 (4), Thr Val 1.0 (1), 0.94 (2), Lys

1.97 (2), Arg. 2.94 (3).

EXAMPLE 1 a d 3

Preparation of Trifluoroacetate Thr ² A and ¹⁵ Va ^{19 19} Leu ²⁷ bGRF (1-29) NH 2, Mp.3.

Synthesis of GRF Analog Peptide of Formula H-Tyr-Thr-Asp-Ala-11e -Phe-Thr-Asn-Ser-Ty-Arg-Lys-Va1-Leu-A1 and Gn-Leu-Ser-Leu-Arg-Lys -Leu-Leu-Gln-Asp-III-I.eu-Asn-Arg-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), Clay 1.88 (2), Leu 6.11 (6), Tyr 1.90 (2), Phe 0.93 (1), Lys 2.03 (2), Arg 3.09 (3).

IN

Example 4

Preparation of three fluoroacetate Val ² Ala ¹⁵ Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH 2>

Compound No. 4.

Synthesis of GRF Analog Peptide of Formula H-Tyr-Va1-Asp-A1a-II and -Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu-A1 and G1n-Leu-Ser-Leu-Arg-Lys -l.eu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH2 (as trifluoroacetate) was carried out sequentially according to Procedure A. Mass spectral analysis (by Cf252 plasma desorption method) m / z for (M + H) * Observed 3454.6, theory 3452.1. Amino acid analysis results, theoretical values in parentheses: Asp 4.01 (4), Thr 0.93 (1), Ser 00 (2), Val 1.85 (2), Clay 1.88 (2), Phe 0.94 (1), Lys

1.68 (2), Glu Leu 6.11 (6), 3.11 (3).

2.03 (2), Ala 2 Tyr 1.94 (2)

1.97 (2), Arg

Example 5

Preparation of Trifluoroacetate I 1 e ² A 1 and ¹⁵ Leu ¹⁹ Leu ²⁷ hGRF (1-29) NH 2, Compound No. 5.

Synthesis of GRF Analog Peptide of Formula Η-Tyr-11e-Asp-A1-a-11e-Phe-Thr-As n-Ser-Tyr-Arg-Ly s-Va1-Leu-A1 and G1n-Leu-Ser -Leu-Arg - Lys-Leu-Leu-G 1 n -As p-Ie-LeuA with n-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A. Mass spectral analysis (Cf252 plasma desorption method) ) m / z for (M + H) ⁺ : observed 3460.6, theory 3466.1. The results of the analysis and less okin, the theoretical values in brackets: Asp 4.02 (4), Thr 0.94 (1), Ser

1.67 (2), Glu Leu 6.12 (6), 3.13 (3).

2.05 (2), Ala 2.00 (2), Val 0.94 (1), Clay 1.83 (2), Tyr 1.94 (2), Phe 0.95 (1), Lys 1.99 (2), Arg

Example 6

Preparation of trifluoroacetate Va 1 ² A 1 and ^{1 5} 11 e ^{1 P 9 * * *} Leu ^{2 7} bGRF (1-29) NH 2, compound c.6.

Synthesis of GRF Analog Peptide of Formula H-Tyr-Nal-Asp-A1a-11e-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Nal-Leu-Ala-Gln-Leu-Ser-1 T e-Arg. -Lys-Leu-Leu-Gln-Asp-11e-Leu-Asn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A. Mass spectral analysis (plasma desorption method Cf252) m / z for ( M + H ^{+ +} : observed 3453.4, theory 3452.1. Amino acid analysis results, theoretical values in parentheses: Asp 4.03 (4), Thr 0.94 (1), Ser 2.03 (2), Ala 2.01 (2), Val 1.89 (2), Clay 2.85 (3), Phe 0.95 (1) , Lys

1.69 (2), Glu Leu 5.11 (5), 3.08 (3).

1.99 (2), Arg

Tyr 1.95 (1),

Example 7

Preparation of three fluoroacetate 11 e ² A1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ bGRF (1-29) NH 2, compound 7.

Synthesis of GRF Analog Peptides of Formula vzorca-Tyr-11e-Asp-A1 and -11e -Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ile-Arg -Lys-Leu-Leu-Gln-Asp-Tle-Leu-Asn-Arg-NH2 (as trifluoroacetate) is carried out sequentially according to Procedure A. Mass spectral analysis (by plasma desorption method Cf252) m / z for (M + H) *: observed 3469.7, theory 3466.1. Amino acid analysis results, theoretical values in brackets: Asp 4.03 (4), Thr 0.94 (1), Ser 2.06 (2), Ala 2.04 (2), Val 0.94 (1), Clay 3.78 (4), Phe 0.95 ( 1), Lvs

1.69 (2), Glu Leu 5.11 (5), 3.09 (3).

Tyr 1.94

1.98 (2), Arg

Example

Preparation of tr 2 -turate of Val ² A 1 and ¹⁵ V and ¹ V ⁹ Leu ^{2 7} bGRF (1- 29) NH 2, Compound No. 8.

Synthesis of GRF Analog Peptide of Formula Η-Tyr-Va1-Asp-A1a-11e

-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-V and 1-Leu-A 1a-G 1n-Leu-Ser-Va1-Arg

- Ly s - Leu - Ie uG 1 n -As p-I 1 e - leu - A sn - A rg - NH 2 (as trifluoroacetate) is carried out sequentially according to procedure A. Mass spectral analysis (by the CI method ^; 252 desorption) plasma) m / z for (M + II) ⁺ : observed 3440.6, theory 3430.1. Results of amino acid analysis, theoretical values in 1.67 (2), Glu Leu 5.09 (5), 3.05 (3).

0.93 (1), Ser

Asp, 4.02 (4), Thr 2.03 (2), Ala 2.00 (2), Val 2.78 (3), Clay 1.86 (2), Tyr 1.92 (2), Phe 1.04 (1), Lys 1.98 (2) , Arg

Example 1

Preparation of 1 n e c o n i n ad 9 t ri fluoroacte n tate n. 9.

Ile ² Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-29) NH2

Synthesis of GRF Analog Peptide of Formula H-Tyr-11e-Asp-Ala-11e -Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Val-Arg -Lys- I.eu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A. Mass spectral analysis (by plasma desorption method Cf252) m / z for (M + H) ⁺ : observed 3454.9 theory 3452.1. Amino Acid Analysis Results, Theoretical Values in Parentheses: Asp 4.02 (4), Thr 0.93 (1), Ser 1.69 (2), Glu 2.04 (2), Ala 2.06 (2), Val 1.86 (2), Clay 2.84 (3) , Leu 5.10 (5), Tyr 1.94 (2), Phe 0.94 (1), Lys 1.98 (2), Arg 3.07 (3).

Using the sequential synthesis of Procedure A, the following peptides can also be prepared:

I le ² Ser-8AIa 5val i ⁹ Leu ^{2 7} S er ^{2 8} Hs e ³ ° bGRF (1-30) NH2

II e ² S er ⁸ A 1 a ^{1 5} Va 1 ^{1 9} Leu ^{2 7} S er ^{2 8} H with ^{3 3} bGRF (1-33) NH 2

I le ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2

Ile ² Ser ⁸ Ala ¹⁵ Val ' ⁹ Leu ²⁷ Ser ²⁸ Hs e ⁴ ' bGRF (1-44) NH2

Va 1 ² Ser ⁸ Ala ¹⁵ Va 1 ¹⁹ Leu ^{2 7} Ser ^{2 8} Hse ³ ° bGRF (1-30) NH2

Va1 ² Ser ⁸ A 1 and ¹⁵ Va1 ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NH2

T le ² Ser ⁸ Ala

Ile ² Ser ⁸ Ala

T 1 e ² Ser ⁸ A la J 1 e ² S er ⁸ A 1 a Val ² S er ⁸ A 1 a V a 1 ² S er ⁸ Λ 1 a ⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37 ) NH 2 ⁵ Val ^{1 9} Leu ^{2 7} Ser ^{2 8} lis e ^{4 4} bGRF (1-44) NH 2 ⁵ Va 1 1 ⁹ Ie u ^{2 7} S er ^{2 8} H with ^{5 0} bGRF (1-30) NH 2 ⁵ Va 1. ^{1 9} l.eu ^{2 7} Ser ^{2 8} Hs e ^{3 3} bGRF (1-33) NH2 ⁵ Va 1 ^{1 9} Le u ^{2 7} S er ^{2 8} H with ^{3 7} 1 »GRF (1-37) NH 2 ⁹ V a 1 ^{1 9} Leu ^{2 7} Ser ² lise ^{4 4} bGRF (1-4 -4) NII2 ⁵ I 1 e ^{1 9} Leu ^{2 7} Ser ^{2 8} Hse ³ <'bGRF (1-30) N 1 2 ⁹ 1 I e ^{1 9 7} Ser Leu ^{2 2 3 3 8} Hse bGRF (1-33) NH2

0

Va 1 ² S er ⁸ A1 a ^{1 5} 11 e ^{1 9} L eu ^{2 7} S e: r ^{2 8} H e e ^{3 7} _b GR p (i - 3 7) NH 2

V a 1 ² S er ⁸ A 1 a ^{1 5} U e ^{1 9} Le u ^{2 7} S er ^{2 8} H with ^{4 4} bG RF (1 - 4 4) NH 2

Ile ² Ser ⁸ Ala ^{1 5} Val ¹⁹ Leu ^{2 7} S e: r ^{2 8} H e ³ ObGRF (1-30) NH 2

² Thr Ser ⁸ Ala ¹⁵ Leu ¹⁹ Val ²⁸ 7series ² hours _to 3 ° _bGRF (I-30) _NH2

Thr ² Ser ⁸ Λ1 and ¹⁵ Va 1 1 ⁹ Leu ²⁷ Ser ² 8Hse ^{3 3} bGRF (1-33) NH2

Thr ² Ser ⁸ Ala ¹⁵ V and 1 ¹⁹ L eu ^{2 7} S e: r ^{2 8} H with ^{3 7} bG RF (1 - 3 7) NH 2

Thr ² Ser ⁸ A 1 and ¹⁵ Va ^{1 19} Leu ^{2 7} S e: r ^{2 8} Hs e ^{4 4} bGRF (1-4 4) NH 2

Thr ² Ser ⁸ A1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NH2

Thr ² Ser ⁸ Ala ^{1 5} il ¹⁹ Leu ²⁷ s er ^{2 8} hs e ^{3 3} bGRF (1-33) NH 2

Thr ² Ser ⁸ Ala ¹⁵ Ile ⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH2

Thr ² Ser ⁸ A to ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-44) NH 2

Thr ² Ser ⁸ Ala ¹⁹ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NHEt Thr ² Ser ⁸ Ala1 ⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NHEt Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NHEt Ile ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NHEt Val ² Ser ⁸ Ala ^{] 5} Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH-n-propyl.

PROCEDURE

In addition to the preparation of GRF analogs by solid phase technique, these analogs can be obtained by recombinant DNA method according to the procedure described for Leu ²⁷ bGRF (1-44) OH (European patent application 0212531) with subsequent modifications in the DNA segment encoding Leu ²⁷ bGRF (1-44). 0H, the codons for Ala ² , Asn ⁸ , Gly ¹⁵ and Asn ²⁸ are replaced by codons: for example, (ACT) Thr ² or (ATT) Ile ² or (GTT) Val ² , (AGT) Ser ⁸ , (GCT) Ala ¹⁵ , (AGT) Ser ^28th

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: extension: at the N-terminus are added as follows: (TATACT) for Tyr-Thr, (TATACT) n for (Tyr-Thr) n, (TATAGT ) for (Tyr-Ser) n or (TATAGTTATACT) for Tyr-Ser-Tvr-Thr or (TATACTTATAGT) for Tyr-Tlir-Tvr-Ser, (GATGCT) for Asp-Ala, etc.

The precursor protein gene is inserted into an 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 performed. Formic acid is removed under reduced pressure and the crude polypeptide is then purified by the methods described.

The compounds of the invention can be prepared by known recombinant DNA technology, since these peptides are composed of only the amino acids present in addition. This is in contrast to known analogs that contain unencoded DNA moieties such as D-Ala and / or desaminoTyr and will utilize costly chemical synthesis or a combination of genetic engineering with chemical procedures for any large-scale production.

The recombinant starting microorganisms used in this invention are prepared by recombinant DNA techniques well known to those skilled in the art and 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)). By this reference, these books are included in the description.

Analogues of IIse lactone, HseOH, HseNRaRb at the C-terminus can be prepared by the methods described by Kemp et al. [BIOTECHNOLOGY, Vol. 4, p. 565-568 (1986)].

PROCEDURE

This procedure relates to alkylated on nitrogen.

GRF analogs of the invention. The peptides are prepared by either a chemical or a biochemical method (procedure A or B). The N-alkylation is then achieved by known methods, e.g. Murphy, W.A. and Coy D. H (Long-term Effective Growth Hormone Releasing Factor, Peptide Research 1, 36-41 (1988)), V.

Sythyammorthy et al. [Reducing Inetylation of Botulinum Neurotoxin 11 in A and B, Mol. Cell, B o c h e m. 83, 65-72 (1988)].

The following peptides can also be prepared by Procedure C:

N - eps 1 l on - i P r ^- - Th r ¹ T hr ² S er ^H N - epsi 1 on - j Pr - l s ^{1 2 2 1} V a 1 ^{1 5} ]] _e 1 9

2

Leu ²⁷ Ser ²⁸ bGRF (1-29) NH 2

N -eps i 1 on-i Pr-Tyr ¹ Thr ² S er ⁸ N-eps i 1 on-i Pr-Ly with ^{1 2} ' ^{2 1} A 1 and ^{1 5} Va 1 ¹⁹ Leu ²⁷ bGRF (1-4O) NIÍ2

N-epsilon-Bzl-Tyr ¹ 'hrhr ² Ser ⁸ N-epsilon-Bzl-Lys ¹² Val ²¹ Val ¹⁵ Ile ¹⁹ e ²⁷ Ser ²⁸ bGRF (1-32) NH2

N-epsilon-iPr-Tyr ¹ Ile ² Ser ⁸ N-epsilon-.iPr-Lys ^{12 '21} Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-40) NH2

N-epsilon-Bzl-Lys ¹ Val ² Ser ⁸ N-epsilon-Bzl-Lys ^{1 2 2 1} Va 1 ^{1 5} Val ¹⁹ 11 e ²⁷ S er ²⁸ bGRF (1-32) NH2

All synthetic GRF peptides of the invention, including those prepared in the examples, are considered 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 at 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 GH levels. In addition, administration could alter the body fat content and regulate other GH-dependent metabolic, immunological and developmental processes. For example, these analogs may be useful as agents to stimulate anabolic processes in humans in burn-like conditions. In another example, these analogs can be administered to commercial warm-blooded animals such as chickens, turkeys, pigs, goats, cattle and sheep, and can be used in fish farming and other cold-blooded marine animals, e.g. sea turtles and eels, 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 function in humans and animals to treat diabetes resulting from abnormal growth hormone production, to improve bone, wound and burn healing, or in osteoporosis. It is also possible to use these analogs to stimulate hair growth.

Daily allowances between. 10 nanograms / kg and about. 50 micrograms / kg body weight are considered to be particularly effective in increasing. 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 to form a pharmaceutical composition, preferably a sustained release, are administered to animals and humans either intravenously,

subcutaneously, through the skin or nasal mucosa. They may be used by the physician to stimulate the release of GH if the treated patient in need of such therapy. Appropriate dosages 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, e.g. with zinc, iron and the like (they are considered as salts for application). As addition salts there may be mentioned hydrochloride, hydrobromide, sulfate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc. Where the active ingredient is to be administered intravenously in isotonic saline, phosphate or other buffers may be used.

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

Although the invention has been described with respect to the preferred forms, it is 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, it is possible to modify the peptide chain by omitting one or two residues from the C-terminus of the peptide according to currently known experimental practice, resulting in peptides that retain a considerable amount of glycosylation. n n osti. Therefore, a t o t o r o z. síi l'i u t o b o t o n e n e. The C-terminus and / or C-terminus of the peptide is characterized. s u b s t. i t. The equivalents are generally known in the peptide. chemistry. Thus, peptides falling within it can be substituted and / or unsubstantiated.

for example, to produce other analogs with increased resistance to proteolysis and at. having at least a substantial portion of the activity of the polypeptides of claim without departing from the scope of the invention illustrated by compounds 1 to 15. Also known substitutions of the carboxyl group at the C-terminus, e.g. lower alkylamidines, lead to equivalent molecules.

Spôsob'mi which have been described above, it is ^- possible to produce GRF peptides of the formula

R'-R ¹ -R'2-R 3 -A 1 a-11 e-Phe-Thr-R 6 -Ser-Tyr-Arg-R 11 -R 13-Leu-R 15 -Gln-Leu -Ri8 ~ Ri9-Arg-R'2i-R22-Leu-Gln-Ile-R25-R27-R28-Arg-Gln

-Gln-Gly-Glu-R34-R35-Gln-Glu-R3 8-R39-R4O-Arg-R.5 -Arg-Leu-Z, preferably 0-10 and X is naturally alanine, serine, treo where R 'is (XY) - (X'-Y) n with 0-20, a divalent amino acid, nin or proline, and if n = 0, then Y is selected from the group consisting of alanine, serine or threonine, X' is any naturally occurring amino acid, with the exception of proline or hydroxyproline,

ri is a Tyr or His, R ’ 2 is A1 a , Gly, Thr , Val and White or White, R3 is a Asp, Glu or Cya, Rs is a same time or Ser,

R 't 2 is Lys or Arg,

R 1 3 is a Val or Ile, R1 5 is a Ala, Val, Leu, Ile or Gly, R 1 8 is a Ser or Tyr, R 1 9 is a Val or ile, R ’2: 1 is ! Lys and 1ebo Arg, R2 2 is a Ala or Leu, R2 5 is a Asp, Glu or Cya, R 2 7 is a Met, clay ale bo L e u, R 2 8 is a Asn or Ser. R 3 4 is a Ser or Arg, R 3 5 is a Asn but b 0 S t: r, R 3 8 is a Arg or Gin, R 3 9 is a G 1 y or Arg, R 4 0 is a A 1 a a 1 e b 0 Ser, R í 2 is a Ala, Val, or ľ h e,

Z represents the carboxyl terminus of the amino acid residue at the C-terminus and is -COORa, -CR _and O, -CONHNHRa, -CON (R _a ) (Rb) or -CH 2 ORa with R _a and R t, which are C 1 -C 6 alkyl or hydrogen or a biologically active fragment thereof extending from the R at the N-terminus to any residue at any position between 27 to 44 as its C terminus, or Hse (lactone) ', HseOH or HseN (R _a ) (R bj) and non-toxic salts thereof. These modifications can be prepared according to currently known experimental procedures, since they retain a substantial part of the biological activity of the parent peptide, these modifications are considered to be part of the invention.

The extending portion of the GRF peptide of the invention has an amino acid sequence corresponding to formula (X-Y) - (X'-Y) n, wherein n represents the number of linearly linked X-Y groups and is 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, serine, and threonine, and only when n = 0 is Y selected from the group consisting of alanine, serine, or threonineine, and

X 'is selected from the group consisting of any naturally occurring amino acid s. with the exception of proline or, hydroxyproline.

According to this formula, if n = 1, there are two Y groups. Further, it is possible to have up to twenty-one Y groups and twenty X-groups in one embodiment. The individual residues Y and X 'may be any amino acid from the appropriate group from which to be selected. This means that 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 'group, each individual X' group may be any amino acid except proline and hydroxyproline, regardless of the nature of the other X 'residues. All the individual Y and X residues must conform to the rules for that particular (and it is sufficient, and one or more of the residues at the designated positions would correspond to the higher rules).

Table I

Effect of bGRF analogues on excretion of in vitro rat GH from cultures of rat anterior pituitary *

Bile. Title concentration ana1óga n M number. (as trifluoroacetate.) 0.01 0.1 1 10 without GRF dosing 1964 «1964« 1963 « 1963 « Leu ²⁷ bGRF (1-29) NH 2 3474® · ^d 75614 15172® 16506® 9 11 e ² A 1 and ¹⁵ Va ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 55988 120828 17230 ^f 7 11 e ² A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ bGRF (1-29) NH ₂ 5162 f - 811006i 816980 ^f 8 Val ² Ala 15 Val ¹⁹ Leu ²⁷ bGRF (1-29) NH2 4629® · f 11832i 8165788 6 Va1 ² A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ bGRF (1-29) NH2 . 4141 ^d . c ₁₀ 686 ^f 16370® F 1 Thr ² Ala * ⁵ Va ^{11 9} Leu ²⁷ bGRF (1-29) NH 2 3852 ^d 9252 ⁽ R) 16138® F 2 Thr ² Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1- 29) NH 2 2541 «· b 6263® ^d 13122 ^d Thr ² Ala * ⁵ Leu ²⁷ bGRF (1-29) NH 2 2 8 3 3 ^b · ® 5 8 31 ^c 13378 ^d 5 11e ² Ala> ⁵ Leu ¹⁹ Leu ²⁷ bGRF (1- 29) NH 2 ....... 5835® 12298 ^d 17272 ^d 4 Val ² Ala ' ⁵ Leu > ⁹ Leu ²⁷ bGRF (1-29) NH2 3976b ....... 9540® 16455® 3 Thr ² A 1 and ¹⁵ Leu ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 ......... 2443 « • 4483 ^b 13012 ^b

* The amount of rGH secreted from pituitary cell cultures was measured by radioinotest and is expressed in ng per sample over 4 hours. Assay conditions are described in Frohman et al., Methods Enzymol. 124, 371 (1986).

a, b. c, de, f, e _{in a} given column, the results are different from other indices at a significance level of p <0.05.

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

Table II

In vitro stability of GRF peptides substituted at position 19 in plasma

compound number Title (as trifluoroacetate) 11/2 [h] Leu ²⁷ bGRP (1-29) NH 0.6 * 1 Thr ² Ala> 5 Val ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 4.9® 2 Thr ² Ala ¹ 5Val ⁹ Leu ^{2 7} bGRF (1-29) NH 2 14.7 « Thr ² Ala ¹ Mal ^{1 9} I.eu ²⁷ bGRF (1-29) NHg 6.6 ^p 3 Thr ² Alai M.eu ¹⁹ Le u ²⁷ bGRF (1-29) NH 2 12.3 «

* Average! the value of three independent experiments (each performed three times).

7

9 a, b, c compounds with different indices are significance p <0.05. different on the surface Table III. Serum GH Ho 1 Stein Castor Wate Response to Analogs with Different Substitution at Positions 2 and 19 GRF scheme infections

X ² A1 and ¹⁵ X ¹⁹ Leu ²⁷ bGRF (1- 29) NH2

Dishes. number Bile. with 1 o Title * * ·' (as t ri f 1u ó r a e e t a t share [%] Max iraum [ng / ml] 1 Water 0 ^a 11.7 ^a 2 A bGRF (1-44) NH2 83 ^p 73.2 ^a 3 B Thr ² Ala ¹⁵ Leu ²⁷ bGRF (1-29) NH 100 ^p 75.7 ^a 4 3 Thr ² Ala ¹⁵ Leu ¹⁹ Leu ^{2 7} bGRF (1-29) NH 2 • 100 ^p 83.5 ^a 5 4 Val ² Ala ¹⁵ Leu ¹⁹ Leu ^{2 7} bGRF (1-29) NH 2 92 ^p 70.9 ^a 6 5 Ile ² Ala ¹⁵ Leu ¹⁹ Leu ^{2 7} bGRF (1-29) NH 2 92 ^p 42.8 ^a 7 1 ThHAla ^{1 5} Val ^{19 9} Leu ^{2 7} bGRF (1-29) NH 2 ₉₂ p 79.6 ^a 8 8 Val Ala ¹ 5 ² to ⁹ _and if the Leu ^{2 7} bGRF (l-29) NIl2 100 ^p 69.4 ^a 9 9 Ile ² Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-29) NH2 92 ^p 72.7 ^a 10 2 Thr ² Ala ¹⁵ 11 E ¹⁹ Leu ²⁷ bGRF (1-29) NH ₂ 92 ^p 60.7 ^a 11 6 Val ² Ala ¹⁵ 11e ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 92 ^p 78.7 ^a 12 7 Ile ² Ala ¹⁵ Ile ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 100 ^p 49.5 ^a 13 PULL 5 12.7f 14 P value 0.0001 0.75

Data presented include only animals that responded to GRF injections * * GRF analogs were given as intravenous injections pmol GRF / kg 2 hours for feeding * * * Heterogeneity of variation (Hartley F-max test), P value and index are calculated from logarithmic data transformation. a, b.c.d, e values with different indexes in the column are significantly different (P <0.05).

^f SEM for GRF holo 12.7 ng / ml, while SEM for water control was 45.2 ng / kg ⁶ SEM for GRF was 4 minutes, while SEM for co 111 with water holo 14 minutes.

16000-I

1400012000 • ο ο

(-1 η

Ν \

C) c

10000Compound • Uz7-bCRr (1-29) NH2- □ T2A1SL27- bGRFIL-2fl) NH2

O T2AlSU9L27-bCR (X1-29) NH2

M T2AJSV10L27-bGRF (1-29) NH2

X T2A1SI19UU7 — bGRF (1-29) NH2

C Basal ___ • 000 □

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k.0 IIIII ll | - ^ 1- T ¹ » ¹ » o · ''

ANALOG, nM

Figure 1

Effect of Thr ² A 1 and ¹⁵ Leu ²⁷ Analogs ⁷ bGRF (1-29) N1 2 on GH release in rat pituitary cell cultures in vitro. The experiment was performed according to the procedure of Frohman and Downs: Methods Enzymol. 1.2A, 371-309 (1,986). The Leu ¹⁹ substance had a deleterious effect on the ability of GRF analogs to release GH, while the V a 1 ¹⁹ and 13 e ¹⁹ modifications resulted in analogs with either increased or unchanged biactact in vitro compared to Thr ² A1 and ¹⁵ Leu. ²⁷ bGRF (1-29) NH 2. beu ^{2 7} bGRF (1 - 29) NH 2 served as experimental standard.

00-1

700050-29 CD

C x

to>

o (-1 'OJ tn

302010-

Compound X bCRF (I-44) NH2 • T2AlSL27-bCl RF (1-20) NH2

O T2A15LH> L27-bGRrtl-28) NH2 □ T2AlSV19L27-bGRF (1-28) NH2

T2AlS118L27-bGRF (l-281WH2

—T * .o

T

Minute

Figure 2

Mean serum growth hormone concentrations of GH in bran stein castrates fed by bran after intravenous injection of 10 pmol / kg GRF analogs. The experiment was performed as described by Moseley et al., J. Endocrinol. 117: 252-259 (1988). Bovine GRF (1-44) NH 2 was used as an experimental standard.

Claims (5)

GRF peptide with Val or clay at the amino acid residue site normally found at position 19, Gly, Thr, Val or clay at the amino acid residue site normally found at 2, and with one amino acid selected from the group consisting of Ala, Val, Leu, Ile, or Gly at position 15. The GRF peptide of claim 1, wherein the amino acid at The GRF peptide of claim 1, -A1 and -11e-Phe-Thr-Re-Ser-Tyr 1, characterized by position 2 is Thr, Val or Ile. of the formula R-R1-R2-R 3 Arg-Ri2 ~ RL3-Leu-Ri5-Gln-Leu-RI8-Ri9-Arg-R 2 -L-R22-Leu-Gln-R25-Ile-R27-R> 8-Arg-Gln-Gln-Gly-Glu-R 34 -R35-Gln-Glu-R38-R39-R4O-Arg-R12-Arg-Leu-Z where R is a H, C 1 -C 5 alkyl or benzyl ri is a Tyr and 1ebo His, R 2 is a Gly, Thr, Val or Ile, R 3 is a Asp, Glu or Cya, R 8 is a 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, Rt 3 is a wall or clay, R 1 5 is a Ala. Val, Leu, clay or Gly, R 1 8 is a Ser or Tyr, R 1 9 is a wall or clay, R2 1 is a Lys or Arg or N-epsi 1on-a1ky1 or N-epsi- 1 on -benzy1- Lys in case when R is C 1 -C 5 alkyl or benzyl R 2 2 is a Ala or Leu, R25 is a Asp, Glu, R 2 7 is a M e t., goals but b 0 L e u, R 2 8 is a same time a 1 e b 0 S e r, R i 4 is a S e r a 1 e b 0 Arg, R 3 5 is a same time or Ser, R 3 8 is a Arg or Gin, R 3 9 is a G 1 y a 1 e b o Arg, -: π » R 4 o is Ala or Ser, R 42 is Ala, VIIa or Phe and Z represents the carboxyl portion of the amino acid residue at the C-terminus and is -COORa, -CRaQ., -CONHNHRa, -CONRaRb or -CH 2 ORa, wherein R _a and R b 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-terminal, or Hse (lactone), HseOH or HseNRaRb, and / or non-toxic salts thereof. The GRF peptide of claim 3, wherein R 2 is Thr, Val or Ile. The GRF peptide of claim 4, selected from the group consisting of Thr2Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 Thr ² Ala ¹ Sil ¹⁹ Leu ²⁷ bGRF (1-29) NH2 Val ² Ala ¹⁵ 11e ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 Ile ² Ala ¹⁵ I1 e ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 Val ² Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-29) NH 2 Ile ² Ala ¹⁵ Val ¹⁹ Leu ²⁷ bGRF (1-2 9) NH2 Thr ² Ser ⁸ Ala ¹ 5 Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NH 2 Thr ² Ser ⁸ Ala ¹ 5 Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NHEt Thr ² Ser ⁸ Ala ¹⁸ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NH 2 Thr ² Ser ⁸ Ala ¹⁵ Val ¹⁹ Leu ²⁷ Ser ² Hse ^{3 3} bGRF (1-33) NHEt Thr ² Ser ⁸ Ala ¹⁸ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2 Thr ² Ser ⁸ Ala * ⁸ Val ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-44) NH 2 Thr ² Ser ⁸ Ala ¹⁸ 11 e ¹⁹ Leu ^{2 7} Ser ²⁸ Hse ³ ° bGRF (1-30) NH2 Thr ² Ser ⁸ Ala ¹ Sil ¹⁹ Leu ²⁷ Ser ²⁸ Hse ^{3 3} bGRF (1-33) NH 2 Thr ² Ser ⁸ Ala ¹⁸ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2 Thr ² Ser ⁸ Ala ¹⁸ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-44) NH 2 11 e ² Ser ⁸ A1 and ¹⁸ Va1 ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³ ° bGRF (1-30) NH2 11 e ² Ser ⁸ A to ¹⁸ Va1 ¹⁹ Leu ²⁷ Ser ^{2 8} Hs e ³⁰ bGRF ( ^1-30 ) NHEt 11 e ² S er ⁸ Λ 1 and ^{1 8} V a 1 ^{1 9} L eu ^{2 7} S er ^{2 8} 11 with ^{3 3} bG RF (1 - 3 3) NH 2 I 1 e ² S er ⁸ A 1 and ¹⁸ V and ^{11 9} l, f; u ^{2 7} 3 er ^{2 8} H with ^{3 3} bG RF (1 - 3 3) NHE t. I 1 e ² Ser ⁸ A 1 a ^{1 8} Va 1 ^{1 9} Le u ^{2 7} S er ^{2 8} Hs e ^{3 7} bGRF (1-37) N11 2 Γ1 e ² S er ⁸ A 1 and ¹⁵ Va1 ¹⁹ Leu ^{2 7} Ser ^{2 8} H with ⁴⁴ bGRF (1-44) NH 2 Va 1 ² Ser ⁸ Ala ^{1 r,} Va 1 ^{1 9} Leu ^{2 7} S er ^{2 8} H with ³ ° bGRI · '(1-3O) N1i2 3 2 Val ² Ser ⁸ A la ^{1 5} Val ^{1 9} Leu ^{2 7} S er ^{2 8} Hse ^{3 3} bGRF (1-33) NH2 Va1 ² Ser ⁸ A 1 and ¹⁵ Va1 ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2 Val ² Ser ⁸ Ala ¹⁵ Val ^{1 9} Leu ^{2 7} S er ^{2 8} lis ^{4 4} bGRF (1-4 4) NH 2 11 e ² Ser ⁸ A1 and ^{1 5} 11 e ^{1 9} Leu ^{2 7} Ser ^{2 8} Ilse ³ ° bGRF (1-30) NH 2 Ile ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NH2 11 e ² Ser ⁸ A1 and ¹⁵ 11 el ⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH ₂ 11 e ² Sef ⁸ A 1 & ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-44) NH2 Va1 ² Series ⁸ A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ Series ²⁸ Hse ³ ° bGRF (1-30) NH2 Va1 ² Ser ⁸ A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³³ bGRF (1-33) NH2 Va1 ² Ser ⁸ A1a ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NH 2 Va1 ² Ser ⁸ A 1 and ¹⁵ 11 e ¹⁹ Leu ²⁷ Ser ²⁸ Hse ³⁷ bGRF (1-37) NHnPr or Val. ² Ser ⁸ Ala ¹⁵ Ile ¹⁹ Leu ²⁷ Ser ²⁸ Hse ⁴⁴ bGRF (1-4) NH 2 or non-toxic salts thereof. 6. GRF p e p t i d by claim 3 'WITH t ý m that R 1 5 'Ala, wall 7. GRF pept i d by claim 6. t ý m that R is H and R15 is a 8. GRF t ý peptide m that by R1 is claim Tyr. 7 9. GRF peptide by claim 8 t ý m that Re a R 28 are Ser 10. . GRF peptide pod ľa claim 6. * t ý m that R2 is Val. 11, . GRF t ý peptide m that p 0 d ¾ a R2 is n and r 0 k u clay. 6. .12. . GRF t ý p e. p t i d m that according to claim R19 is V and 1. 6. 13th GRF p e p t i d p 0 d ¾ a n a r 0 k u 6. t. is that R 9 is white. characterized by Leu or clay. characterized by Ala. characterized by characterized by characterized by characterized by characterized by and 14. A method of stimulating growth hormone release in an animal, comprising administering to said animal an effective amount of a GRF peptide having a Val or clays at position 19, a Thr, Val or clays residue in place of the amino acid residue normally found at position 2, and one. an amino acid selected from the group consisting of Ala, Val, Lcu, Clay or Gly at position 15. / 15. The stimulation method of claim 14, wherein the GRF peptide has the general formula R-R-R2-R3-Ala-Ile-Phe-Thr-R8-Ser-Tyr-Arg-Ri2-Ri3-Leu-Gln-Ri5-Leu-RI8 ~ R'9-Arg-R 2i-R22-Leu-Gln R25-Ile-R27-R28-Arg-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R38-R39-R40-Arg-R42-Arg-Leu-Z where R is H, C 1 -C 5 alkyl or benzyl, ri is a Tyr or His, R 2 is a Gly, Thr, Val or clay R3 is a Asp, Glu á 1ebo Cya, re is a same time 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, R 13 is Val or clays, R 15 is Ala, Val, Leu, Ile or Gly, R 18 is Ser or Tyr, R1 9 is Val or clays, R 21 is Lys or Arg, or N-epsilon-alkyl or N-epsilon-benzyl-Lys when R is C 1 -C 5 alkyl or benzyl, R2 2 is a Ala or Leu, R2 5 is a As p, Glu, R 2 7 is a M e t, I 1 e or R 2 8 is a A s n and 1 ebo Ser, R 3 4 is a Ser a 1 e b 0 Arg, R 3 5 is a same time and 1 e 1) 0 Ser. R 3 8 is a Arg and 1 e bo G 1 n, R39 is a G 1 y a 1 e b 0 Arg, R 4 n is a A 1 a and 1 ebo · Ser. R A 2 is a Λ I a, V a 1, and 1 e bo Z represents the carboxyl portion of the amino acid residue at the C-terminus is -COORa, - CR _and O, -CONHNHRa, -CONRaR t> or -CH 2 ORa, wherein R _a and R b are C 1 -C 6 alkyl or hydrogen, or biologically active thereof a fragment extending from the R at the N-terminus to a residue at any position between 27 to 44 as its C-terminus, or Hse (lactone), HseOH or IlseN-R _and Rb and / or non-toxic salts thereof. 16. A composition for release of growth hormone in an animal comprising an effective amount of a GRF peptide of the formula: R-R1-R2-R3-Ala-Ile-Phe-Thr-R8-Ser-Tyr-Arg-Si2-R13-Leu-R15-Gl-Leu-R18-R19-Arg-R21-R22-Leu- Gln-R25-Ile-R27-R28-Arg-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R38-R39-R40-Arg-R42-Arg-Leu-Z where R is H, C 1 -C 5 alkyl or benzyl, ri is a Tyr or Hi s, R 2 is a Gly, Thr wall or clay R3 is a A with p, G 1 u or Cya, re is a Asn and 1ebo Ser. R 1 2 is Lys or Arg or N-e-alkyl- or N-eps i- lon-benzyl- Lys v if R is C 1 -C 5 alkyl or benzyl, R 13 is wall or Ile, R15 is Ala , Val, Leu, clay or Gly, R18 is Ser or Tyr, R19 is wall or Ile, R 21 is I.ys or Arg, or N-e-alkyl, or N-eps i - 1on-benzyl-1 Lys v if R is C 1 -C 5 alkyl and 1 ebo benzyl, R '2 2 is Ala or Leu, R2 5 is Asp , G 1 u, R2 7 is Met , clay a 1 e b 0 L e u, R2 8 is same time or Ser. R3 4 is Ser a 1 e b 0 Arg, R 3 5 e same time a 1 e b 0 Ser. R 3 8 e A r g and 1 e bo Gin, R 3 9 e G) y and 1 ebo Arg, R 4 0 j 0 Ala and 1 e 1> 0 S e r, R 4 2 is Ala, Val but: b o P h e, a Z is carboxyl moiety of the amino acid at the C-terminus and is a group -COOR a, -CR _a O, -CONHNHRa, -CONR a R b or -CH 2 OR _a, wherein R _a and R b are C -C ^ alkyl or hydrogen, or a biologically active a fragment extended from the R at the N-terminus to a residue at any position between 27 to 44 as its C-terminus, or Hse (lactone), HseOH or HseN-R _and Rb and / or r / their non-toxic salts in combination with a pharmaceutically acceptable carrier.