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WO1997017367A1 - Technique de synthese de peptides grf - Google Patents

Technique de synthese de peptides grf Download PDF

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Publication number
WO1997017367A1
WO1997017367A1 PCT/CA1996/000712 CA9600712W WO9717367A1 WO 1997017367 A1 WO1997017367 A1 WO 1997017367A1 CA 9600712 W CA9600712 W CA 9600712W WO 9717367 A1 WO9717367 A1 WO 9717367A1
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WO
WIPO (PCT)
Prior art keywords
ala
grf
segments
ser
gly
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.)
Ceased
Application number
PCT/CA1996/000712
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English (en)
Inventor
Michel Ibea
Paul Brazeau
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.)
Theratechnologies Inc
Original Assignee
Theratechnologies Inc
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 Theratechnologies Inc filed Critical Theratechnologies Inc
Priority to AU72731/96A priority Critical patent/AU7273196A/en
Publication of WO1997017367A1 publication Critical patent/WO1997017367A1/fr
Anticipated expiration legal-status Critical
Ceased 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 invention relates to a high yield synthesis methodology of GRF peptides .
  • GH Growth hormone
  • IGF-I insulin-like growth factor-I
  • EGF epidermal growth factor
  • GH Through its action on IGF-I (somatomedin C) synthesis and secretion, GH stimulate the growth of the cartilage and the bones (structural growth), the protein synthesis and the cellular proliferation in multiple peripheral organs, including muscles and the skin. Through its biological activity, GH participates within adults at the maintenance of a protein anabolism state, and plays a primary role in the tissue regeneration phenomenon after a trauma.
  • IGF-I somatomedin C
  • GH secretion with the age, demonstrated in humans and animals, favors a metabolic shift towards catabolism which initiates or participate to the aging of an organism.
  • GH is thus a physiological anabolic agent absolutely necessary for the linear growth of children and which controls the protein metabolism in adults.
  • the secretion of GH by the pituitary gland is principally controlled by two hypothalamic peptides, somatostatin and growth hormone-releasing factor (GRF).
  • Somatostatin inhibits its secretion, whereas GRF stimulates it.
  • the human GH has been produced by genetic engineering for about ten years. Until recently most of the uses of GH were concerned with growth delay in children and now the uses of GH in adults are studied. The pharmacological uses of GH and GRF may be classified in the following three major categories.
  • Recombinant human growth hormone Treatments with recombinant human growth hormone have been shown to stimulate growth in children with pituitary dwarfism, renal insufficiencies, Turner's syndrome and short stature.
  • Recombinant human GH is presently commercialized as an "orphan drug" in Europe and in the United States for children's growth retardation caused by a GH deficiency and for children's renal insufficiencies. The other uses are under clinical trial investigation.
  • Preliminary studies of one-year treatment with recombinant human GH reported an increase in the muscle mass and in the thickness of skin, a decrease in fat mass with a slight increase in bone density in a population of aged patients.
  • Short term treatment in adults and elderly patients
  • GH and GRF are also intended for veterinary pharmacological uses. Both GH and GRF stimulate growth in pigs during its fattening period by favoring the deposition of muscle tissues instead of adipose tissues and increase milk production in cows, and this without any undesired side effects which would endanger the health of the animals and without any residue in the meat or milk being produced.
  • the bovine somatotropin (BST) is presently commercialized in the United States.
  • GRF is considered as a second generation product destined to replace in the near future the uses of GH in most instances. Accordingly, the use of GRF presents a number of advantages over the use of GH per se.
  • GRF Growth hormone
  • the recombinant GH which is presently commercialized is the 21.5 kDa form whereas GRF induces the synthesis and secretion from the pituitary gland of all the chemical isomers of GH which participate in a wider range of biological activities.
  • a treatment with GH results in a decreased capacity of the pituitary gland to secrete endogenous growth hormone, and the GH response to GRF is diminished after such a treatment.
  • a treatment with GRF does not present this disadvantages, its trophic action on the pituitary gland increases this gland secreting capacity in normal animals and in patients with somatotroph insufficiency. Economical advantages
  • GH proliferative growth factor
  • genetic engineering is very expensive for clinical use.
  • These bacterial contaminants may be pyrogens or may result in immunogenic reactions in patients.
  • the purification of the recombinant product is effected by following a plurality of successive chromatography steps. The drastic purity criteria causes multiple quality control steps.
  • the synthesis of GRF is of chemical nature.
  • the synthesis effected in a solid phase and its purification is carried out in a single step using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the quantity of GRF to be administered is much less than the quantity of GH for the same resulting biological activity.
  • the human GRF is a peptide of 44 amino acids of the following sequence :
  • Solid phase step by step chemical synthesis is the most useful way known to date for synthesis of many peptides, including GRF peptides.
  • the step by step chemical synthesis involved reagent consumption and side reactions such that a single dose may become of a very high cost.
  • the step by step synthesis of hGRF(1-29)NH 2 is well known to yield about 20% of pure material, while the synthesis of hGRF(1-44)NH 2 allow only to a 5% to 10% yield. This constitute one of the main disadvantages in the commercial availability of GRF peptides.
  • the GRF peptides segment coupling methodology of the present invention provides the ultimate solution of this fundamental problem.
  • the method of the present invention particularly allows the coupling of long segments such as GRF peptide segment (1-15)-OH + segment (1-29)NH-; segment (1-15)-OH + segment (16-32) + segment (33-44)-NH-, among others.
  • the step by step method of the prior art uses only shorter segments of 5, 6 or 7 residues.
  • the present invention relates particularly to a high yield manufacturing process of (Gly or Ala) 15 or 32 GRF containing peptide comprising:
  • the present invention relates to a segment coupling process of GRF peptides.
  • the invention relates to the coupling of GRF peptide segments comprising the equations (1) to (4):
  • amino acids are identified by the conventional three-letter abbreviations as indicated below, which are as generally accepted in the peptide art as recommended by the IUPAC-IUB commission in biochemical nomenclature.
  • 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.
  • GRF peptides as used herein is intended to means a known polypeptide which is between about 25 and 44 residues, preferably between 27 and 44 residues in length, which contains gly or Ala in position 15 and/or 32 and which polypeptide promotes the release of growth hormone by the pituitary gland.
  • Illustrative GRF peptides include the natural or synthetic polypeptides disclosed in U.S. Patents Nos.
  • GRF peptides are synthesized in accordance with the present invention:
  • Fig. 1 is a schematic representation of the synthesis of hGRF(1-29) in accordance with the present invention.
  • the present invention relates to a segment couplings process of GRF peptides.
  • the invention relates to the coupling of GRF peptide segments comprising the sequence:
  • Q is 3-(5-hydroxyindolyl)-methyl or an omega or alpha-omega substituted alkyl of the following structure: (HO) m -[ ⁇ ]-(CH 2 ) n -C(R 4 )(R 5 )- wherein,
  • [ ⁇ ] is phenyl
  • R 4 is H, NH 2 , CH 3 -CO-NH-, CH 3 -NH-;
  • R 5 is H or CH 3 -;
  • n 1 or 2;
  • n 0,1 or 2;
  • Q0 is hydrogen or any hydrophobic tail selected from the following formula:
  • G is preferably a carbonyl, but may also be a phosphonyl, a sulfuryl or a sulfinyl group, with "a" being zero or 1;
  • X is an oxygen atom, or an amino group, with b having the value of zero or 1;
  • R 1 , R 2 and R 3 are identical or different, and are either hydroxyl groups, hydrogen or lower linear or branched alkyl groups; c and g can be
  • R 6 is an hydroxyl group, hydrogen or C 4 -C 9 alkyl, with e varying from zero to 6;
  • Z is an amino group -NH-; h varies from zero to
  • Q 1 is Tyr, His, des-amino Tyr, D-Tyr, Met, Phe,
  • Q 2 is Ala, D-Ala, (D or L) N- ⁇ Me-Ala, Val, D- Val, Abu, Aib or D-Arg;
  • Q 3 is Asp or D-Asp
  • Q 7 is Thr, Aib, Leu, Trp, ⁇ -Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO, -OMe, or Me;
  • Q 8 is Asn, D-Asn, Ser, D-Ser, Abu, Ala, Aib,
  • Trp Trp
  • ⁇ -Nal Trp
  • p-X-Phe in which X is H, F, Cl, Br, NO 2 , -OMe, or Me;
  • Q 9 is Ser, Thr, Ala, Aib, Leu, Trp, ⁇ -Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO 2 , -OMe, or Me;
  • Q 10 is Tyl or D-Tyr
  • Q 12 and Q 21 are Lys, Arg, or N e -I-Lys in which
  • I is lower alkyl, acyl, alkenyl, acenyl or cycloalkyl, Q 12 an Q 21 may be the same or different;
  • Q 13 is Ile or Val
  • Q 14 , Q 17 , Q 23 and Q 26 are Leu, Ile, D-Leu or D- Ile and may be the same or different;
  • Q 15 is Gly or Ala
  • Q 18 is Tyr or Ser
  • Q 24 is His or Gln
  • Q 25 is Glu, Asp, D-Glu or D-Asp
  • Q 27 is Met, D-Met, Ala, Nle, Ile, Leu, Nva,
  • Q 28 is Asn, Ser or Asp
  • Q 29 is Arg, D-Arg
  • R is of the following general formula:
  • R 9 is hydrogen, lower alkyl, lower alkenyl, lower aryl , aralkyl or lower alkylcarboxamide;
  • Q 30 , Q 31 are Gln or Asn and may be identical or different
  • Q 32 is gly or Ala, preferably Gly;
  • Q 34 is ser or Arg
  • Q 35 is Asn or Ser
  • Q 38 is Arg or Gln;
  • Q 39 is Gly or Arg;
  • Q 40 is Ala or Ser
  • Q 42 is Ala, Val or Phe
  • R 3 represent the whole carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COOR 4 , -COR 4 , -CO-NHNH-R 4 , -CO- N(R 4 )(R 5 ) or -CH 2 -OR 4 , with R 4 and R 5 being hydrogen or C 1 -C 8 alkyl or a biologically active fragment thereof extending from Q at the N-terminus to a residue in any of position 20 to 44 at its C-terminus; or a Hse(lactone), a
  • One of the preferred embodiment of the high yield method of synthesis of the present invention relates to the coupling of GRF peptide segments as shown in Fig. 1.
  • the N-alpha amino group of Tyr 1 as well as the side chain of Lys 12 are protected as Boc group.
  • Side chains of Tyr 1,10 , Ser 9 and Thr 7 are protected as tert-butyl ether.
  • the side chain of Arg 11 is protected as Pmc or Pbf group.
  • Side chain of Asn 8 is protected as trityl and side chain of Asp is protected as tert-butyl ester.
  • Another embodiment of the high yield method of synthesis of the present invention relates to the coupling of GRF peptide segments coupling shown in equation (1),
  • a preferred embodiment of the present invention relates to the segment coupling shown in equation (5) above, wherein the N-terminal Boc group in Tyr 1 is replaced by the group hexanoyl. This will result in a final peptide as an hexanoyl-Tyr 1 hGRF(1-29) amide after final cleavage in TFA.
  • the most preferred embodiment of this invention relates to the coupling shown in equation (5), wherein the N-terminal Boc group of Tyri is substituted by the group hexenoyl (cis or trans)-3-. This will afford a final peptide as a (cis or trans)-3-hexenoyl-Tyr 1 hGRF(1-29) amide, after cleavage in TFA .
  • Another embodiment of the high yield method of synthesis of the present invention relates to any of the foregoing embodiments wherein Met is substituted for He in position 27 and/or Tyr is substituted for His in position 1.
  • GRFs peptides segments as well as GRFs peptides using the strategy of the present invention when compared to the step by step coupling strategy of the prior art, is illustrated in Table 1 below.
  • C-terminal acidic segments are synthesized manually on sasrin resin (Bachem California, catalog part number RMIS45). In these conditions, the first amino acid is linked to the resin using DIC/DMAP methodology. C-terminal amide segments are synthesized on Pal-PEG-PSTM support (Perspective biosystem, part number GEN 913383), utilizing a Millipore 9050TM plus peptide synthesizer and synthesis cycle supplied by Millipore.
  • Solvents such as DMF and DCM are of HPLC grade and are purchased from Anachemia Sciences. Fmoc amino acid and other reagents are supplied by Synpep Corporation and other commercial sources. Sequential Fmoc Chemistry using double couple protocols are applied to the starting Pal-PEG-PS.
  • Another embodiment of the high yield method of synthesis of the present invention relates to resin for the production of C-terminal carboxamide. Both, aminoacid and segment are coupled using HBTU/HOBt or other coupling methodology. The following side chain protection is used.
  • the minimum segment length is of 14 residues for segments 1 and 2, and of 12 residues for segments 3, 4 and 5.
  • the fully protected acidic segment e.g. (S 1 )-OH or (S 2 )-OH
  • S 1 )-OH or (S 2 )-OH is then afforded by selective cleavage of the peptide resin bond in extremely mild conditions, usually 0.5% TFA in dichloromethane.
  • the coupling efficiency for the synthesis of hexanoyl-Tyr 1 hGRF(1-29) NH 2 is determined by weight , while the purity is determined by HPLC or by TLC for protected individual segments.
  • the mixture CHCl 3 /MeOH/80% Ac-OH (16:1:1) side chain protected acidic segments hexanoyl-Tyr 1 hGRF (1-15)-OH and trans-3-hexenoyl-Tyr 1 hGRF(1-15)-OH gave respectively in TLC, the RF value of 0.47 and 0.49. Again the RF value is the ratio between the migration level of the compound versus the migration level of the eluent.
  • Segment couplings can be performed either by solid phase or in solution synthesis, both resulting in a high yield average of about 65-68% for any 29 or 32 amino acids GRF peptides.
  • the methodology provides also another advantage characterized in that, excess of acidic segment to be coupled can be quantitatively regenerated after coupling by precipitation and washings with water.
  • the side chain protected C-terminal amide segment (e.g. segment 3 for hGRF(16-29)amide) is appropriately made using the step by step methodology and kept as a N-terminal free amine on resin. Then acidic GRF peptide segment bearing protecting groups for its alpha amino group (and, where appropriate, for its amino acid side chain) is coupled in a minimum amount of DMF. After completion of this coupling step, the N-alpha amino protecting group (if any), is removed from this newly anchored segment and the next segment (if any), suitably protected, is added and so forth.
  • the N-terminal protecting groups are removed after each residue is added, but the side chain protecting groups are not yet removed.
  • the peptide is cleaved from the support and then freed from any side chain protecting groups.
  • Such cleavage is performed under conditions that are minimally destructive toward residue in sequence (e.g. in TFA/H 2 O/scavenger), as in procedure supplied by Millipore.
  • the suspension is filtered off and the solution is precipitated , washed with ether and dried. Example of improved yield using this methodology is shown in Table 1 above.
  • Purification is carried out by reverse phase chromatography on a WatersTM prep. 4000 equipped with 3 cartridges (25 ⁇ 100mm, DeltaTM pak C 18 ) at a flow rate of 50 ml per min.
  • the peptide is applied using TEAP buffer at pH 2.5 and eluated with a 90 min. gradient consisting of: Buffer A: TEAP pH 2.5; Buffer B: 80% CH 3 CN/TEAP and starting from 20% B under the isocratic conditions (analytical column DeltaTM pak C18 6 ⁇ 250mm) and ending at 10 % B above.
  • Fractions with minimum purity of 95 % are then pooled and desalted using buffers C: 0.1% TFA in H 2 O and D: 0.1% TFA in CH 3 CN / H 2 O(80:20).
  • the free acid segment to be coupled is properly protected during the coupling step with appropriate acid or base sensitive protecting groups.
  • protecting groups should have the properties of being stable in the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained herein.
  • the in vivo growth hormone releasing activities for hexanoyl-Tyr 1 hGRF(1-29) amide synthesized by solid segment coupling methodology outlined for this invention is summarized in Table 2 and 3 in comparison to hGRF(1-29) and hexanoyl-Tyr 1 hGRF (1-29) amide synthesized using solid phase step by step methodology.
  • the Table 2 is shown for intravenous injection while the Table 3 for subcutaneous injection.

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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)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention porte sur un procédé de production à haut rendement d'un peptide GRF contenant (Gly ou Ala)?15 ou 32¿, ce qui débouche sur une synthèse de peptides GRF d'un rendement moyen compris entre 65 et 68 %. La technique consiste: a) à faire la synthèse de 14 à 15 restes de segments acides de peptide GRF entièrement protégés (S¿1?)-OH et (S2)-OH provenant de résine sasrine, en faisant intervenir la chimie du fluoromethoxycarbonyle, b) à faire la synthèse de 12 à 15 restes de segments amides de peptide GRF protégés à chaîne latérale (S3)-NH-, (S4)-NH- et/ou (S5)-NH- en phase solide en faisant appel à n'importe quelle résine réagissant à l'acide trifluoroacétique, et c) à effectuer une ou deux étapes de couplage en phase solide des segments du peptide GRF de synthèse des étapes a et b.
PCT/CA1996/000712 1995-11-03 1996-10-28 Technique de synthese de peptides grf Ceased WO1997017367A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU72731/96A AU7273196A (en) 1995-11-03 1996-10-28 Method of grf peptides synthesis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55259695A 1995-11-03 1995-11-03
US08/552,596 1995-11-03

Publications (1)

Publication Number Publication Date
WO1997017367A1 true WO1997017367A1 (fr) 1997-05-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027897A1 (fr) * 1997-12-03 1999-06-10 Applied Research Systems Ars Holding N.V. Preparation specifique a un site de conjugues de polyethylene glycol-grf

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0193910A2 (fr) * 1985-03-06 1986-09-10 Sumitomo Pharmaceuticals Company, Limited Synthèse d'un dérivé du "GRF" et peptides intermédiaires
FR2599038A1 (fr) * 1986-05-26 1987-11-27 Sanofi Sa Procede de preparation de nonacosapeptides et peptides intermediaires
WO1994011397A1 (fr) * 1992-11-13 1994-05-26 The Administrators Of The Tulane Educational Fund Agonistes de somatostatine
EP0606816A1 (fr) * 1992-12-10 1994-07-20 Lipotec, S.A. Procédé de préparation de la calcitonine du saumon

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0193910A2 (fr) * 1985-03-06 1986-09-10 Sumitomo Pharmaceuticals Company, Limited Synthèse d'un dérivé du "GRF" et peptides intermédiaires
FR2599038A1 (fr) * 1986-05-26 1987-11-27 Sanofi Sa Procede de preparation de nonacosapeptides et peptides intermediaires
WO1994011397A1 (fr) * 1992-11-13 1994-05-26 The Administrators Of The Tulane Educational Fund Agonistes de somatostatine
EP0606816A1 (fr) * 1992-12-10 1994-07-20 Lipotec, S.A. Procédé de préparation de la calcitonine du saumon

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027897A1 (fr) * 1997-12-03 1999-06-10 Applied Research Systems Ars Holding N.V. Preparation specifique a un site de conjugues de polyethylene glycol-grf
EP0922446A1 (fr) * 1997-12-03 1999-06-16 Applied Research Systems Ars Holding N.V. Préparation des conjugués du GRF-PEG par un méthode en solution spécifique à un site
US6869932B2 (en) 1997-12-03 2005-03-22 Applied Research Systems Ars Holding N.V. Site-specific preparation of polyethlene glycol-GRF conjugates
US7317002B2 (en) 1997-12-03 2008-01-08 Applied Research Systems Ars Holding N.V. Site-specific preparation of polyethylene glycol-GRF conjugates

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Publication number Publication date
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