EP0457786A1 - Stabilisierte wirksame grf-analoge - Google Patents

Stabilisierte wirksame grf-analoge

Info

Publication number
EP0457786A1
EP0457786A1 EP19900902439 EP90902439A EP0457786A1 EP 0457786 A1 EP0457786 A1 EP 0457786A1 EP 19900902439 EP19900902439 EP 19900902439 EP 90902439 A EP90902439 A EP 90902439A EP 0457786 A1 EP0457786 A1 EP 0457786A1
Authority
EP
European Patent Office
Prior art keywords
arg
leu
ala
ser
gln
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.)
Withdrawn
Application number
EP19900902439
Other languages
English (en)
French (fr)
Inventor
Alan R. Friedman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmacia and Upjohn Co
Original Assignee
Upjohn Co
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Upjohn Co filed Critical Upjohn Co
Publication of EP0457786A1 publication Critical patent/EP0457786A1/de
Withdrawn legal-status Critical Current

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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

Definitions

  • the present invention relates to a peptide having influence on the function of the pituitary gland in humans and other animals, particularly mammals.
  • 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.
  • hypothalamus controls the secretory functions of the adenohypophysis with the hypothalamus producing special substances which stimulate or inhibit the secretion of each pituitary hormone.
  • human pancreatic hormone In 1982, human pancreatic hormone
  • hpGRF growth hormone releasing factors
  • 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
  • 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-NH 2 /OH.
  • GH growth hormone
  • 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- ⁇ osition, Phe in the 10-position, or Ala in the 15-position. Asn or Ser may be present in the 28-position.
  • Native GRF sequences have a Gly residue at the 15-position.
  • 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 residues, 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.
  • GRF PEPTIDE 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.
  • GRF PEPTIDE includes nontoxic salts thereof.
  • GRF PEPTIDE The nomenclature used to define the GRF PEPTIDE is that specified 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-meric forms, the L-form of the amino acid is being represented unless otherwise expressly indicated.
  • the residue N- [ (beta(para-hydroxyphenyl) ⁇ ropionyl]- is identified herein as DesaminoTyr (or PHPP) .
  • the residue N-[alpha(para-hydroxyphenyl)acetyl]- is identified herein as PHPA.
  • GRF PEPTIDES synthetic GRF peptide analogs having the following formula:
  • R 1 is PHPP (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 allqrl or C 1 -C 4 alkoxy), PHPA (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy) , N- [para- hydroxy benzoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy), N-[para-hydroxy cinnamoyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy) or N-[(4-hydroxy phenoxy)acetyl] (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 alkyl or C 1 -C 4
  • R 15 is Ala or Leu
  • R 22 is Ala or Leu
  • R 2 5 is Asp or Glu
  • R 34 is Ser or Arg
  • R 35 is Asp or Ser
  • R 38 is Arg or Gin
  • R 39 is Gly or Arg
  • R 40 is Ala or Ser
  • R 42 is Ala, Val or Phe; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COOR a , -CR a O, -CONHNHR a , -CON(R a )(R b ) or -CH 2 OR a , with R a and R b being lower alkyl or hydrogen; or a biologically active fragment thereof extending from R at the N-terminus to a residue in any of positions 27 through 44 as its C-terminus; or a Hse(lactone), HseOH or HseN(R a ) (R b ) of the foregoing and/or a nontoxic salt of the foregoing.
  • Y signifies the carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COOR a , -CR a O, -CONHNHR a , -CON(R a )(R b
  • R 1 substituents (optionally substituted with 1 or 2 members selected from the group consisting of halogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy) 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 cinnamoyl and p-hydroxyphenyl propionyl.
  • C 1 -C 4 alkyl includes methyl, ethyl, propyl, butyl and isomers thereof.
  • C 1 -C 4 alkoxy includes methoxy, ethoxy, propyloxy, butoxy and isomers thereof.
  • halogen includes chloro, fluoro, 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:
  • 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:
  • the carboxy terminal residue is preferably homoserine, homoserine lactone, homoserine amide, or a lower C 1 -C 4 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
  • Procedure A sets forth a preferred method for synthesizing GRF peptide analogs of the subject invention.
  • the peptides are synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, 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 corresponding 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.
  • Boc deprotection is accomplished with trifluoroacetic acid (TFA) in methylene chloride.
  • TFA trifluoroacetic acid
  • the peptides are deprotected 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 0oC or below, preferably -20oC for thirty minutes followed by thirty minutes at 0°C.
  • 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.
  • 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.
  • 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
  • PHPP-Ala-Asp-Ala-lle-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.
  • the compound gave the expected amino acid analysis and gave by Fast atom bombardment mass spectroscopy (FAB-MS) the expected monoisotopic (M+H) + of 3681.
  • FAB-MS Fast atom bombardment mass spectroscopy
  • Example 2 PHPA-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 hGRF(2-32)NH 2 ; Cpd. No . 2
  • 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-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 3667.
  • 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 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 hGRF(2-32)NH 2 ; Cpd. No. 8
  • 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-Asp-lle-Leu-Ser-Arg-Gln-Gln-Gly-NH 2 is conducted in a stepwise manner as in procedure A.
  • PHPP-Ala 2 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Ser 28 Arg 41 bGRF(2-44)OH can be prepared by a combination of biosynthetic and synthetic methodology by the procedure described for Leu 27 bGRF(1-44)OH (European Patent Application 0212531) with the following modifications:
  • 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.
  • Hse(lactone), HseOH and HseN(R a ) (R b ) analogs can be prepared by the methods disclosed in Kempe, et al., BIO/TECHNOLOGY, Vol 4, pp 565-568 (1986) .
  • Dosages between about 50 nanograms and about 5 micrograms of these peptides per Kg of body weight are considered to be particularly 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.
  • administration should alter body fat content and modify other GH-dependent metabolic, immunologic and developmental processes.
  • these analogs may be useful as a means of stimulating anabolic processes in human beings under circumstances such as following the incurring of burns.
  • 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.
  • commercial warm-blooded animals such as chickens, turkeys, pigs, goats, cattle and sheep
  • fish and other cold-blooded marine animals e.g., sea turtles and eels, and amphibians
  • Daily dosages of between 50 nanograms/Kg and about 50 micrograms/Kg body weight are considered to be particularly effective in increasing lactation and growth.
  • these synthetic peptides should have a purity of at least about 93% and preferably at least 98%.
  • These synthetic peptides or the nontoxic salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition 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).
  • acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
  • 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.
  • the parental dosage will be from about 100 nanograms to about 50 micrograms of the peptide per kilogram of the body weight of the host.

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  • 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)
EP19900902439 1989-01-27 1990-01-03 Stabilisierte wirksame grf-analoge Withdrawn EP0457786A1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30351889A 1989-01-27 1989-01-27
US303518 1989-01-27
US32395589A 1989-03-14 1989-03-14
US323955 1994-10-17

Publications (1)

Publication Number Publication Date
EP0457786A1 true EP0457786A1 (de) 1991-11-27

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Family Applications (1)

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Country Status (4)

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EP (1) EP0457786A1 (de)
AU (1) AU4955490A (de)
CA (1) CA2026776A1 (de)
WO (1) WO1990008776A1 (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2063360T3 (es) * 1989-06-16 1995-01-01 Upjohn Co Analogos potentes de grf, estabilizados.
ZA914983B (en) * 1990-06-29 1992-03-25 Hoffmann La Roche His-grf-analogs
AU662731B2 (en) * 1991-04-09 1995-09-14 F. Hoffmann-La Roche Ag Growth hormone releasing factor analogs

Family Cites Families (3)

* Cited by examiner, † Cited by third party
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
AU617426B2 (en) * 1988-01-29 1991-11-28 Upjohn Company, The Growth hormone releasing factor analogs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9008776A1 *

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Publication number Publication date
AU4955490A (en) 1990-08-24
WO1990008776A1 (en) 1990-08-09
CA2026776A1 (en) 1990-07-28

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