CA2092906A1 - Stabilized, potent grf analogs - Google Patents

Abstract

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

Description

wo 92/08481 ~ 9 2 9 ~ ~ Pcr/usg1/08248 STABILIZED, POIENT GRF ANALOGS
INTRODUCTION
The present invention relates to a peptide having influence on the function of the pituig gland in humarls and other animals, particularly marnmals. In particular, the present S invention is directed to peptides which promote the release of growth hormone by she pituitary gland. The peptides of the present invention are potent in YiVo, more stable in plasma and selected peptides are more stable in an aqueous environment at neutral pH than native GRF
sequences.
BACKGROUND OF THE IN~ENTION
Physiologists have long recognized that the hypothalamus controls the secretory functions of the adenohypophysis with the hypothalamus producing special substances which stimulate or inhibit the secretion of each pituitary hormone. In 1982, human pancreasic (tumor) : -releasing factors ~pGRF) were isolated from extracts of human pancreatic tumors, purified, characterized, synthesized, tested, and found to promote release of growth hormone (GH) by thepituitary. Guillemin, R., et al., Science218, 585-585 (1982). Since then, corresponding hypothalamic GH releasing factors from other species including the rat species, the porcine species, the ovine species, the boviDe and caprine species and from the human species have also been characterized and synthesized.
Human hypothalamic GRF ~GRF) has been found to have the same formula as hpGRF, namely: H-Ty~-Ala-Asp-Ala-lle-Phe-Thr-Asn-Se~-Tyr-Arg-~ys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-lle-Met-Ser-Arg-Gln-Gln-Gly-Glu-Se~-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH2.
Rat GRF (rGRF) has been found to have a Ser residue at position 8 and the formula:
H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-lle-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH. (See for example US Patent 4,S95,676).
Bovine GRF (bGR~;) has been found to have the formula: H-Tyr-Ala-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Atg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Giu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH2.
Porcine GRF has been found to have a Ser residue at position 28.
- ~ It has been reported that natiYe GRP sequences are subject to rapid inactivation by blood plasma enzymes. The rapid breakdown involves cleavage of the 2-3 bond of the pèptide by a dipeptidylpeptidase, Type lV ~PP-IV), which in the past was also named - -dipeptidylaminopeptidase-IV. Frohman, L.A. et al., J. Clin. ~nvest., 78, 906 913 (1986).
The metabolic stability of GRF and various methods for protecting GR~: peptides against dipeptidylpeptidase cleavage have been proposed, including Felix et al., Synthesis and SUBSTITUT~ SHEE~

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WO 92/084Bl 2 0 9 ~ ~ PCr/US91/û8248 biological activity of novel linear and cyclic GRF analogs, in Peptides. Chemistry and Biology, Proc. 10th Am. Peptide Symposium, Ed. G.R. Marshall, ESCOM Sci. Publishers, Leiden, The Nether!and, pp.465 467, (1988), who reported on GRF analogs substituted with desNH2-Tyr at position 1, or/and D-Ala at position 2 which had enhanced stability of their N-terrnini to 5 enzymatic degradation. This information was recently confirmed by Frohman et al., Dipeptidylpeptidase-IV and trypsin-like enz~matic degradation of human gro~ hormone-releasirlg 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 residue or substitution with D-Tyr-1 in GRF completely inhibited cleavage at the 2-3 position. On the 10 other hand, alpha-methylation of Tyr-1, only partially blo~ked degradation by DPP-IV.
Murphy, W.A. and Coy, D.H., Potent long-acting alkylated analogs of growth hormone-releasing factor, Peptide Research 1, 36~1 (1988), describe analogs of &RF which show enhanced resistance to enzymatic degradation as a result of N-alkylation or N-arylalkylation of the N-terminal amino acid with or without concomitant N-alkylation of the side groups of 15 lysines within the peptide chain.
Native GRF sequences have a Gly residue at the 15-position. Analogs with Ala or Leu at the 15-position are known to have increased GH releasing potency. See for exarnple US
Patents 4,649,131 and 4,734,399 as well as Ling, N., et al., Quo Vadis?, Symposium, Sanofi Group, May 29-30, 198~, Toulouse-Labege, France (pp. 309-322). Substitutions of Gly-15 20 with Val or alpba-amino-isobutiric acid also resulted in enhanced potency of GRF analogs, Feli~ et al., Synthesis and biological activity of novel growth honnone releasing factor analogs, in Peptides 1986, Walter de Gruyter & Co., Berlin-New York, pp. 481~84 (1987).
A.M. Felix has reported on a prograrn to synthesize analogs with enhanced and/orprolonged biological activity, including the preparation and testing of Ala15 h-GRF(1-29)NH2 25 and desNH2-Tyr1, D-Ala2, Ala15 hGRF(1-29)NH2. See, for example, U.S. Patents 4,649,131 and 4,734,399 as well as A.M. Felix, E.P. Heime~, T.F. Mowles, H. ~3isenbeis, P. Leung, T.J. Lambros, M. Ahmad, C.T. Wang & Paul Brazeau: Synthesis and biological activity of novel growth hormone releasing factor analogs. in Peptides 1986, pp. 481-484 (1987); Felix, A.M., Wang, C.T., Heimer, E., Fournier, A., Bolin, D., Ahmad, M., Lambros, T., Mowles, 30 T., and MDler, L.: Synthesis and biological activity of novel linear and cyclic GRF analogs. in Peptides. Chemistry and Biology. Proceedings of the 10th American Peptide Symposium, E~d.
G.R. Marshall, Escom Science Publishers, Leiden, The Netherlands (1988), pp. 465~67;
- D.Peticlerc, H. Lapierre, G. Pelletier, P. DubreuD, P. Gaudreau, T. Mowles, A. Felix and P.
Brazeau: Effect of a potent analog of human growth hormone-releasing factor (hGRF) on 35 growth hormone (GH) release and mDk production of dairy cows. Meeting Abstract P223, 82nd Meeting American Dairy Sci. Assn., Columbia, MO, June 21-24 (1987).
SUBSTITUTE SHEET

WO ~2/08481 2 ~ ~ 2 9 ~ ,~ PCl/US91/08248 A GRF analog that was modified with Ser2, in addition to eight other modifications in the same molecule, is described by Tou et al., Amphiphilic Growth Hormone-Releasing Factor (GRF) analogs: peptide design and biological activity in vivo. Biochem. Biophys. Res.
Com~nun. 139, 763-770 (1986). This analog was reported to have 16~% activity in ViYo in S sheep as compared to bGRF(l 44~NH2.
U.S. Patene 4,734,399 discloses GRF analogs having Ala, N-Methyl-D-Ala or D-Ala at position 2 and Ala, Leu, Val, lle, Nle, Nval or B-Ala i~t position 15. See also U.S. Patent 4,649,131.
European Patent Application of Coy and Murphy, Publication Number 0 188 214, 10 Application Number 86100127.9, discloses GRF analogs with Leu or Phe at position 2, in addition to GRP peptides having various unnatural amino acids of L or D-configuration as substituents at position 2.
GRF analogs with very low bioactivity having Sar2 or pro2 are described by Coy et al., Strategies irl the design of synthetic agonists and antagonists of grow~ hormone-releasing 15 factor, Peptides, vol. 7, Suppl. 49-52 (1986).
Lin~ et al., Growth hormone-releasing factor analogs with potent antagonistic actiYity. in Peptides. Chemistry and Biology. Proceedings of the 10th American Peptide Symposium, Ed.
G.R. Marshall, Escom Science Publishers, Leiden, 'rhe Netherlands (1988), pp. 484 486, reported on a series of GRF analogs substituted with either Arg or a variety of D-amino acids 20 at position 2. All of them are less potent than the parent horTnone and some of them displayed antagonistic activity.
A prior invention provides synthetic GRF polypeptides having a Ser residue in place of the amino acid residue norrnaily found at position 8 and 28 of the polypeptide as a means of inhibiting chemical breakdown (deamidation) in aqueous environrnents. See U.S. Patent 25 Application Serial No. 07/303,518, filed 27 lanuary 1989 and Serial No. 07/323,955, filed 15 March 1989. See also A.R. Friedman, A.K. Ichhpurani, D.M. Brown, R.M. Hillman, L.F.
Krabill, R.A.Martin, H.A. Zurcher-Neely, and D.M. Guido. The Degradation of Growth Horrnone Releasing Factor Analogs in Neutral Aqueous Solution is Related to Deamidation of Asparagine Residues. Replacement of Asparagine Residues by Serine Shbilizes. Int. J.
30 Peptide Protein Res., 37, 1~20 (1991).
- A prior invention provides synthetic GRF polypeptides having a cysteic acid residue (Cya) substituted for the arnino acid residue in position R3 andlor R2~; See U.S. Patent Application Serial No. 07/150,301, filed 29 3anuary 1988 and Serial No. 89/00245, filed 27 January 1989.
A 29-amino acid analog of hGRF was designed by G. Velicelebi, et al., Proc. Natl.
Aca. Sci USA, Vol 83, 5397-5399 (1986), in which the sequence of the first six amino acids at SlJ135TITUTE SHE~

wo 92/08481 2 0 9 2 9 0 6 ~ PCI/US91/08248 the amino terminus, and differing from the natural peptide by 13 amino acid in ~he rest of the sequence including incorporation of ~ Ser residue at position 8. The amide and free acid f~rms of the analog had the forrnula: H-Tyr-Ala-Asp-Ala-Ile-Phe-Ser-Ser-Ala-Tyr-Arg-Arg-Leu-Leu-Ala-Gln-Leu-Ala-Ser-Arg-Arg-Leu-Leu-Gln-Glu-Leu-Leu-Ala-Arg-NH2/OH. When assayed for S the abili~ to stimulate growth hormone (GH) secretion in primary cultures of rat arlterior pituitary cells, the amide analog was 1.57 times æ potent as hGRF(1~0)OH, while the free acid fonn was reported to be 1/6th as potent in the same assay.
Vale, et al., (US Patent Application Serial No. 053,233, filed May 22, 198~) describe 31-residue hGRF analogues which utilize a 31-position residue possessing a functional side 10 chain group which may be conjugated to a separate protein. The 31-residue hGRF analogues may also have substitutions for other residues which appear in a natural &RF sequence, such as Asn or Ser in the 8-position, Phe in the lQposition, or Ala in the 15-position. Asn or Ser may be present in the 28-posidon.
Asn residu~s in polypeptides are reported to be the subject, under some circumstances, 15 to deamidation in the presence of water. However, the rules governing the rates of deamidaffo~ are not clear. For e~ample, in the polypeptide trypsin only some of the Asn resi-dues, with the partial se~uence Asn-Ser, are deamidated while others are not. See Kossiakoff, M, Science 240, 191-194 (1988).
European Patent Application Number 86308337.4, Publication number 0 220 958, 20 discloses a class of compounds having the formula H-X-Pro-Peptide in which X is the residue of a naturally occurring amino acid, Pro refers to the naturally occurring amino acid proline aod Peptide is a sequence of arnino acid residues defining that of a biologically active peptide or protein, Examples of H-X-Pro-Peptide include Met-Pro-(growth hormone-releasing factor) which carl be chemically converted to GRF.
N-terminally extended analogs on non-GRF peptides have been reported for variouspurposes including, for example:
M.A. Tallon et al., Biochem., 26:7767-7774 (1987), made synthetically a series of N-terminally extended analogs of yeast alpha-mating factor with Ala, Glu-Ala, Ala-Glu-Ala or Glu-Ala-Glu-Ala in the extensioQ part. These peptides were used in strucnlre-activity relationship studies.
~ D. Aodreu et al., 20th Eur. Pepîide Symp., Tubiogen, GFR, September ~9, 1988, Syrnposium Abstracts, p. 33, synthesized the entire 64 amioo acid sequence of the precursor form of cecropin A along with several shorter peptides corresponding to potential processing intermediat~s. Among them there was a full cecropin sequence extended with Ala-Pro-Gly-Pro at its N-terrninus which was used to show that the extension part was indeed cleaved by a par-tially purified dipeptidylpeptidase-like enzymatic preparation obtained from the cecropia SUBSTITlJ'rE SHEE:l wo 92/08481 PCI/US91/OX248 -5- 2~29~'~
silkmoth pupa. See also H. Boman et àl. J. Biol. Chem. 264:5852-~860 (1989).
a. Kreil et al., Eur. J. Biochem., 111 49-~8 (1980) reports that melittin, ehe main constituent of honeybee venom, is derived from pro-melittin. The pro-sequence of pro-melittin consists of six X-Pro and five X-Ala repetitive dipeptidyl residues. The resul~s presented by 5 Kreil et al. suggest that the precursor-product conversion may proceed via stepwise cleavage of - dipeptide units by a dipeptidylpeptidase IV type enzyme present in e3~tracts from venom glands.
C. Mollay et al., Eur. J. Biochem., 160:31-35 (1986). Caerulein and xenopsin are two peptides found in slcin secretion of Xenopus laevis. The former has a sequence of Phe-Ala-Asp-Gly and the latter Ser-Ala-Glu-Ala in the N-terminal extensions in their respective 10 precursor forms. A dipeptidylpeptidase of type IV, isolated from frog skin secretion, has the specificity required for the cleavage of these N-terminal extensions leading to the formation of the mature products.
I:). Julius et al., Cell, 32:839-852 (1983). Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cervisiae alpha cells Nonmating alpha-cell 15 mutants, which lack a membrane-bound dipeptidylpeptidase, do not produce normal alpha-factor, but releass a collection of incompletely processed forms with structures Glu-Ala-Glu-Ala-alpha-factor or Asp-Ala-Glu-Ala-alpha-factor that have a markedly reduced biological activity. It has been shown that the membrane-bound dipeptidylpeptidase is required for normal alpha-factor precursor processing and this process may be rate-limiting for alpha-factor 20 maturation in ~ormal yeast alpha cells.
C.L. Choy et al:, Eur. J. Biochem., 160:267-272 (1986). The prosequence of the antifreeze protein from the Newfoundland winter flounder contains four X-Pro and seven X-Ala repetitive sequences in its N-terrninal part. Although the processing of this precursor has not been investigated, the authors speculate that such a conversion might take place in serum by a 25 dipeptidylpeptidase-line enzyme which would sequentially cleave the dipeptidyl units in the extension part to release the mature antifreeze protein.
Subsequent to the priority date of the parent application, Suhr et. al. reported the isolation and characterization of a full-lengtb cDNA clone encoding mouse GRF. The mature mouse GRF was predicted to be a 42 amino acid residue peptide with a free carboxyl-terrninus.
30 . This peptide has a valine residue at position 2 which makes it unique among the GR~s from other species all having Ala at position 2. See Mol. Endocrinology 3:1693-1700, 1989.
: Based on considerations proposed by others, GRF analogs with Leul9 substitution [namely, Thr2 Alal~ Leul9 Leu27-bGRF(1-29~NH2, trifluoroacetate salt Compound No. 3;
Leu19 Leu27 bGRF(1-29)NH2 trifluoroacetate salt; Thr2 Leul9 Leu27 bGRF(1-29)NH2 35 trifluoroacetate szlt; Ala15 Leul9 Leu27 bGRF(1-29)NH2 trifluoroacetate salt] were prepared.
When tested in virro in a growtb hormone (GH) release assay using rat anterior pituitary cells, SU?BST~TUTE SHEET

- - - . , , :, ' :': . ' , ' ' " :. ' " ' ' , ' .
. 1, . . ~ , . . " ' , .. , , ~ . , ' j: . .
- , . ..

:
WO 92/08481 P~/US91/08248 20~290~ ~ -the Leul9 analogs (namely, Thr2 Ala1s Leul9 Leu27-bGRF(1-29)NH2, trifluoroaceta~e salt Compound No. 3, Leul9 Leu27 bGRF(1-29)NH2 trifluoroacetate salt, Thr2 Leul9 Leu27 bGRF(1-29)NH2 trifluoroacetate salt, and Alals Leul9 Leu27 bGRF(1-29)NH~ trifluoroacetate salt were significantly less active than the corresponding Ala19 allalogs. When tested ~n ViYo, S in growi~g steers at 10 pmol/kg, the amount of GH released over the 2 hour test period by the Leul9 analogs was not significantly different (p > 0.05) from the Alal9 compounds. When tested for stability ill bovine plasma, the Leu19 compounds were more stable than the Alal9 analogs.
In the GRF molecule, residue 19 is in a region which is helical in membrane like10 enYironments lClore,G.M., Martin,S.R., and Gronenborn, A.M., J.Mol. Biol. I9l, 553-561 (1986)]. It is not known if the role of Alal9 residue in GR~: molecules is structural or whether it is a receptor contact residue [Sato,K., HoKa,M., Kagegama,J., Chiang,TC., Hu,HY., Dong,MH., and Ling,N., Biochem. Biophys. Res. Commun. 149 (2) 531-537 (1987).
Schiffer,M., and Edmundson,A.B., Biophys. J. 7, lZ1 (1967)]. Substin~tion of Alal9 by D-15 Alal9 i~ hGRF(1-29)NH2 reduced in vitro St release potency to 6% of the L-Alal9 analog in a rat pituitary ce]l assay. This same substitution in Nlæ rGRF(1-29)NH2 reduced potency to 10% of the standard in a bovine pituitary cell test system. This same substitution in Nle27 rGRF(1-29)NH2 reduced potency tO 30% of standard in a rat pituitar~Y cell test system [Rivier, J. Second International Symposium on Vasointestinal Peptide and Related Peptides, Cap d'Agde 20 France June 1985]. However, a Serl9 GRF analog was fully as potent as hG~F(140) in a rat pituitary cell St release assay ~Velicelebi,G., Patthi,S., and Kaiser,T.E., Design and biological activity of analogs of growth hormone releasing factor with potential amphiphilic helical carbo~cy termini, Proc. Natl. Acad. Sci. 83, 5397-5399 (1986)].
Getzoff reported that in a peptide epitope of myohemerythrin, Ala, Ile, Ser and Thr 25 could be substituted for Val without loss of antibody binding, but that a Leu substituted peptide had reduced binding [Getzoff,E.D., Gysin,H.M., Rodda,S.J., Alexander,H., Tainer,J.A., and Lerner,R.A., Mechanism of antibody binding to a protein. Science, 235, 1191-1196 (1987)].
WO 90/15821 (published 27 December 1990 after the priority date of the subject appli~ation) discloses various GRF PEErrlDES haYing Thr, Val or lle residue in place of the 30 amino acid residue normally found at position 2, Ala at position 19, in combination with one of the following amino acids Ala, Val, Leu, Ile or Gly at position 15. Optionally, the GRF
PEPIIDE can have a Ser residue in place of the amino acid residue norrnally found at position 8 and 28 of the polypeptide. , ~ .
- In addition, the GRF PEPIIDES could be optionally be N-terminally extended with Cl-35 Cs allcyl, benzyl, H-(Y-X)Q or H (Y-X)m(Y'-X')p wherein Y and Y', being the sarne or different, is a naturally occurring amino acid, preferably Tyr or Asp; X and X', being the same WO 92/08481 2 ~ 9 2 9 a ~ PCI/US91/08248 or different, is selected from Thr, Ser or Ala, p~eferably Thr or Ser; n is 1-10; m is 1-5; p is 1 -s.
EP Heilmer et al., (12th Am. Peptide Symposium, Carnbridge, MA June 16-22, 1991)Abstract #P-32 in the symposium ~Program and Abstract~ repoIted that the Val2 renders mGRF
s resistant to cleavage by dipeptidylpeptidase lV. A series of hGRF analogs incorporating - position 2-modificatiorls, with emphasis on Val2, were ~eported. At the poster session; Gly2, Ile2, and Leu2 GRF analogs were also disclosed.
SUMMARY C~F T~IE IN~ENTION
The present invention provides a polypeptide which promotes the release of growth hormone by the pituitary gland (GRF PEErIlDE) and having Val or Ile at position 19. The GRF PEPTlD~ will have Gly, Thr, Val or lle residue in place of the arnino acid residue nor nally found at position 2 in combination with one of the following amino a~ids Ala, Val, Leu, Ile or Gly at position 15. Optionally, the GRF PEPIIDE can have a Ser residue in place of the amino acid residue normally found at position 8 and 28 of the polypeptide. In addition, the GRF PEPIIDES of the present in~vention can optionally be N-terminally extended with Cl-C5 alkyl, benzyl, ~ (X'-Y)n wherein n is G20, preferably 0-10, X is any naturally occuring arnino 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' is any naturally occuring amino acid e~cept proline or hydroxyproline.
The peptides of the present invention are potent in v~vo and more stable than native GRF sequences against breakdown by blood plasma enzymes. In addition, compounds substituted with Ser8 and Ser28 are protected against deamidation in aqueous environments and are chemically more stable.
DETAILED DESCRIPllON Ol: THE INVENTION
2~ The term "&R~7 PEPIlDEn, as used in the speciffcation and claims, means a known polypeptide which is between 27 and 44 residues in length and that promotes the release of growth honnone by the pituitary gland. Illustrative GRF PEPIIDES include the natural or synthetic polypepddes disclosed in US Patent Nos. 4,517,181, 4,518,586, 4,528,190, 4,529,595, 4,563,352, 4,585,756, 4,595,676, 4,605,643, 4,610,976, 4,626,523, 4,628,043, 4,689,318, 4,784,987, 4,843,064 and U.S. Patent Application Serial No. 89l00245, filed 27 lanuary 1989; all of which are incorporated herein by reference. Felix, A., Wang, C.T., Heimer, E., Fournier, A., Bolin, D., Ahmed, M., Lambros, T., Mowles, T., and Miller, L., "Synthesis and Biological Activity of Novel Linear & Cyclic GRF Analogsn, in ieptides.
Chemistry and Biology, Proc. 10th Am. Peptide Symposium, Ed. G.R. Marshall, ESCOM Sci.
Publishers, Leiden, The Netherland, pp.465 467, (1988); Tou, J.S., Kaempfe, L.A., Vineyard, B,D" Buonomo, F.C., Della-Fera, M.A., and Baile, C.A., "Amphiphilic Growth Hormone SUlBS~ S~;~ d , WO 92/08481 2 ~J 9 2 9 ~ ~ -8- Pcr/VS9~/~8248 Releasing Factor Analogs. Peptide Design and Biological Activity in vivo Biochem. Biophys Res. Commun. 139 #2, pp. 763-770 (1986); Coy, D.H., Murphy, W.A., Sueires-Diaz, J., Coy, E.J., Lance, V.A., 'Structure Activity Studies on the N-Tenninal Region of Growth Ho~none Releasing Factorn, J. Med. Chem. 28, pp. 181-185 (1985); Felix, A.M., Heimer, 5 E.P., Mowles, T.F., Eisenbeis, H., Leung, P., Larnbros, T.J., Ahmed, M., and Wang, C.T., "Synthesis and Biological Activiey of Novel Growth Hormone Releasing Factor Analogs", in Peptides 1986, Walter de Gruyter & Co., BerliD-New York, pp. 481~84 (1987); Velicelebi, G., Patthi, S., and Kaiser, E.T., ~Design and Biological Activity of Analogs of Growth Hormone Releasing Factor with Potential Amphiphilic Helieal Carboxyl Termini", Proc. Natl.
10 Acad. Sci. U.S.A., 85, pp. 5397-5399 (1986); Ling., N., Baird, A., Wehrenberg, W.B., Munegumi, T., and Ueno, N., "Synthesis GRF Analogs as Competitive Antagonists of GRF
Therapeutic Agents Produced by Genetic Engineeringn, Quo Vadis Symposium, Sanofi Group, May 29-30, 1985, Toulouse-Labege, France, pp. 309-329. Murphy, W.A. and Coy, D.H., Potent long-acting alkylated analogs of growth horrnone-releasing factor, Peptide Research 1, 15 36~1 (1988). 3.C Tou, L.A. Kaempfe, B.D. Yineyard, F.C. Buonomo, M.A. Della-Fera and C.A. Baile: Amphiphilic Growth Hormone-Releasing Factor (GRF) analogs: peptide design and biological activlty in vivo. Biochem. Biophys. Res. Commun. 139, 763-770 (lg86). The tenn GR}~ PEPTIDE includes nontoxic salts thereof.
The nomenclature used to define the GRF PEPTIDE is that specified by Schroder &
20 Lubke, "The Peptides", Acadernic Press (1965) wherein in accordance with conYentional representation the amino group at the N-terminal appears to the left and the carboxyl group at the C-terminal to the right. Where the amino acid residue has isomeric forms, the L-form of the amino acid is being represented unless otherwise expressly indicated.
The present invention provides synthetic GRF peptide analogs (GRF PEPIIDES) 25 having the following formula:
R-Rl-R2-R3-Ala-lle-Phe-'rbr-R8-Ser-Tyr-Arg-R12-R13-Leu-R15-Gln-Leu-R1 8-Rlg-Arg-R21-R22-Leu-Gln-R25-Ile-R2~ R28-Arg-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R38-R39 R40 Arg R42-Arg-LeU-Z
wherein ~ -R is H, Cl-C5 alkyl or benzyl;preferably H
R1 is Tyr or His, preferably Tyr;
~, R2 is Gly, Thr, Val or Ile, preferably Val or Ile; --. ........ .
R3 is Asp, Glu or Cya, preferably Asp;
- R8 is Asn or Ser, preferably Ser;
35 - R12 is Lys, N-e-allyl- or N-e-benzyl-Lys or Arg, preferably Lys; or N-~-alkyl- or N-~-benzyl-Lys when R is Cl-C5 alkyl or benzyl;

Sl.~ lTUT~ S~
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,, ' , . ' . . ' I;' . ' ,' ' ' . " ` ~ ' '' ' ' ' . , ' ' .' ,` ' ' ~ ~ . ' ' , ., ' ' ' ' ', WO 92/08481 PCr/US91/08248 2~2~
Rl3 is Val or Ile, preferably Val;
~15 is Ala, Val, Leu, lle or Gly (preferably Ala, Val, Leu or Ile, more preferably Ala), R1g is Ser or Tyr, preferably Ser;
S Rlg is Val or Ile; (preferably Val);
R21 is Lys, N-~-alkyl- or N-~-benzyl-Lys or Arg, preferably I,ys or N-~-alkyl- or N-~-berl7yl-Lys when R is Cl-Cs alkyl or benzyl;
R22 is Ala or Leu, preferably Leu;
R25 is Asp or Glu, preferably Asp;
R27 is Met, lle or Leu, preferably Leu;
R28 is Asn or Ser, preferably Ser;
R34 is 5er or Arg, preferably Arg;
R35 is Asn or Ser, preferably Asn;
R3~ is Arg or Gln, preferably Gln;
R39 is Gly or Arg, preferably Gly;
R40 is Ala or Ser, preferably Ala;
R42 is Ala, Val or Phe, preferably Val; and Z signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COORa, -CRaO, -CONHNHRa, -CON(R~)(Rb) or -CH20Ra, with Ra aod Rb being Cl-C8 alkyl or hydrogen; or a biologically active fragment thereof extending frotn R at the N-terrn~nus to a residue in any of positions 27 through 44 as its C-terminus; or a Hse(lactone), HseOH or HseN(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing. Ra is preferably hydrogen (H).
Rb is preferably Ethyl Examples of Cl-C8 alkyl are methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl and isomeric ~orms thereof.
The terrn iPr refers to isopropyl.
Tbe term Bzl refers to benzyl.
An embodiment of this invention are GRF peptides where Rlg is Val or lle, including the peptides Thr2 Ala15 Vall9 Leu27 bGRF(1-29)NH2, Thr2 Ala15 Ilel9 Leu27 bGRF(l-29)NH2, Val2 Ala15 Ilel9 Leu27-bGRFtl-29)NH2, 11e2 Ala15 11el9 Leu27-bGRF(1-29)NH2, - ' Val2 Ala15 Vall9 Leu27-bGRF(1-29)NH2, lle2 Ala15 Vall9 Leu27-bGRF(1-29)NH2; Gly2 Ala15 Vall9 Leu27-bGRF(1-29)NH2, Gly2 Aia15 Vall9 Leu27-bGR~ 29)NH2, preferably the --peptides Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse30 bGRF(1-30)NH2, Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse33 bGRF(1-33)NH2, Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse37 bGRF(1-37)NH2, Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse44 bGRF(1~4)NH2, Thr2 Serg Ala15 11el9 ` ` - 5~ UTE SHEET

WO 92/08481 PCr/US91/082~8 2~929~
Leu27 Ser28 Hse30 bGRF(1-30)NH2, ~r2 Ser8 Alal5 Ilel9 Leu27 Ser28 Hse33 bGRF(1-33)NH2, Thr2 Ser8 Ala15 Ilel9 Leu27 Ser28 Hse37 bGRF(1-37)NH2, or lhr2 Ser8 Alals Ilel9 7 eu27 Ser28 Hse44 bGRF(1 44)NH2; more preferably Ile2 Ser8 Ala15 ~all9 Leu27 Ser28 Hse30 bGRF(1-30)NH2, Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse30 bGRF(1-30)NHEt, lle2 Ser8 Alal5 S Vall9 Leu27 Ser28 Hse33 bGRF(1-33)N~I2, Ile2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse33 bGRF(I-33)NHEt ~2 Ser8 A~a15 ~allg LeU27 Ser28 Hse33 bGRF(1-33)NHEt Ile2 Ser8 Ala1s Yall9 Leu27 Ser28 Hse37 bGRF~1-37)NH2, Ile2 Ser8 Ala15 Yall9 Leu27 Ser28 Hse44 bGRF(l-44)NH2, Val2 Ser8 AlaI5 Val19 Leu27 Ser28 Hse30 bGRF(1-30)NH2, Val2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse33 bGRF(1-33)NH2, Val2 Ser8 Ala1s Vall9 Leu27 Ser28 Hse37 bGR~
10 37)NH2, Val2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse44 bGRF(I 44)NH2, lle2 Ser8 Alals llel9 Leu27 Ser28 Hse30 bGR~(1-30)NH2, 11e2 Ser8 Ala15 llel9 Leu27 Ser28 Hse33 bGRF(I-33)NH2, 11e2 Ser8 Ala15 llel9 Leu27 Ser28 Hse37 bGRF(1-37)NH2, lle2 Ser8 Alal5 Ilel9 Leu27 Ser28 Hse44 bGR~(l 44)NH2, Val2 Ser8 Ala15 Ilel9 Leu27 Ser28 Hse30 bGRF(l-30)NH2, Val2 Ser8 Ala15 llel9 Leu27 Ser2g Hse33 bGRF(1-33)NH2, Val2 Ser8 Ala15 Ilel9 15 Leu27 Ser28 Hse37 bGRF(1-37)NH2, Val2 Ser8 Ala15 Ilel9 Leu27 Ser28 Hse44 bGRF(1-44)NH Val2 Ser8 Ala15 Ilel9 Leu27 Ser28 Hse37 bGRF(1-37)NH-n-Propyl; or a non-toxic salt thereof.
Most preferred GRF peptide of the subject invention is lle2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse30 bGE~F(1-30)NHEt.
Still another embodiment of this inventiorl are N-terminally e~tended peptides, including for e~ample N-cY-(Tyr-Ala-Phe-Pro-Phe-Ala)-Tyrl 'rhr2 Ser8 Alal,S Ilel9 Leu27 Ser28 bGR~(l-29)NH2; N-~Y-tLeu-Pro-Gly-Pro-Tyr-Ala)-Tyr1 Thr2 Ser8 Ala15 Vall9 Leu27 Ser28 bGRF(I-29)NH2; N-o~-(Ala-Pro-Gly-Pro-Tyr-Ser)2-Tyrl Val2 Ser8 Ala15 llel9 Leu27 Ser28 Hse 32 bGRE7(1-32)NH2; N-c~-(Leu-Pro-Tyr-Ala-Tyr-Ala)-Tyrl Ile2 Ser8 Alal5 Vall9 Leu27 Ser28 25 bGR~ 29)NH2; N-cY-tHis-Ala-Tyr-Pro-Tyr-Ala)-Tyrl lle2 Ser8 AlalS llel9 Leu27 Ser28 bGRF(1-29)NH2; N-a-(C~lu-Pro-Phe-Ala-Tyr-Pro-His-Ala)-Tyrl lle2 Ser8 Ala15 Vall9 Leu27 Ser28 bGRF(1-29)NH2; N-a-(His-Pro-His-Pro-His-Ala-Tyr-Ala)-Tyrl Thr2 Ser8 Alals Vall9 Leu27 Ser2g bGRF(1-37)NH2; N-~Y-(Tyr-Ala-Gly-Pro-Leu-Pro-Phe-Ala)2-Tyrl lle2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse 32 bGRF(1-32)NH2; N~-tVal-Pro-Arg-Pro-Phe-Pro-Tyr-Se})-Tyrl 30 Val2 Serg Ala15 llel9 Leu27 Ser28 Hse33 bGRF(1-33)NHEt; N-~-(Arg-Pro-Tyr-Ala-lle-Pro-Phe-Ala)-Tyrl lle2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse30-bGRF(1-30)NHEt; N-a-(Tyr-Ala)~
: Tyrl Yal2 Ser8 Ala15 Val19 Leu27 Ser28 Hse34 bGRF(1-34)NHCH3; N-~-(IIe-Pro-Glu-Ala-Tyr-Ala)-Tyrl De2 Ser8 Alal5 Vall9 Leu27 Ser28 bGRF(1-37)NH2.
Another embodiment of thiS inVention iS any of the foregoing embodiments wherein 3S Cya is substituted for Asp in position 3 and/or 25, prefe~ably in position 3.Val19 and llel9 compounds of the subject invention are as active (and sometimes more SlJBSTlTVTE SHI~

. .... ......

WO 92/08481 PCr/US91/08248 active) in releasing GH in vi~ro (rat pituitary cells) ~an their corresponding Alal9 counterparts ~able I and Fi~ure 1). They are more stable to proteolysis when incubated in bovine plasma.
Evidence of the improved metabolic stability of the compo~mds of this invention (when compared to a native GRF sequence as embodied by the compound Leu27 bGRF(1-29)NH2) is S illustrated in Table II by the in vitro stability data provided. The compounds of this invention are more active in vivo at 10 pmol/kg ~han the Leul9 analogs Figure 2) and generally release more grow~h ho~none and with a sustained effect over the Alal9 and Leul9 an~logs when tested in steers at 30 pmol/kg. CI able m). In Figures 1 and 2 (the compounds were tested as ~/
the trifluoroacetate salt); T2 means Thr2, A15 means Alal~, L27 means Leu27, L19 means Leul9, V19 means Vall9, and Il9 means lle19.
The enhanced in vivo potency of the compounds of this invention where R19 is Val or Ile is illustrated in Table III and Figures 2. For example, a person skilled in the art would recognize from the relative in ~vo potency (at a dose of 10 pmol/kg, Figure 2) ~hat Thr2 AlalS
Vall9 Leu27 bGRF (1-29)NH2, trifluoroacetate salt and Thr2 Ala15 Ilel9 Leu27 b&R~
29)NH2, trifluoroacetate salt and similarly (at a dose of 30 pmollkg, Table III) the Vall9 and Del9 compounds are more bioactive than the native GRF.
For pu~poses of commercial produetion methodology, the carboxy tenninal residue is ~ `
preferably homoserine, homoserine lactone, homoserine amide, or a Cl-Cg alkyl (preferably Cl-C4 alkyl), secondary or tertiary amides of homoserine.
The synthetic GRF peptide analogs are synthesized by a suitable method, including for example the methods disclosed in U.S. Patent 4,529,595 (Col 2, In 35 to Col 5, In 64) and US
Patent 4,689,318 (Col 2, In 23 to Col 9, In 13), each of which are incorporated herein by reference.
Procedure A sets forth a method for synthesizing GRF peptide analogs of the subject invention.
PROCEDURE A
The peptides are synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosysterns, Foster City, California) and synthesis cycles supplied by Applied Biosystems~ Boc Amino acids and other reagents were supplied by Applied Biosysterns and other commercial sources. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resin for the-production of C terminal carboxamides. Por the production of C terminal acids, the corresponding PAM resin is used. AsparagiDe, glutamine, and arginine are coupled using preformed hydroxy benztriazole esters. All other amino acids are coupled using the preforrned symmetrical Boc amino acid anhydrides.
The following side chain protection is used:
S~JE~T~E. ~EE7~
~ . .~.. ..

WO 92/084~l ~ V ~ ~ ~ O ~ Pcr/US~ 8~48 Arg, Tosyl Asp, Benzyl Cys, 4-Methyl Benzyl Glu, Benzyl Ser, Benzyl Thr, Benzyl Tyr, 4-Bromo Carbobenzoxy Lys, 2-Chloro Carbobenzoxy Boc deprotection is accomplished widl trifluoroacetic acid CI'FA) in methylene chloride. When Hse containing analo~s are desired, Met should be incorporated by solid phase and then modified with cyanogen bromide after HF cleavage by methods well known in the art.
This cyanogen bromide cleavage converts the Met to the C-terminal Hse lactone peptide. This can be converted to the Hse amide peptide by treatment with the appropriate arnine in a solvent such as methanol or dimethyl formamide. [C-terminal Hse~actone), HseOH and HseN(Ra)(Rb) analogs can be prepared by the methods disclosed in Kempe et al, BIOITECHNOLOGY, Vol 4, pp 565-568 (19B6).] Following completion of the synthesis, the peptides are deprotected and cleaved from the resin widl anhydrous hydrogen fluoride containing 10% p-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at 0DC or below, preferably -20C for thirty minutes followed by thirty minutes at 0C. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and Iyophilized. Before purification, crude cysteine containing peptides are then oxidized to the corresponding cysteic acid containing compound using performic acid at -10C to 10C, preferably at 0C, as described by Stewart et al., Solid Phase Peptide Synthesis, pg. 113, Pierce Chemical Company, Rockford, Illinois, I984. Conversion to C-terminal Hse lactones and Hse arnides is carried out as described above.
Purification is carried out by ion exchange chromatography on a Synchroprep S-300 (SynChrom lnc. Linden, Indiana) cation exchange column. The peptide is applied using a buffer of 20 millimolar TRIS (pH 6.8) in 20% acetonitrile and eluted using a gradient of 0~0.3 molar sodium chloride in the same solvent. Compounds are further purified and desalted by reverse phase liquid chromatography on a Vydac C-18 (Separations Group, Hesperia, -California) column using water: acetonitrile gradients, each phase containing 0.1% TFA. ~he desired fractions are pooled and Iyophilized yielding the desired GRF PEPIIDE as its - ' I
trlfluoroacetate salt. The trifluoroacetate salt can be converted, if desired to other suitable salts, by well known ion e~change methods.
Peptides are hydrolyzed under vacuum by a vapor phase method in a Pico-Tag Work Station (Waters) using constant boiling HCI (Pierce) in the presence of phenol as scavenger at 5~B~ITV'rE S~IEE~
., ........... , .,.... ,.. ,....... :
. .

WO 92~08481 13 2 ~ 9 2 ~ 0 6 110 C for 24 hrs. Hydrolysates are analyzed on a Beckman Amino Acid Analyzer, Model 6300. Peptide content is calculat~d using norleucine at a known concentration as an internal standard.
Mass ~pectra were obtained using an Applied Biosystems Time of Flight Mass S Spectrometer.
EXAMPLES
E~cample 1: Preparation of Thr2 Ala15 Vall9 Leu27-bGRF(1-29)NH2, trifluoroacetate salt Compound No. 1.
The synthesis of the aRF analog peptide having the formula:
10 H-Tyr-Thr-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Yal-Arg-Lys-Leu-Leu-Gln-Asp-lle-Leu-Asn-Arg-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Amino acid analysis, theoretical values in parantheses: Asp 4.06 (4); Thr 1.57 (2); Ser 1.64 (2); Glu 2.10 (2); Ala 2.25 (2); Val 2.00 (2); lle 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 Thr2 Ala15 11el9 Leu2~-bGRF(1-29)NH2, trifluoroacetatesalt Compound No. 2.
The synthesis of the GR~: analog peptide having the formula:
H-Tyr-Thr-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-lle-Arg-Lys-Leu-Leu-Gln-Asp-lle-Leu-Asn-Arge-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Amino acid analysis, theoretical values in parantheses: Asp 4.09 (4); Thr 1.98 (2); Ser 1.64 (2); Glu 2.05 (2); Ala 2.25 (2); Val 1.0 (1); lle 2.84 (3); Leu 5.07 (5); Tyr 1.87 (2); Phe 0.92 (1); Lys 1.97 (2); Arg 2.94 (3).
Example 3: Preparation of Thr2 Alals Leul9 Leu27-bGRF(1-29)NH2, trifluoroacetatesalt Compound No. 3.
The synthesis of the GRF analog peptide having the formula:
H-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Se~-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Leu-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arge-NH2 (as the CF3COOH salt) is conducted i~l a stepwise manner as Ln procedure A. Arnino acid analysis, theoretical Yalues in parantheses: Asp 4.08 (4); Thr 1.87 (2); Ser 1.72 (2); Glu 2.07 (2); Ala 1.93 (2); Val 1.04 (1); lle 1.~8 (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 Yal2 Ala15 Leul9 Leu27-bGRF(1-29)NH2, trifluoroacetatesalt Compound No. 4. ` ~ - ~
The synthesis of the GR~; analog peptide having the forrnula:
H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Leu-Arg-Lys-Leu-Leu~ln-Asp-lle-Leu-Asn-Arg-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption me~od), m/z for SUBSTITUT~ SHEET

.

wo 92/08481 PCr/US91/08~48 ~Q~29~
[M+H]+: observed 3454.6; theoretical 3452.1. Amino acid analysis, theoretical values in pareDtheses: Asp 4.01 (4); Thr 0.93 (1); Ser 1.68 (2); Glu 2.03 (2) Ala 2.00 (2); Val l.BS
(2); lle 1.88 (2); Leu 6.11 (6); Tyr 1.94 (2); Phe 0.94 (1); Lys 1.97 (2); Arg 3.11 (3) E~ample S: Preparation of lle2 Alals Leul9 Leu27-bGRF(1-29)NH2, trifluoroacetate5 salt Compound No. 5.
The synthesis of ~he GRF aDalog peptide having the formula:
H-Tyr-Ile-Asp-Ala-Ile-phe-Thr-Asn-ser-Tyr-Arg-Lys-val-Leu-Ala-Gln-Leu-ser-I~eu-Arg-L
Leu-Leu-Gln-Asp-lle-Leu-Asn-Arg-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption method), m/z for 10 ~M+H]+; observed 3468.6; theoretical 3466.1.
Amino acid analysis, theoretical values in parentheses: Asp 4.û2 (4); Thr 0.94 (1); Ser 1.67 (2); Glu 2.05 (2); Ala 2.00 (2); Val 0.94 (1); lle 2.83 (3); Leu 6.12 (6); Tyr 1.92 (2); Phe 0.95 (1); Lys l.99 (2); Arg 3.13 (3).
Example 6: Preparation of Val2 Ala15 Ilel9 Leu27-bGRF(1-29)NH2, trifluoroacetate15 salt Compound No. 6.
The synthesis of the 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-lle-Arg-Lys-Leu-Leu-Gln-Asp-De-Leu-Asn-Arg-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption method), m/z for 20 lM~H]+: observed 3453.4; theoretical 3452.1.
Amino acid analysis, theoretical values in parentheses: Asp 4.03 (4); Thr 0.94 (1); Ser 1.69 (2); Glu 2.03 (2); Ala 2.01 (2); Val 1.89 (2); Ile 2.85 (3); Leu 5.11 (5); Tyr 1.95 (2); Phe 0.95 (1); Lys 1.99 (2); Arg 3.08 (3).
Exarnple 7: Preparation of lle2 AlalS llel9 Leu27-bGRF(1-29)NH2, trifluoroacetate 25 salt Compound No. 7.
The synthesis of ~he GRF analog peptide having the fo~nula:
H-Ty~-Ile-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-lle-Arg-Lys-Leu-Leu-Gln-Asp-lle-Leu-Asn-Arg-NH2 (as the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption method), m/z for 30 [M+H]+: observed 3469.7; theoretical 3466.1.
Amino acid analysis, theoretical values in parenthesesi: Asp 4.03 (4); Thr 0.94 (1); Ser 1.69 (2); Glu 2.06 (2); Ala 2.04 (2); Val 0.94 (1); lie 3.78 (2); Leu 5.12 (5); Tyr 1.94 (2); Phe 0.95 (1); Lys 1.98 (2); Arg 3.09 (3).
~ ample 8: Preparation of Val2 Ala15 Vall9 Leu27-bGRF(1-29)NH2, trifluoroacetate 35 saltCompoundNo. 8.
The synthesis of the GRF aDalog peptide haYing the fonnula:

.... .;.... ., .. ~ .. , -.. ..... : : . ` ....... : , - -WO 92/08481 ;~ ~ 9 2 ~ a ~ PCT~us91~o8248 H-Tyr-Val-~sp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Ar~-Lys-Val-Leu-Ala-Gln-Leu-Ser-Val-Arg-Lys-Leu-Leu-Gln-Asp-lle-Leu-Asn-Arg-N~I2 (as ~he CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption method), m/z for [M+H]~: observed 3440.6; theoretical 3438.1.
S Amino acid analysis7 theoretical values in parentheses: Asp 4.02 (4); Thr 0.93 (1); Ser 1.67 (2); Glu 2.03 (2); Ala 2.00 (2); Yal 2.78 (3); lle 1.86 (2); Leu 5.~9 (5); Tyr 1.92 (2); Phe 1.04 (1); Lys 1.98 (2); Arg 3.05 (3).
Example 9: Preparation of lle2 Ala1s Vall9 Leu27-bGRF(1-29)NH2, trifluoroacetatesalt Compound No. 9.
The synthesis of the GRF analog peptide having the formula:
H-Tyr-Ile-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 the CF3COOH salt) is conducted in a stepwise manner as in procedure A. Mass spectral analysis (Cf-252 plasma desorption method), m/z for [M+H]+: observed 3454.9; theoretical 3452.1.
15 Amino acid analysis, 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); lle 2.84 (3); Leu 5.10 (5); Tyr 1.94 (2); Phe 0.94 (1); Lys 1.98 (2); Arg 3.07 (3).
Following the stepwise manner as in procedure A, the following peptides can also be prepared:
I~e2 Ser8 Ala1S Vall9 Leu27 Ser28 Hse30 bGRF(1-30)NH2 2 Ser8 Alals Vall9 Leu27 Ser28 Hse33 bGRF(1-33)NH2 2 Sers AlalS ~all9 Leu27 Ser28 Hse37 bGRF(1-37)NH2 le2 Ser8 AlalS Vall9 Leu27 ser28 Hse44 bGRF(l 44)NH2 Va~2 Ser8 A~alS Vall9 Leu27 Se~28 Hse30 bGRF(1-30)NH2 Val2 Ser8 AlalS Vall9 Leu27 ~er28 Hse33 bGR~:(1-33)NH2 Val2 Ser8 Alal5 Vall9 Leu27 Ser28 Hse37 bGRP(1-37)NH2 Val~ Ser8 Ala15 Vall9 Leu27 Ser28 Hse44 bGRF(1-44)NH2 le2 Ser8 AlalS Del9 Le*7 Ser28 Hse30 bGRF(1-30)NH2 lle2 Sers A~a15 1~el9 Leu27 S~ r28 Hse33 bG~ -33)NH2 I~e2 Ser8 A~a15 Del9 Leu27 Ser28 Hse37 bGRF(1-37)NH2 De2 Ser8 Ala15 Del9 Leu27 Ser28 Hse44 bGR~ 14)NH2 -- -Va~2 Ser8 AlalS Del9 Leu27 Ser28 Hse30 bGRF(1-30)NH
Val2 Ser8 A~alS llel9 Leu27 Ser28 Hse33 bGRF(1-33)NH2 -ya~2 Ser8 AlalS I~e19 Leu27 Ser28 Hse37 bGRF(l-37)NH2 Val2 Ser8 AlalS Ilel9 Leu27 Ser28 Hse44 bGRF(l 44)NH2 Thr2 Sers A~alS Vallg Leu27 Ser28 Hse30 bGRF(l-3o)NH2 SUBSTITUTE SHEET

, .. . , ~ . . ,- : - . ` . : .
. . . ~ `, . ..

.: " . . . `

Wo 92~08481 2 0 9 2 9 ~ Pcr/US9l/08248 ~hr2 Ser8 Ala15 Vall9 Leu27 Ser28 Hse33 bGRF(1-33)NH2 ~2 se~8 Ala15 Val19 Le*7 S~28 Hse3~ bGRF~1-37)NH2 Thr2 Ser8 ~la15 Val19 Leu27 Ser28 Hse44 bGRF(l 44)NH2 Thr2 SerS Alal5 Ilel9 ~,eu27 Ser28 Hse30 bGRF(1 30)NH2 T~,r2 Ser8 Ala15 Ilel9 Leu27 Ser28 Hse33 bGRF(1-33)NH2 Thr2 Ser8 Alal5 llel9 Leu27 S~28 Hse37 bGRF(1-37)NH2 Thr2 Ser8 Alal5 ~el9 Leu27 Ser28 Hse44 bGRF(144)NH2 Thr2 Ser8 Alals Vall9 Leu27 Ser28 Hse30 bGRF(1-30)NHEt Thr2 Ser8 Alal5 Va~l9 Leu27 Ser28 Hse33 bGRF(1-33)NHEt Ile2 Ser8 A~alS vallg Leu27 Ser28 Hse30 bGRF(1-30)NHEt s Ala15 va]l9 LeU2? ser28 Hse33 bGRF(l-33)NHEt ~al2 Sers Alal5 llel9 Leu27 Ser28 Hse37 bGRF(l-37)NH-n-pr PROCEDURES
PROCEDURE B
In addition to preparation of GRF analogs by solid phase methods, the analogs can be obtained by recombinant DNA methodology by the procedure described for Leu27 bGRF(I-44)OH ~uropean Patent Application 0212531) with the following modifications in the segment of DNA coding for bGRF(1 44)OH the codons for Ala2, Asn8, Glyls and Asn28 are replaced by the codons: for example (ACI) Thr2 or (ATT~ Ile2 or (GTT) Val2, (AGT) Ser8, (GCT) 20 Alal5, (AGT) Ser28, respectively.
Additionally, for Cya3 containing GRF analogs, tbe codon GAT (Asp3) is replaced by the codon TGT for Cys3. After expression of the protein and cleavage with cyanogen bromide in formic acid as described in the above European Patent Application, hydrogen peroxide is added to the solution at about 0C to effect oxidation of the Cys residues to Cya. The peptide 2S is then purified by the methods described.
Additionally, for N-terminally extended GR~ analogs, the DNA segments coding forthe extension are added to the N-terminus as follows: (TATACT) for Tyr-Thr, (TATACT)n for ~yr-Thr)D or (TATAG~ for Tyr-Ser, (IA~AGT)~I for (Tyr-Se-)n, or (TATAGTTATACT) - .
for Tyr-Ser-Tyr-Thr or (TATACTTATAGT) for Tyr-Thr-Tyr-Ser, (GATGCT) for Asp-Ala etc.
The gene for the precursor protein is inserted into an E. coli expression vector. After expression of the protein isolation of the inclusion bodies and then cleaving them with cyanogen bromide in formic acid as described i`n the above Europèan Patent Application, the formic acid is removed under reduced pressure; The crude peptide is then purified by the methods described. - `
The ability to produce the compounds of the subject invention by known recombinant DNA technology is possible since the daimed peptides are constituted entirely of naturally RSTITU~E SI~EE~ l WO 92J0~481 PCI/US91/08248 -17- ~290~' occurring amino acids. This is in contrast to known analogs which contain non-DNA coded components, such as D-Ala and/or desaminoTyr, and will require for any large scale production a costiy chemical synthesis or a combination of genetic engineering with chemical procedures.
S Recombinant host microorganisms used ~n ~is invention are made by recombinant DNA techniques well known to dlose skilled in ~e art and set forth, for example, in Molecular Cloning, T. Maniatis, et al., Cold Spring H~bor Laboratory, (1982) and B. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984), which are incorporated herein by reference.
C-terminal Hse(lactone), HseOH and HseN(Ra)~Rb) analogs can be prepared by the me~hods disclosed in Kempe et al, BIO/TECHNOLOGY, Vol 4, pp 565-568 (1986).
PROCEDURE C
This procedure deals with the preparation of N-alkylated GRF analogs described in the subject invention. The peptides will be made using either chemical or biotechnology 15 procedures (Procedure A or Procedure B, respectively). N-alky3ation will then be achieved by known methods e.g. Murphy, W.A. and Coy, D.H., Potent long-acting alkvlated analogs of growth hormon~releasing factor, Peptide Research l, 36~1 (1988), V. Sythyamoor~y et al.
Reductive methylation of botulinum neurotoxin types A and B. Mol. Cell. Biochem. 83, 65-72 (198~).
Following Procedure C, the following peptides can also be prepared:
N-e-iPr-Tyrl Thr2 Ser8 N-e-iPr-Lysl2,21 Val15 llel9 Leu27 Ser28bGRF(1-29)NH2 N e iPr-Tyrl Thr2 Ser8 N e ipr Lys12~21 Ala15 V~l9 LeU27 bGRF(1 40)NH2 N-e-Bzl-Tyrl Thr2 Ser8 N-e-Bzl-Lysl2,21 Valls llel9 11e27 Ser28 bGRFtl 32)NH
N e ipr Tyrl lle2 Ser8 N-e-iPr-Lysl2,21 AlalS Vall9 Leu27 bGRF(l~O)NH2 N e Bzi Tyr1 Val2 Ser8 N-~-Bzl-Lys12,21 Val15 Val19 lle27 Ser28 bGRF(1-32)NH2 All of the synthetic GRF peptides of the subject invenlion, including the peptides prepared in the Examples, are considered to be biologically active and useful for stimulating the release of GH by the pituitary.
Dosages between about 10 nanograms and about 5 micrograms of these peptides per 30 ~ilogram ~g) of body weight are considered to be particulary effective in uusing GH
secretion.
- - Stirnulation of GH secretion by such peptides should result in an attendant increase in growth for humans, bovine and other animals with normal GH levels~ Moreover, administration should alter body fat content and modify other GH~ependent metabolic, 35 immunologic and developmental processes~ For e~ample, these analogs may be useful as a means of stimulating anabolic processes in hurnan beings under circumstances such as following SU~3STITUTE SHEE~I

.. . . .

~.

. , ~ . . ~ . , .
` .: . ~ ~. . . -: . `

wo 92/08481 PCl/US91/08248 2~929~ -18-the incurring of burns. As another example, these analogs may be administered to eom~nercial warm-blooded animals such as chickens, turkeys, pigs, goats, cattle and sheep, and may be used in agriculture for raising fish and other cold-blooded marine animals, e.g., sea tur~les and eels, and amphibians, to accelerate growth and increase the ratio of pro~ein to fat gained by S feeding effective amounts of the peptides. These analogs may be used for stimulation of the immune functions in human and animal for the treatment of diabetes resulting from abnormalities in growth honnone production or for the improvement of bone, wound or burn healing, or osteoporosis. These analogs may be used to enhance hair grow~h.
Daily dosages of between I0 nanograrns/Kg and about 50 micro-grams/Kg body weight 10 are considered to be particularly effective in increasing lactation, grow~h and stimulating the 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 the nontoxic salts thereof, combined with a pharmaceutically 15 or veterinaray acceptable carrier to form a pharmaceutical composition, preferably as sustained release formulations, may be administered to animals, including humans, either intravenously, subcutaneously, intrarnuscularly, percutaneously, e.g. intranasally. The administration may be employed by a physician to stimulate the release of GH where the host being: treated requires such therapeutic treatment. The required dosage will vary with the particular condition being 20 treated, with the severity of the condition and with the duration of desired treatment.
Such peptides are often administered in the form of nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, 25 ascorbate, tartrate and the like. If the active ingredient is to administered by intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
The peptides should be administered to humans under the guidance of a physician, and ph~maceutical compositions wDI usually contain the peptide in conjunction with a conventional, solid or liquid, pharmaceutically-acceptable carrier. Usually, the parental dosage will be from 30 about lO0 nanograms to about 50 micrograms of the peptide per kilogram of the body weight of the host.
Although the invention has been described with regard to its preferred embodiments, it should be understood that various changes and modifications as would be obvious to one having the ordinary skDI in this art may be made without departing from the scope of the invention 35 which is set forth in the claims appended hereto. Por example, modifications in the peptide chain, par~cularly deletions of one or two residues beginning at the C-terminus of the peptide, SUBSTITUl_ SHEET

. ~ ` , .

WO 92/08481 ~ 2 9 0 ~ ' PCr/US91/û8248 can be made in accordance with known experimental practices to date to create peptides that retain very substantial portions of the biological potency of the peptide, and such peptides are corlsidered as being within the scope of the invention. Moreover, additions may be made to the C-terminus, andlor to the N-terrninus, and/or generally e~uiYalent residues can be substituted 5 for naturally occurring residues, as is known in the overall art of peptide chemistry, to produce other analogs, having increased resistance to proteolysis, for example, and also having at least a substantial portion of ~he potency of the claimed polypeptide, without deviating from the scope of the invention, such as ~ose illustrated by S~ompounds 1-15. Likewise known substitutions in the carboxyl moiety at ~e C-ter~ninus, e.g. a lower alkyl amide, also produce equivalent 10 molecules.
In the same manner as disclosed herein, GRF PEPrIDES of the formula R'-RlR'2-R3-Ala-lle-Phe-Thr-R8-Ser-Tyr-Arg-R'12-R13-LeU-RlS-GIn-LeU-R18-Rlg-Arg-R 2l-R22-Leu-Gln-R2s-De-R27-R2g-Arg-Gln-Gln-Gly-G
R34-R35-Gln-Glu-R3g-R3g-R4o-Arg-R42-Arg-Leu-z wherein R' is (X-Y)-(X'-Y)n wherein n is 0-20, preferably 0-10, X is any naturally occuring 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 tbreonine; X' is any naturally occuring amino acid e~tcept prolirle or hydroxyproline.
R1 is Tyr or His;
R'2 is Ala, Gly, Thr, Val or lle;
R3 is Asp, Glu or Cya;
R8 is Asn or Ser;
R'l2 is Lys or Arg;
R13 is Val or lle;
Rl5 is Ala, Val, Leu, Ile or Gly;
R18 is Ser or Tyr;
Rlg is Val or Ile;
R'2l is Lys or Arg;
R22 is Ala or Leu;
:; R25 is Asp, Glu or Cya;
R27 is Met, ne or Leu;
R28 is Asn or Ser;
R34 is Ser or Arg;
R35 is Asn or Ser;
R38 is Arg or Glrl;
SVE35rlrUtE SHEEr . : . ~ ' ' . , ' ' , , :

'' . . .: ' ' '. , ' ` ' '~' ' : ' ' wo 92/08481 PCr/lJS91/08248 2~29~6 -2~ ~
R39 is Gly or Arg;
R40 is Ala or Ser;
R42 is Ala, Val or Phe; and Z signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the S radical -COOR~, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH20R8, with Ra and Rb being Cl-C8 alkyl or hydrogen; or a biologically active fragment thereof extending from R at the N-terminus to a residue in any of positions 27 through 44 as its C-~errninus; or a Hse(lactone), HseOH or HseN(Ra~(Rb) of the ~oregoing and/or a non-toxic salt of the foregoing; can be made in accordance with known experimental practices to date to create peptides that retain very l0 substantiàl portions of ~e biological potency of the peptide, and such peptides are considered as also being another aspect of the invention disclosed herein.
The Extension portion (R') of the GRF peptide according to the present invention has an amino acid sequence according to the fonnula:
(X Y) tx Y)D
Where n represents the number of sequentially linked X'-Y groups, that number representing from 0 to 20 of such groups, preferably 0 to l0 groups.
X is selected from the group consisting of any naturally occuring amino acid;
Y is selected from the group consisting of proline, alanine, serine, and threonine, except when n = 0, then Y is selected from the group consisting of alanine, serine, and 20 threonine;
X' is selected from the group consisting of any naturally occuring amino acid except proline or hydroproline;
According to the formula, when n = l, there are two Y residues. Further, it is possible to have up to twenty one Y residues and twenty X"esidues in a single embodiment.
25 Individual Y residues and X' residues respectively can be any residue of the group from which they are selected. lhat is, all of the individual Y residues do not have to be the same in a given embodiment. Similarly, in an embodiment with more than one X' residue, each individual X' residue present can be any amino acid residue except proline and hydroxyproline irrespective of wha~ residue any other X' residue may be. Each individual Y and X' residue 30 respectively must conform to the rules for that particular group and all that is necessary is that ~e various individual residues at the specific positions follow the rules as articulated above~-~. . ........................... .

SU~TITUTE S~EE7 '~ 0 ~ 0 ~

3 ~ ~ ~ a 'C E _ o~ ~o ~ _ ~ æ ~ _ r7 _ ~ ~ E $

a r L~ 1~
Y ~

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~ _ ~ Z ~ ca SUE3SrlTiUTE SHEET

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' '; '' ' ' ' ',',- ' ~ ' ., '~ ' :
. '. " , ' ~, ~ ;, ' .' , ;, ~' ~ , '~ '' '' .. '" ';' WO 92/08481 PCr/US91/0824 2~2~0~
Table II: IN Vl~O PLASMA SIABILITY OF POSITION I9 SUBSTIlVTED GRFs .

-......... ,, . . ___ .
s I C~MPOUND # CO~POUND tl/2 ~r) l (as its trifluoroacetate salt) ~ ._ _ _ l Leu27 bGRF(1-29)NH 0.6 l . __ . 2 . . ____ .
. . Thr2,Alal5,Leu27,bCiRF(1-29)N~I2 4,9c 3 Thr2 Ala15 Leul9,Leu27,bGRP(l-29)NH2 l4.7a ___ - . _ 1 Thr2,Alal5,Vall9,Leu27,bGRF(1-29)NH2 6.6b _ c Thr2 Alal5 Ilel9,Leu27,bGRF(l-?,9)NH2 12.3a Mean value from three independent experiments (each done in triplicate) ~b~c Compounds with di~ferent superscripts are statistically different at p < 0.05.

:
' i'.~ ..

~;U~S~UTE SWEE~
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WO 92~08481 ~ ~3 3 2 9 ~ ~ p~/US9l/08248 __ _ _ _ _ _ ..
;g 8 o~ v~ ~ ~ o ~o æ ~ 00 ~ ~ _ _ __ _ ~ ~ o _ 8 _ o et o~ c~ ~ ~ ~ o~ _ _ U~ ~ ~ ~ V ~ O .D~ O r ~ ~. r~ ~ ~ _ :~ ~ _ ~ ~ ~ ~ ~ ~ ~ ~t ~ d ~ O O
o .~ ~ ~ ~g ~3 ~ ~ ~ 3~ ~o ~} o~ ~ ~ o .C ~ _ V~ ~ . I~ O~ O~ ~ ~ ~ _. ~ ~ ~ . C~
3 ~: o o _ o o o _ _ _ _ _ _ o o o O C _ ~1 ~ ~ _ D I_ O X O ~ ~ t'~l _ ~J o ~ _ _ O _ ~ ~ C`~ _ ~`i ~ C`i ~ ~i _ O O ~ '8 C

a ~ ~ ~

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`~ SU~3STITUTE SHI~

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YVO 92/0&18il PCI'/US91/08248 2 ~ 9 0 ~ -24-.

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Figllre 1. `. . 1 ~' Effect of analogs Of ~2, Ala15, Leu27-bGRF(1-29)NH2 OD growth hormone (GH) release in rat pituitary cell cultures in v~tro. The assay was performed according to procedure of ' ::
Frohman and Downs, Methods Enzymol. 124, 371-389 (19863. Note that the Leul9 -substitutio~ wæ deleterious to the analog GH-releasing potency while Vall9 and 11el9 -~ ..;
~ modifications resulted in analogs with respectively increased or unchanged biactivity in vf~ro as t~
: compared with Ihr~,- Alal5, Leu27-bGRF(1-29)N~I2. Leu27-bG~F(1-29)NH2 was used as the : ~ :
. assay standard. . ,: . .
.' : ' I .
..... . . .... ... . . . I

~3UES~l~UfE ~S~E~iT
~-!

WO 92/08481 PCl'/U591/08248 ~ -25- ~929~6 ..

Figure 2.
40 Mean concentration of ser~m growth hormone (GH) in meal-fed ~Iolstein steers after in~a~venous injection (10 pmol/kg) of GRl: analogs. The assay was performed as described by Moseley et al., J. Endocrinol. 117j 2~2-259 (1988). Bovine GRF(l 44)NH2 was used as the assay standard.
`

... . . . .. .
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~ .

Claims (16)

-26-

1. A GRF PEPTIDE having Val or Ile in place of the amino acid residue normally found at position 19, having Gly, Thr, Val or Ile in place of the amino acid residue normally found at position 2 and having an amino acid selected from the group consisting of Ala, Val, Leu, Ile or Gly at position 15.

2. A GRF PEPTIDE of Claim 1 wherein the amino acid at position 2 is Thr, Val or Ile.

3. A GRF PEPTIDE of Claim 1 having the formula wherein R is H, C1-C5 alkyl or benzyl;
R1 is Tyr or His;
R2 is Gly, Thr, Val or Ile;
R3 is Asp, Glu or Cya;
R8 is Asn or Ser;
R12 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R13 is Val or Ile;
R15 is Ala, Val, Leu, Ile or Gly;
R18 is Ser or Tyr;
R19 is Val or Ile;
R21 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R22 is Ala or Leu;
R25 is Asp or Glu;
R27 is Met, Ile or Leu;
R28 is Asn or Ser;
R34 is Ser or Arg;
R35 is Asn or Ser;
R38 is Arg or Gln;
R39 is Gly or Arg;
R40 is Ala or Ser;
R42 is Ala, Val or Phe; and Z signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb being C1-C8 alkyl or hydrogen; or a biologically active fragment thereof extending from R at the N-terminus to a residue in any of positions 27 through 44 as its C-terminus; or a Hse(lactone), HseOH or HseN(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing.

4. A GRF PEPTIDE of Claim 3 wherein R2 is Thr, Val or Ile.

5. A GRF PEPTIDE according to Claim 4 selected from the group consisting of ; or a non-toxic salt thereof.

6. A GRF PEPTIDE of Claim 3 wherein R15 is Ala, Val, Leu or Ile.

7. A GRF PEPTIDE of Claim 6 wherein R is H and R15 is Ala.

8. A GRF PEPTIDE of Claim 7 wherein R1 is Tyr.

9. A GRF PEPTIDE of Claim 8 wherein R8 and R28 are Ser.

10. A GRF PEPTIDE of Claim 6 wherein R2 is Val.

11. A GRF PEPTIDE of Claim 6 wherein R2 is Ile.

12. A GRF PEPTIDE of Claim 6 wherein R19 is Val.

13. A GRF PEPTIDE of Claim 6 wherein R19 is Ile.

14. A method of stimulating the release of growth hormone in an animal, which comprises administering to said animal an effective amount of a GRF PEPTIDE having Val or Ile at position 19; Thr, Val or Ile residue in place of the amino acid residue normally found at position 2; and having an amino acid selected from the group consisting of Ala, Val, Leu, Ile or Gly at position 15.

15. A method according to Claim 14 wherein the GRF PEPIIDE has the formula wherein R is H, C1-C5 alkyl or benzyl;
R1 is Tyr or His;
R2 is Gly, Thr, Val or Ile;
R3 is Asp, Glu or Cya;
R8 is Asn or Ser;
R12 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R13 is Val or Ile;
R15 is Ala, Val, Leu, Ile or Gly;
R18 is Ser or Tyr;
R19 is Val or lle;
R21 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R22 is Ala or Leu;
R25 is Asp or Glu;
R27 is Met, Ile or Leu;
R28 is Asn or Ser;
R34 is Ser or Arg;

R35 is Asn or Ser;
R38 is Arg or Gln;
R39 is Gly or Arg;
R40 is Ala or Ser;
R42 is Ala, Val or Phe; and Z signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb being C1-C8 alkyl or hydrogen; or a biologically active fragment thereof extending from R at the N-terminus to a residue in any of positions 27 through 44 as its C-terminus; or a Hse(lactone), HseOH or HseN(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing.

16. A composition for stimulating the release of growth hormone in an animal comprising an effective amount of a GRP PEPTIDE of the formula wherein R is H, C1-C5 alkyl or benzyl;
R1 is Tyr or His;
R2 is Gly, Thr, Val or Ile;
R3 is Asp, Glu or Cya;
R8 is Asn or Ser;
R12 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R13 is Val or Ile;
R15 is Ala, Val, Leu or Ile;
R18 is Ser or Tyr;
R19 is Val or Ile;
R21 is Lys or Arg, or N-.epsilon.-alkyl- or N-.epsilon.-benzyl-Lys when R is C1-C5 alkyl or benzyl;
R22 is Ala or Leu;
R25 is Asp or Glu;
R27 is Met, Ile or Leu;
R28 is Asn or Ser;
R34 is Ser or Arg;
R35 is Asn or Ser;
R38 is Arg or Gln;
R39 is Gly or Arg;
R40 is Ala or Ser;

R42 is Ala, Val or Phe; and Z signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb being C1-C8 alkyl or hydrogen; or a biologically active fragment thereof extending from R at the N-terminus to a residue in any of positions 27 through 44 as its C-terminus; or a Hse(lactone), HseOH or HseN(Ra)(Rb) of the foregoing and/or a non-toxic salt of th foregoing; in association with a pharmaceutically acceptable carrier.