CA2026776A1 - Stabilized, potent grf analogs - Google Patents
Stabilized, potent grf analogsInfo
- Publication number
- CA2026776A1 CA2026776A1 CA 2026776 CA2026776A CA2026776A1 CA 2026776 A1 CA2026776 A1 CA 2026776A1 CA 2026776 CA2026776 CA 2026776 CA 2026776 A CA2026776 A CA 2026776A CA 2026776 A1 CA2026776 A1 CA 2026776A1
- Authority
- CA
- Canada
- Prior art keywords
- arg
- ala
- ser
- leu
- c4alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
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 in~ention are more stable in plasma and in an aqueous en~ironment at neutral pH than native GRF sequences. :' BACKGROUND OF THE INVENTION
Physiologists have long recognized that the hypothala~us 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, corresponting hypothalamic GH releasing factors from other species including the rat speciesj 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 ss 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-Ar -Ala-Arg-Leu-NH2 .
Rat GRF (rGRF) has been found to ha~e a Ser residue at position8 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-30 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-NH2/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(1-40)0H, while the free acid form was reported to be l/6th as potent in the same assay.
Vale, et al., (VS 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-position, Phe in the 10-position, or Ala in the 15-position. Asn or Ser may be present in the 28-position. Certain of these peptides, containing 31 15 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 m8y at least partially resist enzymatic tegradation in the body and exhibit substantially increased potency.
Nati~e GRF sequences have a Gly residue at the 15-position.
Analogs with Ala or Leu at the }5-position are known to have incroased GH releasing potency. See for example US Patents 4,649,131 ant 4,734,399 as well as Ling, N., et al., Quo Vadis?, Symposium, 25 Sanofi Group, May 29-30, 1985, Toulouse-Labege, France ~pp. 309-322).
It has been reported that native GRF sequences are subJect to rspid inactivation by blood plasma enzymes. The rapid breakdown involves cleavage of the 2-3 bond of the peptide by a dipeptidyl-aminopeptidase (DAP). Frohman, LA, J. Clin. Invest., 78, 906-913 30 (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-35 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).
~, .
. ~ . . .
W O 90/08776 PCT/US9~/00014 ~
- 3 2 -l~ L~ 7 i SUMMARY OF T~E 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.
DETAILE~ DESCRIPTION OF THE I~VENTION
The term "CRF PEPTIDE", as used in the specification ant claims, means a known polypeptids which i9 between sbout 27 ant 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, 15 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., Ah~ed, M., Lambros, T., Mowles, T., snd Niller, L., "Synthesis ant Biological Activity of Novel Linear & Cyclic GRF Analogs", Poster Presentation, 10th America Peptide Symposium, St. Louis, May 1987; Tou, J.S., Kaempfe, L.A., Vlneyart, B.D., Buonomo, F.C., Della-Fera, M.A., ant Baile, C.A., "Amphiphilic Growth Hormone Releasing Factor Analogs. Peptide Design ant Biological Activity 1~ vivo", Blochem. 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 Stuties on the N-Terminal Region of Growth Hormone Releasing Factor", J. Med. Chem. 28, pp.
181-185 (1985); Felix, A.N., 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", l9th European Peptide Symposium, Aug. 31-Sept. 5, 1986;
Velicelebi, G., Patthi, S., and Kaiser, E.T., "Design and Biological Activ~ty 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", ~uo 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 gr~up at the C-terminal to the right. Uhere the amino acid residue has iso- ..
meric forms, the L-form of the amino acid is being represented unless .
otherwise expressly indicated. :
The residue N-[(beta(para-hydroxyphenyl)propionyl]- is identi-fied herein as DesaminoSyr (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:
Rl-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-R15-Gln-Leu-Ser-Ala-Arg-Arg-R2z-Leu-Gln-R25-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R3g-R39-R4o-Arg-R42-Arg-Leu-y where Rl is P~PP ~optionally substituted with 1 or 2 members selected :.
from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), N-tpara-hydroxy cinnamoyl] (optionally substituted wlth 1 or 2 members selected from the ~roup consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted w~th 1 or Z members selected from the group consisting of halogen, -Cl-C4alkyl or Cl-C4alkoxy);
Rls is Ala or Leu;
R22 is Ala or Leu;
R2s is Asp or Glu;
R34 is Ser or Arg;
R3s is Asp or Ser;
R38 is Arg or Gln;
R3g is Gly or Arg;
R40 is Ala or Ser;
R42 is Ala, Val or Phe; and -~
. ' '.
..
i ` -W O 90/0877~ PCT/US90/00014 t'q ~ t~t ,''~
Y 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 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(Ra~(Rb) of the foregoing and/or a non-toxic salt of the foregoing.
Examples of Rl substituents (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4slkoxy) 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 p-hydroxyphenyl propionyl.
The term Cl-C4alkyl includes methyl, ethyl, propyl, butyl and isomers thereof. The term Cl-C4 alkoxy includes methoxy, ethoxy, propyloxy, butoxy and isomers thereof. The term halogen includes chloro, fluoro, iodo and bromo atoms.
An embodiment of this invention is the peptide PHPP-Ala2 Ser3 Argl2 Alal5 Arg21 Ala22 Leu27 hGRF~2-44)NH2 having the formula:
PHPP-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Aln-Arg-Arg-Ala-Leu-Gln-Asp-Ile-Leu-Sor-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-Ala2 Ser8 Argl2 Leul5 Arg21 LSU27 hGRF(2 32)NH
having the formula: PHPA-Ala-Asp-Ala-lle-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Leu-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 Another embodiment of this invention is the peptide:
3-methoxy PHPA-Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 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-lpara-hydroxy benzoyl]-Ala2 Ser8 Argl2 Alal5 Arg21 GlU25 LeU27 Hse33 hGRF(2-33)1actone 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 :~, ,' .
.
-: - ,. ~ .
, .,,, , , . ,: . ' ' ' W O 90/08776 PCT/US9OtO0014 h i; 2 i i ~ 6-A preferred embodiment of this invention is the peptide:
PHPP Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Hse33 hGRF(2-33)NH2 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-Hse Another preferred embodiment of this invention is the peptide:
PHPA Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Hse30 hGRF(2-30)NH2 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-Gln-Asp-Ile-Leu-Ser-Arg-Hse NH2 For purposes of co~ercial production methodology, the carboxy terminal residue is preferably homoserine, homoserine lactone, homoserine amide, or a lower 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 V.S. Patent 4,529,595 (Col 2, ln 35 to Col 5, ln 64) and US Patent 4,689,318 (Col 2, ln 23 to Col 9, ln 13~, each of which are incorporated herein by reference.
Procedure A sets forth a preferred method for synthesizing GRF
peptide analogs of the sub~ect invention.
PROCEDURE A
The peptides are synthesized by solid-phase methodology utiliz-ing an Appli~d Biosystems 430A peptide synthesizer (Applied Biosys-tems, Fostqr 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 80c 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:
, .. : ... ,.. , . ..... ..... ; . , .......... . ., ...... .. ~ . .,:
.. ~.. . ~ . . . - -~,m r Arg, 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 deprotected and cleaved from the resin with anhydrous hydrogen fluoride contain~ng 104 snisole. Clesvage of the side chain protecting group(s) snd of the peptide from the resin is carried out at O-C or below, preferably -20C for thirty minutes followed by thirty minutes at O-C. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with 15 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 buf~er of 20 millimolar TRIS (pH 6.8) in 204 ~cetonitrile and eluted using a gradient of 0-0.3 molar sodium chloride in the same solvent. Compounts are further purified and desnlted by reverse phase liquid chromatography on ~ Vydac C-18 (Separations Group, Hesperia, California) column using water:acetonitrile gradients, each phase containing 0.1~ TFA. The tesired fractions are pooled and lyophilized yielding the desired GRF PEPTIDE as its trifluroscetste salt. Th~ trifluoroscetat~ salt csn be converted, if desired to other suitable salts, by well known ion exchange methods.
EXAMPLES
Example 1: PHPP-Ala2 Ser8 Argl2 Alal5 Arg21 LeU27 hGRF(2 32)NH2; Cpd. No. 1 The synthesis of the GRF analog peptide 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 -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.
~ ' .
Exsmple 2: PHPA-Ala2 Ser3 Argl2 Alal5 Arg21 LeU27 hGRF(2 32~NH2;
.
W O ~0/08776 PCT/US90/00014 2 ~- 2; ~ 8- -Cpd. No. 2 The synthesis of the GRF analog peptide having the foroula:
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-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)-Ala2 Ser8 Argl2 Alal5 Arg2l LeU27 hGRF(2-32)NH2; Cpd. No. 3 The synthesis of the GRF analog peptide having the formula:
~3-methoxy~PHPA)-Ala Asp-Als 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 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)-Ala2 Ser8 Argl2 Alal5 Arg2l LeU27 hGRF(2 32)NH2; Cpt. No. 4 The synthesis of the GRF analog peptide hnv~ng the foroula:
(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-Gln-Asp-Il~-Leu-Ser-Arg-Gln-Gln-Cly-NH2 i9 conductet in a stepwise manner as in procedure A. The compound gave the expected am~no acld snalysis and gave by FAB-MS the oxpected monoisotopic (M+H)+ of 3686.
Example 5: (3-chloro-PHPA)-Ala2 Ser8 Argl2 Alal5 Arg2l Leu27 hGRF(2-32)NH2; 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-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 FAB-MS the expected monoisotopic (M+H)+ of 3703.
-Example 6: N-[para-hydroxy Cinnamoyl]-Ala2 Ser8 Argl2 Alal5 Arg2l Leu27 hGRF(2-32)NH2; Cpd. No. 6 :
The synthesis of the GRF analog peptide having the formula:
, . . .
, . . .
g ~ s i N-[para-hydroxy Cinnamoyl]-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 is conducted in a stepwise manner as in procedure A. The compound ga~e the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H)+ of 3679.
Example 7: N-[(para-hydroxy-phenoxy)acetyl]-Ala2 Ser Argl2 Alal5 -, Arg21 Leu27 hGRF(2-32)NH2; 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-Ar~-Arg-Val-Leu-Als~Gln Leu~Ser-Ala-Arg-Arg-Leu Leu~Gln Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-~H2 is conducted in a stepwise manner as in procedure A. ~' Example 8: N-~para-hydroxy-benzoyl]-Ala2 Ser8 Argl2 Alal5 Arg2l ' -Leu27 hGRF(2-32)NH2; Cpd. No. 8 , The synthesis of the GRF analog peptide having the formula:
N-[para-hydroxy-benzoyl]-Ala-Asp-Ala-Ile-Phe-T,hr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Arg-Lesu-Lesu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 is conducted in a stepw~3e 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 mothods, for oxample: ' ' PHpp Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Ser28 Arg41 bGRF(2-44)0H can be prepared by a combination of biosynthetic and synthetic methodol- ' ' ogy by the procedure described for Leu27 bGRF(1-44)0H (European Patent Application 0212531) with the following .
modifications:
In the segment of DNA coding for bGRF(1-44)0H the codons for Tyrl are deleted and the codons for Asn8 Lysl2 Glyl5 Lys21 Asn28 Lys41 are replaced by the codons : (TCT) Ser8, (CGT) Argl2, (GCT) Alal5, (CGT) Arg21, (TCT) Ser28, and (CGT) Arg41 respectively. 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 : , : '' ' . . . : : . . .. . ' W O 90/08776 PCT/US9OtO0014 r~ 10- ' - :
dimethylformamide and the amino terminal Ala of the crude Ser8 Argl2 ~ -Alal5 Arg21 Leu27 Ser28 Arg41 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-Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Ser28 Arg41 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(Ra)(Rb) 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 8nd other animal~
wlth normal GH levels. Mcreover, 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 G~ where the host being treated requires such therapeutic treatment. The required dosage will vary with the particular condition being treated, with the severlty 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 considsred as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydro'orod de, sulphate, phosphate, maleate, acetste, citrate, benzoste, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to sdministered by intravenous administration in isotonic saline, phosphate buffer solutions or the like may be efi'ected.
The peptites should be administered to humans under the guidance of a physician, snd pharmsc9uticsl compositions will ususlly contsin the peptite in conJunction with a conventional, solid or liquit, phsrmsceuticslly-scceptsble carrier. Usually, the parental dosage will be from about 100 nanogrsms to about 50 micrograms of the pep-tide per kilogram of the body wei~ht of the host.
Although the invention has been described with regard to its preferred embodiments, it should be unterstood that various changes and motifications 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 ~ 3 Y i ~ ~ : 12 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 rssidues 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 Cq~:iV~Ibllt mOlbC~llbb.
. ,:"' , .
.: . . . .
, . ' ' ' ', ,... . .' ' ~.~ .. " , . . ', ' . ' .. ' .- ' . ', .. , ., , ' ' .
~' ` ` '-; ' ' '. ' ' '. . '; . ' ' '" .' . ' ,'. '': ''. " ' ' ' . ' '. " ' ' ' ' ' , 1
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 in~ention are more stable in plasma and in an aqueous en~ironment at neutral pH than native GRF sequences. :' BACKGROUND OF THE INVENTION
Physiologists have long recognized that the hypothala~us 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, corresponting hypothalamic GH releasing factors from other species including the rat speciesj 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 ss 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-Ar -Ala-Arg-Leu-NH2 .
Rat GRF (rGRF) has been found to ha~e a Ser residue at position8 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-30 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-NH2/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(1-40)0H, while the free acid form was reported to be l/6th as potent in the same assay.
Vale, et al., (VS 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-position, Phe in the 10-position, or Ala in the 15-position. Asn or Ser may be present in the 28-position. Certain of these peptides, containing 31 15 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 m8y at least partially resist enzymatic tegradation in the body and exhibit substantially increased potency.
Nati~e GRF sequences have a Gly residue at the 15-position.
Analogs with Ala or Leu at the }5-position are known to have incroased GH releasing potency. See for example US Patents 4,649,131 ant 4,734,399 as well as Ling, N., et al., Quo Vadis?, Symposium, 25 Sanofi Group, May 29-30, 1985, Toulouse-Labege, France ~pp. 309-322).
It has been reported that native GRF sequences are subJect to rspid inactivation by blood plasma enzymes. The rapid breakdown involves cleavage of the 2-3 bond of the peptide by a dipeptidyl-aminopeptidase (DAP). Frohman, LA, J. Clin. Invest., 78, 906-913 30 (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-35 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).
~, .
. ~ . . .
W O 90/08776 PCT/US9~/00014 ~
- 3 2 -l~ L~ 7 i SUMMARY OF T~E 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.
DETAILE~ DESCRIPTION OF THE I~VENTION
The term "CRF PEPTIDE", as used in the specification ant claims, means a known polypeptids which i9 between sbout 27 ant 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, 15 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., Ah~ed, M., Lambros, T., Mowles, T., snd Niller, L., "Synthesis ant Biological Activity of Novel Linear & Cyclic GRF Analogs", Poster Presentation, 10th America Peptide Symposium, St. Louis, May 1987; Tou, J.S., Kaempfe, L.A., Vlneyart, B.D., Buonomo, F.C., Della-Fera, M.A., ant Baile, C.A., "Amphiphilic Growth Hormone Releasing Factor Analogs. Peptide Design ant Biological Activity 1~ vivo", Blochem. 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 Stuties on the N-Terminal Region of Growth Hormone Releasing Factor", J. Med. Chem. 28, pp.
181-185 (1985); Felix, A.N., 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", l9th European Peptide Symposium, Aug. 31-Sept. 5, 1986;
Velicelebi, G., Patthi, S., and Kaiser, E.T., "Design and Biological Activ~ty 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", ~uo 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 gr~up at the C-terminal to the right. Uhere the amino acid residue has iso- ..
meric forms, the L-form of the amino acid is being represented unless .
otherwise expressly indicated. :
The residue N-[(beta(para-hydroxyphenyl)propionyl]- is identi-fied herein as DesaminoSyr (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:
Rl-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-R15-Gln-Leu-Ser-Ala-Arg-Arg-R2z-Leu-Gln-R25-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu-R34-R35-Gln-Glu-R3g-R39-R4o-Arg-R42-Arg-Leu-y where Rl is P~PP ~optionally substituted with 1 or 2 members selected :.
from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy), N-tpara-hydroxy cinnamoyl] (optionally substituted wlth 1 or 2 members selected from the ~roup consisting of halogen, Cl-C4alkyl or Cl-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted w~th 1 or Z members selected from the group consisting of halogen, -Cl-C4alkyl or Cl-C4alkoxy);
Rls is Ala or Leu;
R22 is Ala or Leu;
R2s is Asp or Glu;
R34 is Ser or Arg;
R3s is Asp or Ser;
R38 is Arg or Gln;
R3g is Gly or Arg;
R40 is Ala or Ser;
R42 is Ala, Val or Phe; and -~
. ' '.
..
i ` -W O 90/0877~ PCT/US90/00014 t'q ~ t~t ,''~
Y 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 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(Ra~(Rb) of the foregoing and/or a non-toxic salt of the foregoing.
Examples of Rl substituents (optionally substituted with 1 or 2 members selected from the group consisting of halogen, Cl-C4alkyl or Cl-C4slkoxy) 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 p-hydroxyphenyl propionyl.
The term Cl-C4alkyl includes methyl, ethyl, propyl, butyl and isomers thereof. The term Cl-C4 alkoxy includes methoxy, ethoxy, propyloxy, butoxy and isomers thereof. The term halogen includes chloro, fluoro, iodo and bromo atoms.
An embodiment of this invention is the peptide PHPP-Ala2 Ser3 Argl2 Alal5 Arg21 Ala22 Leu27 hGRF~2-44)NH2 having the formula:
PHPP-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Aln-Arg-Arg-Ala-Leu-Gln-Asp-Ile-Leu-Sor-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-Ala2 Ser8 Argl2 Leul5 Arg21 LSU27 hGRF(2 32)NH
having the formula: PHPA-Ala-Asp-Ala-lle-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Leu-Gln-Leu-Ser-Ala-Arg-Arg-Leu-Leu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 Another embodiment of this invention is the peptide:
3-methoxy PHPA-Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 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-lpara-hydroxy benzoyl]-Ala2 Ser8 Argl2 Alal5 Arg21 GlU25 LeU27 Hse33 hGRF(2-33)1actone 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 :~, ,' .
.
-: - ,. ~ .
, .,,, , , . ,: . ' ' ' W O 90/08776 PCT/US9OtO0014 h i; 2 i i ~ 6-A preferred embodiment of this invention is the peptide:
PHPP Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Hse33 hGRF(2-33)NH2 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-Hse Another preferred embodiment of this invention is the peptide:
PHPA Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Hse30 hGRF(2-30)NH2 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-Gln-Asp-Ile-Leu-Ser-Arg-Hse NH2 For purposes of co~ercial production methodology, the carboxy terminal residue is preferably homoserine, homoserine lactone, homoserine amide, or a lower 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 V.S. Patent 4,529,595 (Col 2, ln 35 to Col 5, ln 64) and US Patent 4,689,318 (Col 2, ln 23 to Col 9, ln 13~, each of which are incorporated herein by reference.
Procedure A sets forth a preferred method for synthesizing GRF
peptide analogs of the sub~ect invention.
PROCEDURE A
The peptides are synthesized by solid-phase methodology utiliz-ing an Appli~d Biosystems 430A peptide synthesizer (Applied Biosys-tems, Fostqr 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 80c 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:
, .. : ... ,.. , . ..... ..... ; . , .......... . ., ...... .. ~ . .,:
.. ~.. . ~ . . . - -~,m r Arg, 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 deprotected and cleaved from the resin with anhydrous hydrogen fluoride contain~ng 104 snisole. Clesvage of the side chain protecting group(s) snd of the peptide from the resin is carried out at O-C or below, preferably -20C for thirty minutes followed by thirty minutes at O-C. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with 15 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 buf~er of 20 millimolar TRIS (pH 6.8) in 204 ~cetonitrile and eluted using a gradient of 0-0.3 molar sodium chloride in the same solvent. Compounts are further purified and desnlted by reverse phase liquid chromatography on ~ Vydac C-18 (Separations Group, Hesperia, California) column using water:acetonitrile gradients, each phase containing 0.1~ TFA. The tesired fractions are pooled and lyophilized yielding the desired GRF PEPTIDE as its trifluroscetste salt. Th~ trifluoroscetat~ salt csn be converted, if desired to other suitable salts, by well known ion exchange methods.
EXAMPLES
Example 1: PHPP-Ala2 Ser8 Argl2 Alal5 Arg21 LeU27 hGRF(2 32)NH2; Cpd. No. 1 The synthesis of the GRF analog peptide 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 -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.
~ ' .
Exsmple 2: PHPA-Ala2 Ser3 Argl2 Alal5 Arg21 LeU27 hGRF(2 32~NH2;
.
W O ~0/08776 PCT/US90/00014 2 ~- 2; ~ 8- -Cpd. No. 2 The synthesis of the GRF analog peptide having the foroula:
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-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)-Ala2 Ser8 Argl2 Alal5 Arg2l LeU27 hGRF(2-32)NH2; Cpd. No. 3 The synthesis of the GRF analog peptide having the formula:
~3-methoxy~PHPA)-Ala Asp-Als 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 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)-Ala2 Ser8 Argl2 Alal5 Arg2l LeU27 hGRF(2 32)NH2; Cpt. No. 4 The synthesis of the GRF analog peptide hnv~ng the foroula:
(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-Gln-Asp-Il~-Leu-Ser-Arg-Gln-Gln-Cly-NH2 i9 conductet in a stepwise manner as in procedure A. The compound gave the expected am~no acld snalysis and gave by FAB-MS the oxpected monoisotopic (M+H)+ of 3686.
Example 5: (3-chloro-PHPA)-Ala2 Ser8 Argl2 Alal5 Arg2l Leu27 hGRF(2-32)NH2; 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-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 FAB-MS the expected monoisotopic (M+H)+ of 3703.
-Example 6: N-[para-hydroxy Cinnamoyl]-Ala2 Ser8 Argl2 Alal5 Arg2l Leu27 hGRF(2-32)NH2; Cpd. No. 6 :
The synthesis of the GRF analog peptide having the formula:
, . . .
, . . .
g ~ s i N-[para-hydroxy Cinnamoyl]-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 is conducted in a stepwise manner as in procedure A. The compound ga~e the expected amino acid analysis and gave by FAB-MS the expected monoisotopic (M+H)+ of 3679.
Example 7: N-[(para-hydroxy-phenoxy)acetyl]-Ala2 Ser Argl2 Alal5 -, Arg21 Leu27 hGRF(2-32)NH2; 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-Ar~-Arg-Val-Leu-Als~Gln Leu~Ser-Ala-Arg-Arg-Leu Leu~Gln Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-~H2 is conducted in a stepwise manner as in procedure A. ~' Example 8: N-~para-hydroxy-benzoyl]-Ala2 Ser8 Argl2 Alal5 Arg2l ' -Leu27 hGRF(2-32)NH2; Cpd. No. 8 , The synthesis of the GRF analog peptide having the formula:
N-[para-hydroxy-benzoyl]-Ala-Asp-Ala-Ile-Phe-T,hr-Ser-Ser-Tyr-Arg-Arg-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Arg-Lesu-Lesu-Gln-Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-NH2 is conducted in a stepw~3e 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 mothods, for oxample: ' ' PHpp Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Ser28 Arg41 bGRF(2-44)0H can be prepared by a combination of biosynthetic and synthetic methodol- ' ' ogy by the procedure described for Leu27 bGRF(1-44)0H (European Patent Application 0212531) with the following .
modifications:
In the segment of DNA coding for bGRF(1-44)0H the codons for Tyrl are deleted and the codons for Asn8 Lysl2 Glyl5 Lys21 Asn28 Lys41 are replaced by the codons : (TCT) Ser8, (CGT) Argl2, (GCT) Alal5, (CGT) Arg21, (TCT) Ser28, and (CGT) Arg41 respectively. 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 : , : '' ' . . . : : . . .. . ' W O 90/08776 PCT/US9OtO0014 r~ 10- ' - :
dimethylformamide and the amino terminal Ala of the crude Ser8 Argl2 ~ -Alal5 Arg21 Leu27 Ser28 Arg41 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-Ala2 Ser8 Argl2 Alal5 Arg21 Leu27 Ser28 Arg41 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(Ra)(Rb) 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 8nd other animal~
wlth normal GH levels. Mcreover, 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 G~ where the host being treated requires such therapeutic treatment. The required dosage will vary with the particular condition being treated, with the severlty 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 considsred as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydro'orod de, sulphate, phosphate, maleate, acetste, citrate, benzoste, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to sdministered by intravenous administration in isotonic saline, phosphate buffer solutions or the like may be efi'ected.
The peptites should be administered to humans under the guidance of a physician, snd pharmsc9uticsl compositions will ususlly contsin the peptite in conJunction with a conventional, solid or liquit, phsrmsceuticslly-scceptsble carrier. Usually, the parental dosage will be from about 100 nanogrsms to about 50 micrograms of the pep-tide per kilogram of the body wei~ht of the host.
Although the invention has been described with regard to its preferred embodiments, it should be unterstood that various changes and motifications 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 ~ 3 Y i ~ ~ : 12 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 rssidues 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 Cq~:iV~Ibllt mOlbC~llbb.
. ,:"' , .
.: . . . .
, . ' ' ' ', ,... . .' ' ~.~ .. " , . . ', ' . ' .. ' .- ' . ', .. , ., , ' ' .
~' ` ` '-; ' ' '. ' ' '. . '; . ' ' '" .' . ' ,'. '': ''. " ' ' ' . ' '. " ' ' ' ' ' , 1
Claims (11)
1. A GRF PEPTIDE having the formula where R1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy);
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing.
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing.
2. A GRF PEPTIDE of Claim 1 having the formula .
3. A GRF PEPTIDE of Claim 1 having the formula .
4. A GRF PEPTIDE of Claim 1 having the formula .
5. A GRF PEPTIDE of Claim 1 having the formula .
6. A GRF PEPTIDE of Claim 1 having the formula .
7. A GRF PEPTIDE of Claim 1 having the formula .
8. A GRF PEPTIDE of Claim 1 having the formula.
9. A GRF PEPTIDE of Claim 1 having the formula .
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:
where R1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy);
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing.
where R1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy);
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) 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:
where R1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy);
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing; in association with a pharmaceutically acceptable carrier.
where R1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C1-C4alkyl or C1-C4alkoxy);
R15 is Ala or Leu;
R22 is Ala or Leu;
R25 is Asp or Glu;
R34 is Ser or Arg;
R35 is Asp or Ser;
R38 is Arg or Gln;
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 -COORa, -CRaO, -CONHNHRa, -CON(Ra)(Rb) or -CH2ORa, with Ra and Rb 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(Ra)(Rb) of the foregoing and/or a non-toxic salt of the foregoing; in association with a pharmaceutically acceptable carrier.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US30351889A | 1989-01-27 | 1989-01-27 | |
| US303,518 | 1989-01-27 | ||
| US32395589A | 1989-03-14 | 1989-03-14 | |
| US323,955 | 1989-03-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2026776A1 true CA2026776A1 (en) | 1990-07-28 |
Family
ID=26973496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2026776 Abandoned CA2026776A1 (en) | 1989-01-27 | 1990-01-03 | Stabilized, potent grf analogs |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0457786A1 (en) |
| AU (1) | AU4955490A (en) |
| CA (1) | CA2026776A1 (en) |
| WO (1) | WO1990008776A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0477217T3 (en) * | 1989-06-16 | 1995-04-03 | Upjohn Co | Stabilized potent GRF analogs |
| JPH06500311A (en) * | 1990-06-29 | 1994-01-13 | エフ・ホフマン―ラ ロシュ アーゲー | Histidine-substituted human growth hormone-releasing factor analog |
| EP0525838A3 (en) * | 1991-04-09 | 1993-02-10 | F. Hoffmann-La Roche Ag | Growth hormone releasing factor analogs |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL84758A (en) * | 1987-01-13 | 1992-03-29 | Salk Inst For Biological Studi | Peptides stimulating the release of pituitary growth hormone in fish and amphibians,and pharmaceutical compositions containing them |
| US5002931A (en) * | 1987-05-22 | 1991-03-26 | The Salk Institute For Biological Studies | GRF analogs VII |
| NZ227770A (en) * | 1988-01-29 | 1991-08-27 | Upjohn Co | Growth hormone releasing factor (grf) peptide containing a cysteic acid residue in position 3 or 25 |
-
1990
- 1990-01-03 AU AU49554/90A patent/AU4955490A/en not_active Abandoned
- 1990-01-03 CA CA 2026776 patent/CA2026776A1/en not_active Abandoned
- 1990-01-03 EP EP19900902439 patent/EP0457786A1/en not_active Withdrawn
- 1990-01-03 WO PCT/US1990/000014 patent/WO1990008776A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1990008776A1 (en) | 1990-08-09 |
| EP0457786A1 (en) | 1991-11-27 |
| AU4955490A (en) | 1990-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1341207C (en) | Analogues of insulin-like growth factor-1 | |
| JP2791955B2 (en) | Neuropeptide Y agonist and partial agonist | |
| DK162103B (en) | SYNTHETIC GRF ANALOGUE PEPTIDES AND PHARMACEUTICAL ACCEPTABLE SALTS THEREOF AND PHARMACEUTICAL COMPONENTS CONTAINING PEPTID OR NON-TOXIC SALT THEREOF | |
| HU191263B (en) | Process for producing factor for promoting letting out growth hormone of human pancreatic origine | |
| FI87080C (en) | FREQUENCY REARING FOR GRF ANALOGUE | |
| KR0143913B1 (en) | Synthetic peptides and pharmaceutical compositions comprising them | |
| US5756458A (en) | Stabilized potent GRF analogs | |
| JP2512638B2 (en) | Stabilized and potent GRF analog | |
| JP2974254B2 (en) | GRF analog VIIA | |
| CA2026776A1 (en) | Stabilized, potent grf analogs | |
| AU617426B2 (en) | Growth hormone releasing factor analogs | |
| EP0511003B1 (en) | Superactive GRF analogs | |
| AU655791B2 (en) | Stabilized, potent GRF analogs | |
| JPH04503061A (en) | Stable and powerful GRF analogs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |