WO1997017367A1

WO1997017367A1 - Method of grf peptides synthesis

Info

Publication number: WO1997017367A1
Application number: PCT/CA1996/000712
Authority: WO
Inventors: Michel Ibea; Paul Brazeau
Original assignee: Theratechnologies Inc.
Priority date: 1995-11-03
Filing date: 1996-10-28
Publication date: 1997-05-15
Also published as: AU7273196A

Abstract

The present invention relates to a high yield manufacturing process of (Gly or Ala)?15 or 32¿ GRF containing peptide, resulting in GRF peptides synthesis at a yield averaging 65 to 68 %. The method comprises the steps of: a) synthesis of a 14 to 15 residues of the fully protected GRF peptide acidic segments (S¿1?)-OH and (S2)-OH from sasrin resin, using sequential Fmoc chemistry; b) synthesis of a 12 to 15 residues of the side chain protected GRF peptide amide segments (S3)-NH-, (S4)-NH-, and/or (S5)-NH- on solid phase using any TFA sensitive resin; and c) one or two coupling steps of the synthesized GRF peptide segments of steps a) and b) on solid phase.

Description

METHOD OF GRF PEPTIDES SYNTHESIS

BACKGROUND OF THE INVENTION

( a ) Field of the Invention

The invention relates to a high yield synthesis methodology of GRF peptides .

(b ) Description of Prior Art

Growth hormone (GH) or somatotropin, secreted by the pituitary gland constitute a family of hormones which biological activity is fundamental for the linear growth of a young organism but also for the maintenance of the integrity at its adult state. GH acts directly or indirectly on the peripheral organs by stimulating the synthesis of growth factors (insulin-like growth factor-I or IGF-I) or of their receptors (epidermal growth factor or EGF). The direct action of GH is of the type referred to as anti-insulinic, which favors the lipolysis at the level of adipose tissues. 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.

The decrease of 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. The loss in muscle mass, the accumulation of adipose tissues, the bone demineralization, the loss of tissue regeneration capacity after an injury, which are observed in elderly, correlate with the decrease in the secretion of GH. 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.

Children growth

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.

Long term treatment for adults and elderly patients

A decrease in GH secretion causes changes in body composition during aging. 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

In preclinical and clinical studies, growth hormone has been shown to stimulate protein anabolism and healing in cases of burn, AIDS and cancer, in wound and bone healing.

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.

Most of the clinical studies presently undertaken were conducted with recombinant GH. The 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.

Physiological advantages

Growth hormone (GH) is secreted by the pituitary gland in a pulse fashion, since this rhythm of secretion is crucial for an optimal biological activity. The administration of GH to correspond to its natural mode of secretion is difficult to achieve. When GRF is administered in a continuous fashion as a slow releasing preparation or as an infusion, it increases GH secretion while respecting its pulsatility.

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. On the contrary, 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

The production of GH by genetic engineering is very expensive for clinical use. In particular, there are risks of contamination of these commercial preparation with material from the bacterial strain used. 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). Also 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. However, when synthesizing long polypeptide chain, the step by step chemical synthesis involved reagent consumption and side reactions such that a single dose may become of a very high cost. For example, the step by step synthesis of hGRF(1-29)NH₂ is well known to yield about 20% of pure material, while the synthesis of hGRF(1-44)NH₂ 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. Whereas the step by step method of the prior art uses only shorter segments of 5, 6 or 7 residues.

SUMMARY OF THE INVENTION

The present invention relates particularly to a high yield manufacturing process of (Gly or Ala)¹⁵ or ³² GRF containing peptide comprising:

- synthesis of a 14 to 15 residues of the fully protected GRF peptide acidic segments (S₁)-OH and (S₂)-OH from sasrin resin, using sequential Fmoc chemistry; - synthesis of a 12 to 15 residues of the side chain protected GRF peptide amide segments (S₃)-NH-, (S₄)-NH-, and/or (S₅)-NH- on solid phase using any TFA sensitive resin; and

- a single or two coupling steps of the aforementioned GRF peptide segments on solid phase, giving GRF peptides in yield averaging 65 to 68%.

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):

wherein,

the formulas (S₁)-OH and (S₂)-OH designate respectively, the fully protected GRF peptide acidic segments 1 and 2 which have at least 14 residues and have their C-terminal acidic;

the formulas (S₃)-NH-, (S₄)-NH- and (S₅)-NH- designate respectively, the fully protected GRF peptide amide segments 3, 4 and 5 which have at least 12 residues and have their C-terminal amide properly linked to a solid support;

the formulas H-(S₃)-NH-, H-(S₄)-NH-, and H-(S₅)-NH-designate respectively, the side chain protected GRF peptide amide segments 3, 4 and 5, N-terminated as free amine, and C-terminated as an amide properly linked to the solid support;

the formulas (S'₁)-(S'₃)-NH₂, and (S'₁)-(S'₂)-(S'₅)-NH₂ designate the GRF peptide amides resulting from condensation of segments 1 and 3, and from segments 1, 2 and 5 after complete cleavage in trifluoroacetic acid (TFA).

The abbreviations used herein are defined as follows.

In the present invention the 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.

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.

In all case in which isomeric forms of an amino acid exist, where no letter precedes a named residue, the L-isomer is intended. Unless a specific C-terminus substituent is noted, both the -OH (free acid) and -NH₂ (amide) forms of the peptide are contemplated. Also as used herein, the term "lower" in lower alkyl, alkenyl, or acyl refers to C_1-8. Although, the term "lower" in lower cycloalkyl refers to C_3-6.

The term "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. 4,517,181, 4,728,726, 4,518,586, 4,528,190, 4,529,595, 4,563,352, 4,585,756, 4,595,676, 4,605,643, 4,610,976, 4,628,043, 4,626,523, 4,689,318, which are hereby incorporated by reference and which are given to illustrate the invention, rather than to limit its scope.

Preferably, the following GRF peptides are synthesized in accordance with the present invention:

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic representation of the synthesis of hGRF(1-29) in accordance with the present invention.

DETAILED DESCRIPTION OF THE 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:

wherein

Q is 3-(5-hydroxyindolyl)-methyl or an omega or alpha-omega substituted alkyl of the following structure: (HO)_m-[Փ]-(CH₂)_n-C(R₄)(R₅)- wherein,

[Փ] is phenyl;

R₄ is H, NH₂, CH₃-CO-NH-, CH₃-NH-;

R₅ is H or CH₃-;

m is 1 or 2;

n is 0,1 or 2;

Q0 is hydrogen or any hydrophobic tail selected from the following formula:

R₆-(Z)_h-(CHR₃)_g-(W'=Y')_f-(CHR₂)_e-{W=Y)_d-(CHR₁)_c-(X)_b-(G)_a- wherein,

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;

R₁, R₂ and R₃ are identical or different, and are either hydroxyl groups, hydrogen or lower linear or branched alkyl groups; c and g can be

0, 1, 2, 3, 4, 5 or 6;

-(W=Y)- and -(W'=Y')- represent the cis or trans double bounds -(CH=CR₇)-, and -(CH=CR₈)- with R₇, R₈ = H or lower alkyl; d and f being zero or 1

R₆ is an hydroxyl group, hydrogen or C₄-C₉ alkyl, with e varying from zero to 6;

Z is an amino group -NH-; h varies from zero to

1;

Q₁ is Tyr, His, des-amino Tyr, D-Tyr, Met, Phe,

D-Phe, pCl-Phe, Leu, His, or D-His with or without a CαMe or NαMe substituent;

Q₂ is Ala, D-Ala, (D or L) N-αMe-Ala, Val, D- Val, Abu, Aib or D-Arg;

Q₃ is Asp or D-Asp; Q₇ is Thr, Aib, Leu, Trp, β-Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO, -OMe, or Me;

Q₈ is Asn, D-Asn, Ser, D-Ser, Abu, Ala, Aib,

Leu, Trp, β-Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO₂, -OMe, or Me;

Q₉ is Ser, Thr, Ala, Aib, Leu, Trp, β-Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO₂, -OMe, or Me;

Q₁₀ is Tyl or D-Tyr;

Q₁₂ and Q₂₁ are Lys, Arg, or N^e-I-Lys in which

I is lower alkyl, acyl, alkenyl, acenyl or cycloalkyl, Q₁₂ an Q₂₁ may be the same or different;

Q₁₃ is Ile or Val;

Q₁₄, Q₁₇, Q₂₃ and Q₂₆ are Leu, Ile, D-Leu or D- Ile and may be the same or different;

Q₁₅ is Gly or Ala;

Q₁₈ is Tyr or Ser;

Q₂₄ is His or Gln;

Q₂₅ is Glu, Asp, D-Glu or D-Asp;

Q₂₇ is Met, D-Met, Ala, Nle, Ile, Leu, Nva,

Val, Tbg, or Tba;

Q₂₈ is Asn, Ser or Asp;

Q₂₉ is Arg, D-Arg;

R is of the following general formula:

R = -HN-R₉

wherein,

R₉ is hydrogen, lower alkyl, lower alkenyl, lower aryl , aralkyl or lower alkylcarboxamide; Q₃₀, Q₃₁ are Gln or Asn and may be identical or different

Q₃₂ is gly or Ala, preferably Gly;

Q₃₄ is ser or Arg;

Q₃₅ is Asn or Ser;

Q₃₈ is Arg or Gln; Q₃₉ is Gly or Arg;

Q₄₀ is Ala or Ser;

Q₄₂ is Ala, Val or Phe;

R₃ represent the whole carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COOR₄, -COR₄, -CO-NHNH-R₄, -CO- N(R₄)(R₅) or -CH₂-OR₄, with R₄ and R₅ being hydrogen or C₁-C₈ 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

HseOH or HseN(R₄)(R₅) of the foregoing and or a non-toxic salt of the foregoing.

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. As shown in Fig. 1, the N-alpha amino group of Tyr₁, as well as the side chain of Lys₁₂ are protected as Boc group. Side chains of Tyr_1,10, Ser₉ and Thr₇ are protected as tert-butyl ether. The side chain of Arg₁₁ is protected as Pmc or Pbf group. Side chain of Asn₈ 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),

wherein the group 6-amino hexanoyl is anchored as a spacer between the solid support and the peptide. This will result in a final peptide as a 6-amino hexanoyl ₍₃₀₎hGRF(1-30) amide after cleavage in TFA.

A preferred embodiment of the present invention relates to the segment coupling shown in equation (5) above, wherein the N-terminal Boc group in Tyr₁ is replaced by the group hexanoyl. This will result in a final peptide as an hexanoyl-Tyr₁ 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-Tyr₁ 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.

Evidence of the improved yield in synthesis of

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.

SYNTHETIC METHODS;

Synthesis of GRF peptide segments:

Large scale 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-PS™ support (Perspective biosystem, part number GEN 913383), utilizing a Millipore 9050™ 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.

Arg Pmc or Pbf

Asn Trityl

Asp t-butyl

Glu t-butyl

Gln Trityl

Lys Boc

Ser t-butyl

Thr t-butyl

Tyr t-butyl

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. (S₁)-OH or (S₂)-OH ) is then afforded by selective cleavage of the peptide resin bond in extremely mild conditions, usually 0.5% TFA in dichloromethane.

It is particularly required that the free acid segment to be coupled is properly protected during the coupling step. This could be fulfilled if cleavage of peptide resin bond is alternatively followed by a triethylamine neutralization. In these conditions, a pure segment is obtained after complete evaporation of the solvent and subsequent precipitation and washings with water.

As illustrated in Table 1 above, the coupling efficiency for the synthesis of hexanoyl-Tyr₁ hGRF(1-29) NH₂, using both step by step or segment coupling methodology outlined for this invention, is determined by weight , while the purity is determined by HPLC or by TLC for protected individual segments. Using the mixture CHCl₃/MeOH/80% Ac-OH (16:1:1), side chain protected acidic segments hexanoyl-Tyr₁ hGRF (1-15)-OH and trans-3-hexenoyl-Tyr₁ 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:

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.

In addition to the high yield observed in GRF peptide segment coupling methodology outlined in accordance with the present invention, 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 present invention will be more readily understood by referring to the following solid phase general procedure which is given to illustrate the invention rather than to limit its scope.

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. After all the desired segment have been linked in the proper sequence, 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/H₂O/scavenger), as in procedure supplied by Millipore. After cleavage completion, 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 Waters™ prep. 4000 equipped with 3 cartridges (25 × 100mm, Delta™ pak C₁₈) 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% CH₃CN/TEAP and starting from 20% B under the isocratic conditions (analytical column Delta™ 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 H₂O and D: 0.1% TFA in CH₃CN / H₂O(80:20).

As shown in Tables 2 and 3, this is followed by a scrupulous characterization using mass spectrometry or amino acid analysis, and by a careful biological activity confirmation of the synthetic product, so as to ensure that the desired structure is indeed the one obtained.

It is particularly required that the free acid segment to be coupled is properly protected during the coupling step with appropriate acid or base sensitive protecting groups. Such 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-Tyr₁hGRF(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-Tyr₁hGRF (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.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WE CLAIM:

1. A high yield process of manufacturing (Gly or Ala)¹⁵ or ³²GRF containing peptide, which comprises the steps of:

a) synthesis of 14 to 15 residues of fully protected GRF peptide acidic segments (S₁)-OH and (S₂)-OH from sasrin resin, using sequential Fmoc chemistry;

b) synthesis of 12 to 15 residues of side chain protected GRF peptide amide segments (S₃)-NH-, (S₄)-NH-, and/or (S₅)-NH- on solid phase using a TFA sensitive resin; and

c) one or two coupling steps of the synthesized GRF peptide segments of steps a) and b) on solid phase.

2. A process of claim 1 wherein, GRF peptide segments (S₁)-OH, (S₂)-OH, (S₃)-NH-, (S₄)-NH-, (S₅)-NH-have the following structure:

wherein Q is 3-(5-hydroxyindolyl)-methyl or an omega or alpha-omega substituted alkyl of the following structure:

(HO)_m-[Փ]-(CH₂)_n-C(R₄)(R₅)- wherein,

[Փ] is phenyl;

R₄ is H, NH₂, CH₃-CO-NH-, CH₃-NH-;

R₅ is H or CH₃-;

m is 1 or 2;

n is 0, 1 or 2;

Q0 is hydrogen or any hydrophobic tail selected from the following formula:

R₆-(Z)h-(CHR₃)_g-(W'=Y')_f-(CHR₂)_e-(W=Y)_d-(CHR₁)_c-(X)_b-(G)_a- wherein,

X is an oxygen atom, or an amino group, with b having the value of zero or 1;

0, 1, 2, 3, 4, 5 or 6;

Z is an amino group -NH-; h varies from zero to

1;

Q₁ is Tyr, His, des-amino Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His, or D-His with or without a CαMe or NαMe substituent; Q₂ is Ala, D-Ala, (D or L) N-αMe-Ala, Val, D- Val, Abu, Aib or D-Arg;

Q₃ is Asp or D-Asp;

Q₇ is Thr, Aib, Leu, Trp, β-Nal, or p-X-Phe, in which X is H, F, Cl, Br, NO, -OMe, or Me;

Q₈ is Asn, D-Asn, Ser, D-Ser, Abu, Ala, Aib,

Leu, Trp, β-Nal, or p-X-Phe, in which X is H,

F, Cl, Br, NO₂, -OMe, or Me;

Q₁₀ is Tyl or D-Tyr;

Q₁₂ and Q₂₁ are Lys, Arg, or N^e-I-Lys in which

Q₁₃ is Ile or Val;

Q₁₅ is Gly or Ala;

Q₁₈ is Tyr or Ser;

Q₂₄ is His or Gln;

Q₂₅ is Glu, Asp, D-Glu or D-Asp;

Q₂₇ is Met, D-Met, Ala, Nle, Ile, Leu, Nva, Val, Tbg, or Tba;

Q₂₈ is Asn, Ser or Asp;

Q₂₉ is Arg, D-Arg;

R is of the following general formula:

R = -HN-R₉

wherein,

R₉ is hydrogen, lower alkyl, lower alkenyl, lower aryl , aralkyl or lower alkylcarboxamide;

Q₃₀, Q₃₁ are Gln or Asn and may be identical or different

Q₃₂ is gly or Ala, preferably Gly; Q₃₄ is ser or Arg;

Q₃₅ is Asn or Ser;

Q₃₈ is Arg or Gln;

Q₃₉ is Gly or Arg;

Q₄₀ is Ala or Ser;

Q₄₂ is Ala, Val or Phe;

R₃ represent the whole carboxyl moiety of the amino acid residue at the C-terminal and is the radical -COOR₄, -COR₄, -CO-NHNH-R₄, -CO- N(R₄)(R₅) or -CH₂-OR₄, with R₄ and R₅ being hydrogen or C₁-C₈ 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

3. A process of claim 1, wherein (S₁)-OH and (S₂)-OH have at least 14 residues and have either Gly or Ala at their C-terminal position.

4. A process of claim 1, wherein (S₃)-NH-, (S₄)-NH-, and (S₅)-NH- have at least 13 residues and have either Gly or Ala at their C-terminal position.

5. A process of claim 1 and 2, wherein GRF peptide segments (S₁)-OH and (S₂)-OH have a C_1-9 alkanoyl at their N-terminal.

6. A process of claim 1 and 2, wherein for segments solubility, when any N-terminal free amino GRF peptide is needed for the coupling step c), N-terminal residue of GRF peptide segments (S₁)-OH and (S₂)-OH are N-α protected as Boc.