Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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.
• 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
• GH releasing factors were isolated from extracts of human pancreatic tumors, purified, characterized, synthesized, tested, and found to promote release of growth hormone (GH) by the pituitary. 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-Gin-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gin-Asp-lie-Met-Ser-Arg ⁇
• Synthetic polypeptides have also been synthesized which are disclosed as growth hormone releasing factors (GRF). Although there are a number of cysteic acid containing peptides described in the literature, only two have been reported in which cysteic acid replaced aspartic acid. Konig et al, Peptides 7: Suppl 1, 61-67 (1986) reported that cysteic acid replacement of aspartic acid in secretin resulted in a peptide with 20-40% of the biological activity of the parent peptide. In the second case, Morley J.S., Proc. Roy. Soc.
• the present invention particularly provides a natural or synthetic polypeptide which promotes the release of growth hormone by the pituitary gland (GRF PEPTIDE) and having at least one cysteic acid in place of the amino acid residue normally found at position 3 or 25 of the polypeptide.
• 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.
• 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 isomeric forms, the L-form of the amino acid is being represented unless otherwise expressly indicated.
• the cysteic acid (sulfo alanine) residue is designated by the three letter code Cya.
• the residue [beta(para-hydroxyphenyl)propionyl] - is identified herein as DesaminoTyr.
• the residue [alpha(para-hydroxyphenyl)- acetyl]- is identified herein as Desa ⁇ tino para-hydroxyphenyl glycyl or Desamino PHPG.
• the present invention provides synthetic GRF peptide analogs (GRF PEPTIDES) having the following formula: H-R 1 -R 2 -R 3 -Ala-R 5 -R 6 -R 7 -R 8 -R 9 -R 10 -R 11 -R 12 -R 13 -R 14 -R 15 -Gln-R 17 - R 18 -R 19 -R 20 -R 21 -R 22 -R 23 -R 24 -R 25 -R 26 -R 27 -R 28 -R 29 -Gln-Gln-Gly-Glu-Ser- Asn-Gln-Glu-Arg-Gly-Ala-R 40 -Y wherein R 1 is Tyr, D-Tyr, His, D-His,
• R 2 is Ala or D-Ala optionally substituted with either a C ⁇ Me or N ⁇ Me substituent, or Ser
• R 3 is Asp, D-Asp, Glu or D-Glu
• R5 is lie or Leu
• R 6 is Phe or Tyr
• R 7 is Ser or Thr
• R 8 is Asn, D-Asn, Ser, D-Ser or Thr
• R9 is Ala or Ser
• R 10 is Tyr, D-Tyr or Phe
• R 11 is Arg or Lys
• R 12 is Arg or Lys
• R 13 is lie, Leu or Val
• R 14 is Leu, D-Leu, lie or Val
• R 15 is Ala, D-Ala, Gly, Gin or Leu
• R 17 is Ala, Leu or D-Leu
• R 18 is Ala
• a further embodiment of the invention Is a 32 residue bGRF analog Leu 27 bGRF(1-32)NH 2 having the formula:
• Another embodiment of the invention is the peptide Val 27 hpGRF(1-40)NH 2 having the formula:
• a further embodiment of the invention is a 32 residue bGRF analog DesaminoTyr 1 Ala 15 Leu 27 bGRF(1-32)NH 2 having the formula:
• a preferred embodiment of the invention are the GRF peptides with Cya in position 3 and/or 25 and the following group(s) in the peptide:
• R 1 is DesaminoTyr or Desamino PHPG;
• R 8 and/or R 28 is Ser;
• R 12 and 21 are Arg;
• R 15 is Ala;
• R 27 is Leu; and
• R 33 is Gly-Hse(lactone), Gly-HseOH or Gly-HseN(R a )(R b ).
• a preferred embodiment of the invention is a 33 residue bGRF analog DesaminoTyr 1 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Ser 28 Hse 33 bGRF(1-33)lactone having the formula: DesaminoTyr-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-Hse-lactone; wherein Cya is substituted for Asp in position 3 and/or 25; or a non-toxic salt of the foregoing.
• a preferred embodiment of the invention is a 33 residue bGRF
• a preferred embodiment of the invention is a 33 residue bGRF analog Desamino-PHPG 1 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Ser 28 Hse 33 bGRF(1-33)OH the formula:
• Desamino-PHPG 1 -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-OH; wherein Cya is substituted for Asp in position 3 and/or 25; or a non-toxic salt of the foregoing.
• a further preferred embodiment of the invention is a 30 residue bGRF analog Desamino-PHPG 1 Ser 8 Arg 12 Ala 15 Arg 21 Leu 27 Ser 28 Hse 30 bGRF(1-30)NH(Ethyl) 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 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.
• the peptides are synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Bio ⁇ ystems, 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, Histidine, Desamino para-hydroxyphenylglycine [alpha(parahydroxyphenyl)acetic acid] and Desaminotyrosine [beta(parahydroxy phenyl propionic acid)] 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: Arg, Tosyl Asp, Benzyl Cys, 4-Methyl Benzyl Glu, Benzyl His, Benzyloxymethyl Lys, 2-Chloro Carbobenzoxy Ser, Benzyl Thr, Benzyl Tyr, 4-Bromo Carbobenzoxy.
• the first coupling is carried out in dimethyl formamide (DMA) and the second coupling in methylene chloride.
• DMA dimethyl formamide
• hydroxybenztriazole esters are employed, both couplings are carried out in dimethyl formamide.
• 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 0°C 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 neat TFA. TFA is removed by rotary evaporation and the resulting oil dissolved in glacial acetic acid and lyophilized.
• the crude cysteine containing peptide is then oxidized to the corresponding cysteic acid containing compound using performic acid at -10°C to 10oC, preferably at 0°C, as described by Stewart et al, Solid Phase Peptide Synthesis, pg. 113, Pierce Chemical Company, Rockford, Illinois, 1984. 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 20millimolar 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 Cya 25 , Leu 27 bGRF(1-32)NH 2 ; or Somatolibrin (human pancreatic islet) 25-(3-sulfo-L-alanyl)-27-L-leucyl-28-L-asparaginyl-32-glycinamide-33-de-L-glutamic acid-34-de-L-serine-35-de-L-asparagine-36-de-L-glutamine-37-de-L-glutamlc acid-38-de-L-arginine39-deglycine-40-de-L-alanine-41-de-L-arginine-39-deglycine-40-de-L-alanine-41-de-L-arginIne-42-de-L-alanine-43-de-L-arginine-44-de-L-leucinamide; Cpd. No. 1
• Example 3 Cya 3 , Leu 27 bGRF(1-32)NH 2 ; or Somatolibrin (human pancreatic islet) 3-(3-sulfo-L-alanyl)-27-L-leucyl-28-L-asparaginyl-32-glycinamide-33-de-L-glutamic acid-34-de-L-serine-de-L-asparagine 36-de-L-glutamine-37-de-L-glutamic acid-38-de-L-arginine-39- deglycine-40-de-L-alanine-41-de-L-arginine-42-de-L-alanine-43-de-L-arginine-44-de-L-leucinamide; Cpd.
• the compound gave the expected amino acid analysis and the expected monoisotopic protonated molecular ion (3714.2) by fast atom bombardment mass spectroscopy.
• the compound gave the expected amino acid analysis and the expected monoisotopic protonated molecular ion (3715) by fast atom bombardment mass spectroscopy .
• the compound gave the expected amino acid analysis and the expected monoisotopic protonated molecular ion (3700) by fast atom bombardment mass spectroscopy.
• the compound gave the expected amino acid analysis and the expected monoisotopic protonated molecular ion (3715) by fast atom bombardment mass spectroscopy.
• the compound gave the expected amino acid analysis and the expected monoisotopic protonated molecular ion (3700) by fast atom bombardment mass spectroscopy.
• analogs can be synthesized using the same solid-phase synthesis techniques set forth herein: D-Ala 2 , Arg 21 , Cya 25 , lie 27 rGRF(l-43)0H Cpd,. #16;
• certain analogs can be obtained by recombinant biosynthetic (DNA) methods or a combination of recombinant biosynthetic and synthetic methods, for example:
• Cya 3,25 Leu 27 hGRF(1-44)OH (Compound #18) can be prepared biosynthetically by the procedure described for Leu 27 bGRF(1-44)OH (European Patent Application 0212531) with the following modifications:
• Desamino Tyr 1 , Cya 3 , Arg 12 , Ala 15 , Arg 21 , Leu 27 , Arg 41 b (GRF) 1- 440H (Compound 19) can be prepared by a combination of biosynthetic and synthetic steps as follows:
• DNA coding for Met 1 , Cys 3 , Arg 12 , Ala 15 , Arg 21 , Leu 27 , Arg 41 b(GRF)1-440H is prepared by well known methods in the art and inserted into an E. coli expression plasmid which is used to transform an E. coli host for expression of the desired polypeptide.
• the expressed polypeptides are isolated and cleaved by treatment with cyanogen bromide. After oxidation of Cys 3 to Cya 3 and purification, the Cya 3 , Arg 12 , Ala 15 , Arg 21 , Leu 27 , Arg 41 b(GRF)2-440H are dissolved in dimethylformamide and coupled with desaminotyrosine N-hydroxysuccinimide ester in the presence of a suitable tertiary amine base to give Compound #19.
• 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
• Hse(lactone), HseOH and HseN(R a )(R b ) analogs can be prepared by the methods disclosed in Kempe et al, BIO/TECHNOLOGY, Vol
• bGRF(1-44)NH 2 and Leu 27 bGRF(1- 32)NH 2 were also given to meal-fed Holstein steers by intravenous injection (Procedure B) and the serum growth hormone (GH) response evaluated. No significant differences were observed between bGRF(1-44)NH 2 and Leu 27 bGRF(1-32)NH 2 . between Leu 27 bGRF(1-32)NH 2 and Compounds 1, 2 and/or 3. In comparison to bGRF(1-44)NH 2 (100%), the area under the GH response curve was 122% for Leu 27 bGRF(1-32)NH 2 , 100% for Compound #1, 106% for Compound #2, and 111% for Compound #3. PROCEDURE B
• Holstein steers (265 +/- 5kg) were housed in Individual stalls (18-20oC) and exposed to 16h light: 8h darkness (lights on at 0400h). Steers are meal-fed once daily at 1000h and trained to consume their total daily feed intake in 2 h. The diet consisted of rolled corn plus a mineral/vitamin premix and alfalfa hay cubes fed at 160% maintenance energy requirements on a bodyweight basis. Steers gained approximately 0.75 kg/h/d.
• Cpds 12, 13, 14, and 15 exhibited 0.54, 1.98, 1.21, and 0.97 the potency of bGRF(1-44)NH 2 .
• Leu 27 bGRF(1-32)NH 2 exhibited 0.85 the potency of bGRF(1-44)NH 2 .
• test compounds were tested for growth hormone release in steers. In comparison to the standard bGRF(1-44)NH 2 (Area 100%), the test compounds gave the following relative growth hormone areas : Compound 4, 233%; compound 5, 140%; compound 6, 160%; compound 7, 126%; compound 8, 307%; compound 9, 260%; and compound 11, 173%.
• Compound 8 and 9 Based on the bovine pituitary cell culture assays described herein and a compounds proteolytic stability in bovine plasma, compounds with high potency and good stability such as Compound 8 and 9 are preferred. It is contemplated that Compound 20, which can be prepared by a combination of biosynthetic and synthetic steps, will also exhibit high potency and good stability and is therefor preferred. The most preferred compounds are Cpd. #'s 21, 22, and/or 23.
• All of the synthetic GRF peptides of the subject invention 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 particulary 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. 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.
• 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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• 1989
  • 1989-01-27 EP EP89300827A patent/EP0326418A1/de not_active Ceased
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