STABILIZED, POTENT GRF ANALOGS

Abstract: Novel GRF PEPTIDES with enhanced stability in plasma and aqueous environments at neutral pH.

Description: STABILIZED, POTENT GRF ANALOGS The present invention relates to a peptide having influence on the function of the pituitary gland in humans and other animals, particularly mammals. In particular, the present invention is directed to peptides which promote the release of growth hormone by the pituitary gland. The peptides of the present invention are more stable in plasma and in an aqueous environment at neutral pH than native GRF sequences. BACKGROUND OF THE INVENTION 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 pancreatic (tumor) releasing factors (hpGRF) were isolated from extracts of human pancreatic tumors, purified, characterized, synthesized, tested, and found to promote release of growth hormone (GH) by the pituitary. Guillemin, R. , et al. , Science 218, 585-585 (1982). Since then, corresponding hypothalamic GH releasing factors from other species including the rat species, the porcine species, the ovine species, the bovine and caprine species and from the human species have also been characterized and synthesized. Human hypothalamic GRF(hGRF) has been found to have the same formula as hpGRF, 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- Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu- NH 2 . 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-Ile-Met-Asn-Arg- Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH. (See for example US Patent 4,595,676). Porcine GRF has been found to have a Ser residue at position 28. 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 the amino terminus, and differing from the natural peptide by 13 amino acid in the rest of the sequence including incorporation of a Ser residue at position 8. The amide and free acid forms of the analog had the formula: 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-ϊffl/j/OH. When assayed for the ability to stimulate growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, the amide analog was 1.57 times as potent as hGRF(l-40)OH, while the free acid form was reported to be l/6th as potent in the same assay. Vale, et al. , (US Patent Application Serial No. 053,233, filed May 22, 1987) describes 31-residue hGRF analogues which utilize a 31- position residue possessing a functional side 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 GRF sequence, such as Asn or Ser in the 8-ρosition, Phe in the 10-ρosition, or Ala in the 15-position. Asn or Ser may be present in the 28-position. Certain of these peptides, containing 31 amino acid residues and utilizing a 31-position residue possessing a functional side chain group which may be conjugated to a separate protein, have reportedly been synthesized and demonstrated a high binding affinity for hGRF receptors on cultured pituitary cells and which may at least partially resist enzymatic degradation in the body and exhibit substantially increased potency. 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 example US Patents 4,649,131 and 4,734,399 as well as Ling, N. , et al., Quo Vadis?, Symposium, Sanofi Group, May 29-30, 1985, Toulouse-Labege, France (pp. 309-322). It has been reported that native GRF sequences are subject to rapid inactivation by blood plasma enzymes. The rapid breakdown involves cleavage of the 2-3 bond of the peptide by a dipeptidyl- a inopeptidase (DAP). Frohman, LA, J. Clin. Invest., 78, 906-913 (1996). Asn residues in polypeptides are reported to be the subject, under some circumstances, to deamidation in the presence of water. However, the rules governing the rates of deamidation are not clear. For example, in the polypeptide Trypsin only some of the Asn resi- dues, with the partial sequence Asn-Ser, are deamidated while others are not. See Kossiakoff, AA, Science 240, 191-194 (1988). Synthetic polypeptides have also been synthesized which are disclosed as growth hormone releasing factors (GRF). SUMMARY OF THE INVENTION The present invention provides a synthetic polypeptide which promotes the release of growth hormone by the pituitary gland (GRF PEPTIDE) and having a Ser residue in place of the amino acid residue normally found at position 8 and 28 of the polypeptide. The peptides of the present invention are more stable than native GRF sequences against breakdown by blood plasma enzymes and against breakdown in aqueous environments. DETAILED DESCRIPTION OF THE INVENTION The term "GRF PEPTIDE", as used in the specification and claims, means a known polypeptide which is between about 27 and 44 residues in length and that promotes the release of growth hormone by the pituitary gland. Illustrative GRF PEPTIDES include the natural or synthetic polypeptides 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, and 4,689,318; 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 Analogs", Poster Presentation, 10th America Peptide Symposium, St. Louis, May 1987; Tou, J.S., Kaempfe, L.A. , Vineyard, B.D. , Buonomo, F.C., Della-Fera, M.A. , and Baile, CA. , "Amphiphilic Growth Hormone 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-Terminal Region of Growth Hormone Releasing Factor", J. Med. Chem. 28, pp. 181-185 (1985); Felix, A.M., Heimer, E.P., Mowles, T.F. , Eisenbeis, H. , Leung, P., Lambros, T.J., Ahmed, M. , and Wang, C.T. , "Synthesis and Biological Activity of Novel Growth Hormone Releasing Factor Analogs", 19th European Peptide Symposium, Aug. 31-Sept. 5, 1986; Velicelebi, G., Patthi, S., and Kaiser, E.T. , "Design and Biological Activity of Analogs of Growth Hormone Releasing Factor with Potential Amphiphilic Helical Carboxyl Termini", Proc. Natl. 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 Engineer¬ ing", Quo Vadis Symposium, Sanofi Group, May 29-30, 1985, Toulouse- Labege, France, pp. 309-329. The term GRF PEPTIDE includes nontoxic salts thereof. The nomenclature used to define the GRF PEPTIDE is that speci¬ fied by Schroder & Lubke, "The Peptides", Academic Press (1965) wherein in accordance with conventional 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 iso- eric forms, the L-form of the amino acid is being represented unless otherwise expressly indicated. The residue N-[(beta(para-hydroxyphenyl)ρropionyl]- is identi¬ fied herein as DesaminoTyr (or PHPP) . The residue N-[alpha(para- hydroxyphenyl)acetyl]- is identified herein as PHPA. The present invention provides synthetic GRF peptide analogs (GRF PEPTIDES) having the following formula: Rι-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-R 15 -Gln- Leu-Ser-Ala-Arg-Arg-R22-Leu-Gln-R25-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu- R 34- R 35- Gln - Glu - R 38- R 39- R 40- Ar B-R42- Ar S- Leu " Y where R^ is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C^allqrl or C^-C alkoxy) , PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4al l or C 1 -C4alkox ) , N-[para- hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cι-C4alkyl or C^-C4alkoxy) , N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4alk l or C 1 -C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cι-C4alkyl or C^-C4alkoxy) ; R^5 is Ala or Leu; R22 is Ala or Leu; R25 is Asp or Glu; R34 is Ser or Arg; R35 is Asp or Ser; β g is Arg or Gin; R39 is Gly or Arg; R40 is Ala or Ser; R42 is Ala, Val or Phe; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -C00R a , -CR a 0, -C0NHNHR a , -C0N(R a )(R ) or -CH2θR a , with R a and R^ being lower alkyl or hydrogen; or a bio¬ logically 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(R a )(R^) of the foregoing and/or a non- toxic salt of the foregoing. Examples of R^ substituents (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cχ-C4alkyl or C^-C alkox ) include p-hydroxyphenoxy acetyl, p-hydroxyphenyl acetyl, 3-fluoro-4-hydroxyphenylacetyl, 4-hydroxy-3-methoxyphenyl acetyl, 3-chloro-4-hydroxyphenyl acetyl , p-hydroxy-benzoyl, p-hydroxy cin¬ namoyl and -hydroxyphenyl propionyl. The term includes methyl, ethyl, propyl, butyl and isomers thereof. The term C1-C4 alkoxy includes methoxy, ethoxy, propyloxy, butoxy and isomers thereof. The term halogen includes chloro, luoro, iodo and bromo atoms. An embodiment of this invention is the peptide PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Ala 22 Leu 27 hGRF(2-44)NH 2 having the formula: PHPP-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Ala-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu- Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH2. A further embodiment of this invention is the peptide: PHPA-Ala 2 Ser 8 Arg 12 Leu 15 Arg 21 Leu 27 hGRF(2-32)NH 2 having the formula: PHPA-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg- Arg-Val-Leu-Leu-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-lie-Leu-Ser- Arg-Gln-Gln-Gly-NH Another embodiment of this invention is the peptide: 3-methoxy PHPA-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 hGRF(2-29)NH(Ethyl) having the formula: 3-methoxy PHPA-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser- Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile- Leu-Ser-Arg-NH(Ethyl) A further embodiment of this invention is the peptide: N-[para-hydroxy benzoyl]-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Glu 25 Leu 27 Hse 33 hGRF(2-33)lactone having the formula: N-[para-hydroxy benzoyl]-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg- Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Glu-Ile-Leu-Ser-Arg- Gln-Gln-Gly-Hse lactone A preferred embodiment of this invention is the peptide: PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Hse 33 hGRF(2-33)NH 2 having the formula: PHPP-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gin- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Hse NH 2 Another preferred embodiment of this invention is the peptide: PHPA-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Hse 30 hGRF(2-30)NH 2 having the formula: PHPA-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Hse NH2 For purposes of commercial production methodology, the carboxy terminal residue is preferably homoserine, ho oserine lactone, homoserine amide, or a lower G^-C^ 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 preferred method for synthesizing GRF peptide analogs of the subject invention. PROCEDURE A -The peptides are synthesized by solid-phase methodology utiliz- ing an Applied Biosystems 430A peptide synthesizer (Applied Biosys- tems, Foster City, California) and synthesis cycles supplied by Applied Biosystems. Boc Amino acids and other reagents were supplied by Applied Biosystems 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. For the production of C terminal acids, the corres¬ ponding PAM resin is used. Asparagine, Glutamine, Arginine, [alpha- (para-hydroxyphenyl)acetic acids] , [beta(parahydroxyphenyl propionic acids)], para hydroxy benzoic acids, parahydroxycinnamic acids, and para hydroxy phenoxy acetic acids are coupled using preformed hydroxy benztriazole esters. All other amino acids are coupled using the preformed symmetrical Boc amino acid anhydrides. The following side chain protection is used: Ar , Tosyl Asp, Benzyl Glu, Benzyl Ser, Benzyl Thr, Benzyl Tyr, 4-Bromo Carbobenzoxy. Boc deprotection is accomplished with trifluoroacetic acid (TFA) in methylene chloride. Following completion of the synthesis, the peptides are depro ec ed and cleaved from the resin with anhydrous hydrogen fluoride containing 10% anisole. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at 0 P C or below, preferably -20 β C for thirty minutes followed by thirty minutes at 0°C. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and lyophilized. Purification is carried out by ion exchange chromatography on a Synchroprep S-300 (SynChrom Inc. 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. The desired fractions are pooled and lyophilized yielding the desired GRF PEPTIDE as its trifluroacetate salt. The trifluoroacetate salt can be converted, if desired to other suitable salts, by well known ion exchange methods. EXAMPLES Example 1: PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 L eu 27 h GRF(2- 32)NH 2 ; Cpd. No. 1 The synthesis of the GRF analog peptide having the formula: PHPP-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by Fast atom bom- bardment mass spectroscopy (FAB-MS) the expected monoisotopic (M+H) + of 3681. Example 2: PHPA-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 L eu 27 h GRF(2-32)NH 2 ; Cpd. No . 2 The synthesis of the GRF analog peptide having the formula: PHPA-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-GIn-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H) + of 3667. Example 3: (3-methoxy-PHPA)-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 h GRF(2-32)NH 2 ; Cpd. No. 3 The synthesis of the GRF analog peptide having the formula: (3-methoxy-PHPA) -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val- Leu-Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln- Gln-Gly-NH2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H) + of 3698. Example 4: (3-fluoro-PHPA)-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 hGRF(2- 32)NH 2 ; Cpd. No. 4 The synthesis of the GRF analog peptide having the formula: (3-fluoro-PHPA)-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu- Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln- Gl - H2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H) + of 3686. Example 5: (3-chloro-PHPA)-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 hGRF(2- 32)NH 2 ; Cpd. No. 5 The synthesis of the GRF analog peptide having the formula: (3-chloro-PHPA)-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu- Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln- Gly-NH2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H) + of 3703. Example 6: N-[para-hydroxy Cinnamoyl]-Ala 2 Ser 8 Arg-*- 2 Ala^-* Arg 2 ^- Leu 27 hGRF(2-32)NH 2 ; Cpd. No. 6 The synthesis of the GRF analog peptide having the formula: N- [para-hydroxy Cinnamoyl] -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg- Arg-Val-Leu-Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gin-Asp-lie-Leu-Ser- Arg-Gln-Gln-Gly-NH 2 is conducted in a stepwise manner as in procedure A. The compound gave the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H) + of 3679. Example 7: N- [(para-hydroxy-phenoxy)acetyl]-Ala 2 Ser 8 Arg 12 Ala-'--' Arg 21 Leu 27 hGRF(2-32)NH 2 ; Cpd. No. 7 The synthesis of the GRF analog peptide having the formula: N- [ (para-hydroxy-phenoxy)acetyl] -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser- Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile- Leu-Ser-Arg-Gln-Gln-Gly-NH 2 is conducted in a stepwise manner as in procedure A. Example 8: N- [para-hydroxy-benzoyl]-Ala^ Ser 8 Ar 1 ^ Ala-" Arg 21 Leu 27 hGRF(2-32)NH 2 ; Cpd. No. 8 The synthesis of the GRF analog peptide having the formula: N- [para-hydrox -benzoyl]-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg- Arg-Val-Leu- la-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-lie-Leu-Ser- Arg-Gln-Gln-Gly-NH2 is conducted in a stepwise manner as in procedure A. In addition to preparation of GRF analogs by solid phase meth¬ ods, certain analogs can be obtained by a combination of recombinant biosynthetic and synthetic methods, for example: PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 2 7 Ser 28 Arg 41 bGRF(2-44)0H can be prepared by a combination of biosynthetic and synthetic methodol¬ ogy by the procedure described for Leu 27 bGRF(l-44)0H (European Patent Application 0212531) with the following modifications: In the segment of DNA coding for bGRF(l-44)0H the codons for Tyr- 1 - are deleted and the codons for Asn Lys 12 Gly 15 Lys A sn 28 Lys 41 are replaced by the codons : (TCT) Ser 8 , (CGT) Arg 12 , (GCT) Ala 15 , (CGT) Arg 21 , (TCT) Ser 28 , and (CGT) Arg 41 r espectively. 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 in the above European Patent Application, the formic acid is removed under reduced pressure. The residue is dissolved in Q n dimethylformamide and the amino terminal Ala of the crude Ser° Arg ^- Ala 15 Arg 21 Leu 2 7 Ser 28 Arg 41 bGRF(2-44)0H contained within the reaction mixture is acylated with beta[para-hydroxyphenyl propionic acid N-hydroxysuccinimide ester in the presence of a suitable terti- ary base such as diisopropyl ethyl amine. The peptide is then puri¬ fied by the methods described,to give Compound #9, PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 2 7 Ser 28 Arg 41 bGRF(l-44)0H. Recombinant host microorganisms used in this invention are made by recombinant DNA techniques well known to those skilled, in the art and set forth, for example, in Molecular Cloning, T. Maniatis, et al. , Cold Spring Harbor 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(R a )(R^) analogs can be prepared by the methods disclosed in Kempe, et al. , BIO/TECHNOLOGY, Vol 4, pp 565-568 (1986). All of the synthetic GRF peptides of the subject invention, 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 50 nanograms and about 5 micrograms of these peptides per Kg of body weight are considered to be particu¬ larly effective in causing GH secretion. Stimulation 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-dependent metabolic, immunologic and developmental processes. For example, these analogs may be useful as a means of stimulating anabolic processes in human beings under cir- cumstances such as following the incurring of burns. As another example, these analogs may be administered to commercial warm-blooded animals such as chickens, turkeys, pigs, goats, cattle and sheep, and may be used in aquaculture for raising fish and other cold-blooded marine animals, e.g., sea turtles and eels, and amphibians, to accelerate growth and increase the ratio of protein to fat gained by feeding effective amounts of the peptides. Daily dosages of between 50 nanograms Kg and about 50 micro- grams/Kg body weight are considered to be particularly effective in increasing lactation and growth. For administration to humans and animals, these synthetic pep¬ tides 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 or veterinarily acceptable carrier to form a pharmaceutical composition, preferably as sustained release formula¬ tions, may be administered to animals, including humans, either intravenously, subcutaneously, intramuscularly, 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 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, 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 pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, solid or liquid, pharmaceutically-acceptable carrier. Usually, the parental dosage will be from about 100 nanograms to about 50 micrograms of the pep¬ tide 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 skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. For example, modifications in the peptide chain, particularly deletions of one or two residues beginning at the C-terminus of the peptide, 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 considered as being within the scope of the invention. Moreover, additions may be made to the C-terminus, and/or generally equivalent residues can be substituted 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 the potency of the claimed polypep¬ tide, without deviating from the scope of the invention, such as those illustrated by Compounds 1-9. Likewise known substitutions in the carboxyl moiety at the C-terminus, e.g. a lower alkyl amide, also produce equivalent molecules.

Claims: CLAIMS 1. A GRF PEPTIDE having the formula R -Ala-Asp-Ala-Ile-Phe- Thr-Ser-Ser-Tyr-Arg-Arg- al-Leu-R^ 5 -Gin-Leu-Ser-Ala- rg- rg-R 2 -Leu- Gln-R25-Ile-Leu-Ser-Ar -Gln-Gln-Gly-Glu-R 3 -R35-Gln-Glu-R38- 39-R40- where R^ is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C alkyl or C -C4 lkoxy) , PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4alkyl or C^-C4alko y , N-[para- hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C -C4alkyl or C -C4alkoxy , N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4alkyl or C^-C4alkox ) or N- [(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4alkyl or C^-C4alkoxy) ; Rl5 is Ala or Leu; R22 i Ala or Leu; R 2 5 is Asp or Glu; R34 is Ser or Arg; R35 is Asp or Ser; R38 is Arg or Gin; R39 is Gly or Arg; R 0 is Ala or Ser; R42 is Ala, Val or Phe; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -C00R a , -CR a 0, -C0NHNHR a , -C0N(R a )(R b ) or -CH 2 0R a , with R a and R b being lower alkyl or hydrogen; or a bio- logically 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(R a )(R b ) of the foregoing and/or a non¬ toxic salt of the foregoing. 2. A GRF PEPTIDE of Claim 1 having the formula PHPP-Ala-Asp- Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg- Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2. 3. A GRF PEPTIDE of Claim 1 having the formula PHPA- la-Asp- Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gin-Leu-Ser-Ala-Arg- Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH 2 . 4. A GRF PEPTIDE of Claim 1 having the formula [3-methoxy- PHPA]-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2. 5. A GRF PEPTIDE of Claim 1 having the formula [3-fluoro- PHPA] -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gin-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2. 6. A GRF PEPTIDE of Claim 1 having the formula [3-chloro- PHPA] -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Ty -Arg-Arg-Val-Leu-Ala-Gln- Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asρ-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2. 7. A GRF PEPTIDE of Claim 1 having the formula N-[para- hydroxy Cinnamoyl]-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val- Leu-Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln- Gln-Gly-NH 2 . 8. A GRF PEPTIDE of Claim 1 having the formula N- [ (4-hydroxy- phenoxy) acetyl] -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu- Ala-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln- Gly-NH 2 . 9. A GRF PEPTIDE of Claim 1 having the formula N-[para- hydroxy benzoyl]-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val- Leu-Ala-Gin-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln- Gln-Gly-NH 2 . 10. 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 of the formula: R 1 -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-R 15 -Gln- Leu-Ser-Ala-Arg-Arg-R 2 2-Leu-Gln-R25-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu- R34-R35-Gln-Glu- 3 g-R3g-R4o-Ar -R42-Arg-Leu-Y where R^ is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C -C alkyl or C -C4alkoxy) , PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C alkyl or C^-C4alko y) , N- [para- hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C -C4alkyl or , N- [para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, or C^-C alkoxy) or N- [(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C alk l or C^-C4alkox ) ; R^5 is Ala or Leu; R22 Ala or Leu; R25 is Asp or Glu; R34 is Ser or Arg; R35 is Asp or Ser; R38 is Arg or Gin; R3 is Gly or Arg; R40 is Ala or Ser; R42 is Ala, Val or Phe; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -C00R a , -CR a 0, -C0NHNHR a , -C0N(R a )(R b ) or -CH 2 0R a , with R a and R b being lower alkyl or hydrogen; or a bio¬ logically 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(R a )(R j ,) of the foregoing and/or a non¬ toxic salt of the foregoing. 11. A composition for stimulating the release of growth hormone in an animal comprising an effective amount of a GRF PEPTIDE of the formula: R!-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-R 15 -Gln- Leu-Ser-Ala-Arg-Arg-R22-Leu-Gln-R25-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu- R3 -R35-Gln-Glu-R 3 8-R39-R4 0 -Arg-R42-Arg-Leu-Y where R^ is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cχ-C4al yl or Cχ-C4 lkoxy) , PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, , N-[para- hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4 lk l or C^-C4alkoxy , N- [para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C^-C4alkyl or C -C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C -C4alk l or C^-C4alkoxy) ; 5 is Ala or Leu; 22 is Ala or Leu; R25 is Asp or Glu; R34 is Ser or Arg; R35 is Asp or Ser; R38 is Arg or Gin; R3 is Gly or Arg; R4O is Ala or Ser; R42 is Ala, Val or Phe; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -C00R a , -CR a 0, -C0NHNHR a , -C0N(R a )(R b ) or -CH2θR a , with R a and R D being lower alkyl or hydrogen; or a bio¬ logically 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(R a )(R b ) of the foregoing and/or a non- toxic salt of the foregoing; in association with a pharmaceutically acceptable carrier.

Inventors: FRIEDMAN ALAN R, FRIEDMAN, ALAN, R.
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1990
Main classification: C07K7
IPC codes: A61K38, C07K14