CA1340077C - ¬ser 17, leu 18|grf derivatives - Google Patents

Abstract

Disclosed herein are growth hormone-releasing factor derivatives characterized in that they have a rearranged amino acid sequence at positions 17 and 18 (i.e. Ser at position 17 and Leu at position 18) and that they may be optionally substituted at various positions including 1, 2, 15, 21, 24 and 27.
The derivatives are useful for treating growth hormone deficiences and for promoting growth in animals, including humans.

Description

(Serl7, Leul8)GRF DERIVATIVES

Field of Invention This invention relates to derivatives of the growth hormone-releasing factor (GRF). More speci-fically, this invention concerns synthetic peptides having growth hormone-releasing activity, to proces-ses for producing the peptides, to pharmaceutical compositions of the peptides and to methods of using the peptides in agriculture and medicine.

Background of the Invention GRF causes the release of growth hormone (GH) into the blood and as such plays an important role in animal growth. The source for its first isola-tion and characterization was an islet tumor from a human pancreas, see R. Guillemin et al., Science, 218, 585 (1982). GRF now has been isolated from the hypothalamus of humans and various animal species.
The structures of the GRF's from the various species are similar, differing slightly in their amino acid sequence.

The human GRF, conventionally designated by the acronym "hGRF", has the following structure:
3o l 5 lO
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-NH2 Porcine growth hormone-releasing factor (pGRF) has the same structure as hGRF except that the amino acid residues at positions 34, 38 and 42 are re-placed with Arg, Gln and Val, respectively. Bovine growth hormone (bGRF) has the same structure as pGRF
except that the amino acid residues at positions 28 and 41 are replaced with Asn and Lys, respectively.
Ovine growth hormone-releasing factor toGRF) has the same structure as bGRF except that the amino acid residue at position 13 is replaced with Ile.
The intrinsic biological activity of hGRF has been found to reside in the N-terminal portion of the peptide, see N. Ling et al., Biochem. Biophys.
Res. Commun., 123, 854 (1984); and both the carboxy terminal acids and carboxy terminal amides of the full length GRF's of the aforementioned species, as well as the corresponding fragments thereof contain-ing about the first 27 amino acids, display growth hormone-releasing activity. According to conven-tion, the position numbers of the amino acid 1'340077 residues present in the GRF free acid or amide forms, or their fragments are indicated by numbers in parentheses following "GRF". Thus, the amide fragment of hGRF containing the first 29 amino acids of the N-terminal portion thereof is designated as "hGRF(1-29)NH2" and the corresponding free acid as "hGRF(1-29)OH."

The importance and potential use of GRF and its active fragments are well documented; for example, see F.X. Coude et al., Trends in Biotechnology, 2, 83 (1984). As a result, numerous analogs of GRF
have been made and tested for the purpose of finding more potent, and hopefully, less expensive replace-ments for GRF. For instance, see D.H. Coy et al., J. Med. Chem., 28, 181 (1985), J.S. Tou et al., Biochem. Biophys. Res. Commun., 139, 763 (1~86), U.S. patent 4,518,586, May 21, 1985 and European patent application, publication number 177819, published April 16, 1986.

The present application discloses new GRF pep-tides derivatives which are characterized by having the order of the amino acid sequence at positions 17 and 18 rearranged (i.e. Ser at position 17 and Leu at position 18). The new derivatives possess en-hanced growth hormone-releasing activity and can be prepared economically. These attributes render the derivatives useful agents for ~edicine and agricul-ture.

~, 13 l~O77 Summary of the Invention The GRF peptide derivatives of this invention are represented by formula 1 Rl-R2-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-R3-Leu-R4-Gln-Ser-Leu-Ala-Arg-R5-Leu-Leu-R6-Asp-Ile-R7-Y-Z

wherein Rl is Tyr, (lower alkanoyl)-Tyr, des-NH2-Tyr, His or (lower alkanoyl)-His, R2 is Ala or D-Ala, R3 is Val or Ile, R4 is Gly, Ala, Leu, Ile, Sar, Val or Aib, R5 is Lys or Glu, R6 is Gln, Lys, Ser or Glu, R7 is Met, Nle, Nva or lle, Y is des-Y or an amino acid sequence select-ed from the group of R8-Arg-Gln-Gln-Gly-Glu-R9-Asn-Gln-Glu-R10-Gly-Ala-R11-R12-Arg-Leu wherein R8 is Ser or Asn, R9 is Ser or Arg, R1O is Arg or Gln, Rll is Arg or Lys and R12 is Ala or Val, and fragments thereof wherein from one to sixteen amino acid residues may be deleted serially from the carboxy terminus, and Z is hydroxy, amino, lower alkylamino or Gly-OH;
or a therapeutically acceptable salt thereof.

- 4a -A first preferred group of GRF peptide derivatives, namely a human growth hormone-releasing factor (hGRF) or a bovine growth hormone-releasing factor ~bGRF), is represented by formula 1 ~n which R is Tyr, (lower alkanoyl)-Tyr, des-NH2-Tyr, His or (lower alkanoyl)-His, R is Ala or D-Ala, R is Val or Ile, R is Gly, Ala, Leu, Ile, Sar, Val or Aib, R is Lys or Glu, R is Gln, Lys, Ser or Glu, R is Met, Nle, Nva or Ile, Y is des Y or an amino acid sequence R8-Arg-Gln-Gln-Gly-Glu-R9-Asn-Gln-Glu-Rl0-Gly-Ala-Rll-Rl2-Arg-Leu wherein R8 is Ser or Asn, R9 is Ser or Arg, Rl~ is Arg or Gln, Rll is Arg or Lys and Rl2 is Ala or Val, or Y is a fragment selected from the group of: Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu, Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala, Ser-Arg and Asn-Arg, and Z is hydroxy, amino, lower alkylamino or Gly-OH; or a therapeutically acceptable salt thereof.

13 iO077 A preferred group of peptide derivatives of this invention is represented by formula l wherein Rl to R7, inclusive, are as defined herein-above, Y is des-Y, or an amino acid sequence select-ed from the group of Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu, Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala, Ser-Arg and Asn-Arg, and Z is hydroxy, amino or Gly-OH; or a therapeutically acceptable salt thereof.
A more preferred group of peptide derivatives is represented by formula l wherein R1, R3 and R6 are as defined hereinabove, R2 is Ala, R4 is Gly, Ala, Leu, Ile or Val, R5 is Lys, R7 is Met or Nle, Y is Ser-Arg or Asn-Arg, and Z is amino or Gly-OH; or a therapeutically acceptable salt thereof.

A most preferred group of peptide derivatives is represented by formula l wherein Rl is Tyr or AcTyr, R2 is Ala, R3 is Val, R4 is Gly or Ala, R5 is Lys, R6 is Gln, Lys, Ser or Glu, R7 is Met or Nle, Y is Ser-Arg and Z is NH2; or a thera-peutically acceptable salt thereof.
Included within the scope of this invention is a pharmaceutical composition for stimulating the release of GH in an animal comprising an effective amount of a peptide derivative of formula 1, or a 3o 1~007~

therapeutically acceptable salt thereof, and a pharmaceutically or veterinarily acceptable car-rier.

The administration of the peptide derivatives to animals, including humans, stimulates the release of GH in the animals. Thus, included within the scope of this invention is a method of promoting growth in animals, and/or treating growth related disorders caused by a deficiency of GH in the animals, which comprises administering thereto an effective amount of the peptide derivative of formu-la 1, or a therapeutically acceptable salt thereof.

Processes for preparing the peptide derivatives are described hereinafter.

Details of the Invention For convenience, the peptide derivatives of this application hereinafter are designated simply as peptides.

The term 'residue' with reference to an amino acid means a radical derived from the corresponding ~-amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen of the ~-amino group.

In general, the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB

1~0~77 Commission on Biochemical Nomenclature, see Biochem-istry, 11, 1726-1732 (1972). For instance, Gln, Ala, D-Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Asn, Thr, Lys, Val, Met, His, Nle, Nva, Sar and Tyr represent the 'residues' of L-glutamine, L-alanine, D-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L-leu-cine, L-asparagine, L-threonine, L-lysine, L-valine, L-methionine, L-histidine, L-norleucine, L-norval-ine, sarcosine, and L-tyrosine, respectively.

The symbols "AcTyr", "des-NH2-Tyr" and "Aib"
represent the residues of N-acetyl-L-tyrosine, des-amino-L-tyrosine and L-~-aminoisobutyric acid, respectively.

The term "lower alkanoyl" as used herein means straight chain alkanoyl radicals containing from two to six carbon atoms and branched chain alkanoyl radicals containing from four to six carbon atoms and includes acetyl, l-oxopropyl, 2-methyl-1-oxopro-pyl, l-oxohexyl and the like.

The term "pharmaceutically acceptable carrier"
as used herein means a non-toxic, generally inert vehicle for the active ingredient which does not adversely aff'ect the ingredient.

The term "veterinarily acceptable carrier" as used herein means a physiologically acceptable vehicle for administering drug substances to domestic animals comprising one or more non-toxic pharmaceutically acceptable excipients which do not react with the drug substance or reduce its effec-tiveness.

The peptides of this invention can be prepared according to processes known for the preparation of peptides. Suitable processes include exclusively solid phase techniques, partial solid phase tech-niques and/or fragment condensations, or classical solution couplings. For example, the techniques of exclusively solid phase synthesis are described by J.M. Stewart and J.D. Young in the textbook 'Solid Phase Peptide Synthesis', 2nd ed., Pierce Chem. Co., Rockford, Illinois, 1984. The fragment condensation method is exemplified by the disclosure of Canadian patent 1,178,950, issued December 4, 1984. Other available syntheses are exemplified by U.S. patent 3,842,067, issued October 15, 1974, and U.S. patent 3,862,925, issued January 28, 1975.

The terminal amino acylated derivatives of the peptides of formula 1, i.e. peptides of formula 1 wherein Rl is (lower alkanoyl)-Tyr or (lower alkanoyl)-His, are obtained from the corresponding free terminal amino peptides by treatment with a suitable acylating agent; for instance, the appro-priate acid chloride or acid anhydride in the pre-sence of a strong organic base, e.g. l-oxobutyl .. . . ~ ", 13 4 g) 0 7 ~

chloride with triethylamine. Alternatively, the terminal amino acylated derivatives are obtained by using the appropriate N~-acylated amino acid residue while preparing the peptide by conventional means.

A common feature of the aforementioned proces-ses for the peptides is the protection of the labile side chain groups of the various amino acid residues with suitable protective groups which will prevent a chemical reaction from occurring at that site until the protective group is ultimately removed. Usually also common is the protection of an a-aminO group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the ~-amino protecting group to allow subsequent reaction to take place at that location.
An important intermediate in the processes for preparing the peptides of this invention is the intermediate of formula 2 xl -Rl(X2)-R2-Asp(X3)-Ala-Ile-Phe-Thr-(X4)-Asn-Ser(X4)-Tyr(X2)-Arg(X5)-Lys(X6)-R3-Leu-R4-Gln-Ser(X4)-Leu-Ala-Arg(X5)-R5-(X6orX3)-Leu-Leu-R6(X7)-Asp(X3)-Ile-R7-yl_zl ~3'~0~77 wherein Rl to R7, inclusive, are as defined hereinbefore; Xl is hydrogen, an a-amino protec-tive group, preferably t-butyloxycarbonyl, or des-Xl when R1 is (lower alkanoyl)-Tyr, des-NH2-Tyr or (lower alkanoyl)-His; x2 is a protective group for the hydroxyl of Tyr, (lower alkanoyl)-Tyr or des NH2-Tyr,preferably benzyl or 2-chloro-benzyloxycarbonyl, or a protective group for the imidazolyl group of His or (lower alkanoyl)-His, preferably 2,4-dinitrophenylsulfenyl or tosyl; X3 is a protective group for the ~-carbonyl of Asp or Glu, preferably, benzyl, 2,6-dichlorobenzyl or cyclohexyl; X4 is a protective group for the hydroxyl of Thr or Ser, preferably benzyl; X5 is a protective group for the guanidino group of Arg, preferably tosyl or nitro; x6 is a protective group for the amino group of Lys, preferably 2-chlorobenzyloxycarbonyl or tosyl; X7 is des-X7 when R6 is Gln, or is the protective group X6, X4 or X3 when R6 is Lys, Ser or Glu, respec-tively; yl is des-Yl or an amino acid sequence selected for the group of R8(X8)-Arg-Gln-Gln-Gly-Glu-R9(X4 or X5)-Asn-Gln-Glu(X3)-R10~
(X9 )-Gly -Al a-Rll( X5 o r x6 )-R1 2 -Arg -Leu wherein R8 to R12, inclusive, and X3, X4, X5 and x6 are as defined hereinbefore, x8 is a protective group for the hydroxyl of Ser or is des-R8 when R8 is Asn, and X9 is a pro-tective group for the guanidino group of Arg or is des~R9 when R10 is Gln, and fragments of the 13 1~)07~

amino acid sequence thereof wherein from one to six-teen amino acid residues may be deleted serially from the carboxy terminus; and Z1 is hydroxy, amino, lower alkylamino, Gly-OH, OCH2-(resin sup-port), NH-(resin support) or Gly-OCH2-(resin sup-port).

The radicals OCH2-(resin support) and NH-(resin support) represent radicals derived from solid polymeric supports of the type used in solid phase peptide synthesis and may optionally include a spacer or linking group, such as 4-methylphenyl-acetamido present in ~-(phenylacetamido)benzyl resins (PAB-resins), between the radical and the peptide portion. Such radicals are broadly con-sidered as protective groups.

Hence, the peptides of formula l can be pre-pared by forming the intermediate of formula 2 by the stepwise coupling of the appropriate protected amino acid residues or peptide fragments in the order of the sequence of the intermediate to obtain the intermediate of formula 2, followed by depro-tecting the intermediate, including cleaving the solid resin support, if present, to give the corres-ponding peptide of formula l; and if desired, trans-forming the peptide of formula l into a thera-peutically acceptable salt.

In an embodiment of the exclusively solid phase method, the protected intermediate of formula 2 in ~&~77 which Zl is amino is prepared as follows: an a-amino protected amino acid (with side chain pro-tection, if desired), corresponding to the first amino acid of the carboxy terminus, is coupled to a cross-linked benzyhydrylamine (BHA) resin in the presence of potassium fluoride or cesium chloride to give the corresponding solid support resin having the first amino acid (in protected form) linked thereto. Alternatively, a benzhydrylamine solid resin support with the incorporated protected amino acid may be obtained commercially and used as the starting material. In either event, the next step is the removal of the a-amino protective group of the incorporated amino acid to give the free a-amino group. In the instance where the a-amino protective group is a t-butyloxycarbonyl, trifluoroacetic acid in methylene chloride or chloroform, or hydrochloric acid in dioxane, is used to effect the deprotection.
The deprotection is carried out at a temperature between about 0~ C and room temperature. Other standard cleaving reagents and conditions for remov-al of specific a-amino protective groups may be used as described by E. Schroder and K. L~bke, in "The Peptides", Vol. 1, Academic Press, New York, 1965, PP. 72-75. After removal of the a-aminO protective group from the last mentioned intermediate, the remaining a-amino protected amino acids are coupled stepwise in the desired order to obtain the corres-ponding protected intermediate of formula 2 in which 3o zl is NH-(benzhydrylamine resin). Each protected ~1340 077 amino acid is introduced into the reaction system in a one to four-fold excess and the coupling is car-ried out in a medium of methylene chloride, di-methylformamide, or mixtures of dimethylformamdie and methylene chloride. In cases where incomplete coupling has occurred, the coupling procedure is repeated before removal of the ~-amino protective group, prior to the coupling of the next protected amino acid. The success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as described by E. Kaiser et al., Anal. Biochem., 34, 595 (1970).

The preceding intermediate of formula 2 there-after is simultaneously cleaved from the resin and deprotected by treatment with liquid hydrogen fluor-ide to give the corresponding peptide of formula 1 in which Z is amino.

Alternatively, when it is desired to prepare the peptides in the form of their free acids (I, Z
= OH), the peptide can be prepared by the solid phase method using a chloromethylated resin or a PAB
resin, and incorporating into the process the cleavage of the resulting resin-coupled peptide and any required deprotection according to known procedures such as described by Stewart and Young, supra.

Again, alternatively, the latter resin-coupled peptide can be separated from the resin by trans-esterification with a lower alkanol, preferably methanol or ethanol, in the presence of trietyla-mine. Thereafter, the recovered ester is purified by chromatography. The collected fraction may be subjected to treatment with ammonia or a (lower alkyl)amine to convert the lower alkyl ester to the correspondinK carboxy terminal amide (the interme-diate of formula 2 wherein Zl is amino or lower alkylamino); note that the 2,4-dinitrophenyl-sulfenyl or tosyl, if present on a histidyl residue, will be cleaved under these conditions.
The remaining protective groups then are removed, for example with sodium in liquid ammonia or with hydrogen fluoride, to give the corresponding peptide of formula ] in which Z is amino or lower alkyl-amino. Still again, alternatively, the latter resin-coupled peptide, i.e. intermediate of formula 2 in which zl is OCH2-(resin support), can be cleaved with ammonia to give the corresponding intermediate of formula 2 in which z1 is amino, which in turn, is deprotected to give the desired peptide of formula 1 in which Z is amino.

The peptide of formula 1 of this invention can be obtained in the form of therapeutically accept-able salts.

In the instance where a particular peptide has a residue which functions as a base, examples of such salts are those with organic acids, e.g.
acetic, lactic, succinic, benzoic, salicyclic, methanesulfonic or p-toluenesulfonic acid, as well as polymeric acids such as tannic acid or carboxy-methyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or 13'1G~7-i sulfuric acid, or phosphoric acid. If desired, a particular acid addition salt is converted into another aeid addition salt, sueh as a non-toxic, therapeutieally aeeeptable salt, by treatment with the appropriate ion exehange resin in the manner deseribed by R.A. Boissonnas et al., Helv. Chim.
Aeta, 43, 1849 (1960).

In the instance where a particular peptide has one or more free carboxyl groups, examples of sueh salts are those with the sodium, potassium or cal-cium cations, or with strong organic bases, for example, triethylamine or N-ethylmopholine.

In general, the therapeutically acceptable salts of the peptides of formula 1 are biologically fully equivalent to the peptides themselves.

The outstanding effectiveness of the peptides of formula 1 in stimulating or causing the release of growth hormone can be demonstrated in four-day old rat anterior pituitary cells according to the method of P. Brazeau et al., Proe. Natl. Aead. Sei.
U.S.A., 79, 7909 (1982). For example, when tested aecording to this method, (Serl7, Leul8)-hGRF(1-29)NHC) was found to be four times more potent than hGRF(1-29)NH2, described by N. Ling et al., Biochem. Biophys. Res. Commun., 123, 854 (19-84) at concentrations of 0.5 and 2.0 fentamoles.
3o The effectiveness of the peptides of this invent.on, or their therapeutically acceptable salts, to stimulate the release of GH in animals, incluaing humans, render them useful for treating growth hormone deficiencies and for augmenting the desirable effects of GH. More explicitly, the peptides or their therapeutically acceptable salts are useful for treatinB growth related disorders due to insufficient production of endogeneous GH in animals, for example prepubertal growth hormone deficiency in humans; for nealing wounds; for improving milk production in dairy herds, such as cows and goats; 40r improving the quality of meat in meat-producing animals ti.e. increasing the ratio of meat to fat); for increasing wool growth; and for improving feed efficiency in meat-producing animals and dairy cows. The peptides also can be used diagnostically to evaluate pituitary function.

When the peptides of this invention, or their therapeutically acceptable sai~s, are used to effect the release of GH, they usually are administered systemically to warm-blooded animals, e.g. humans, cattle or pigs, in combination with pharmaceutically or veterinarily acceptable carriers, the proportion of which is determined by the solubility and chemi-cal nature of the peptide, chosen route of adminis-tration and standard biological practice.

For systemic administration, the peptides of formul~ l are administered ~y either intravenous, -~0~77 subcutaneous or intramuscular injection, in composi-tions with pharmaceutically acceptable vehicles or carriers. For administration by injection, it is preferred to use the peptides in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives, as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.

Examples of suitable excipients or carriers for human or veterinary use are found in standard phar-maceutical texts, e.g. in "Remington's Pharmaceuti-cal Sciences", 16th ed, Mack Publishing Company, Easton, Penn., 1980.
The dosage of the peptides will vary with the form of administration and the particular compound chosen. Furthermore, it will vary with the particu-lar host and the particular condition to be treated.
Generally, treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the peptides of this invention are given at a concentration which affords effective results without causing deleteri-ous side effects.

Appropriate dosages can be determined by relating the amount of the peptide to be administer-,., , . , . ~

13~0077 ed to give the desired concentration of circulating GH. Hence, when treating GH deficiencies, a suffi-cient amount of the peptide is given systemically e.g. subcutaneously or intravenously, to the host to maintain the amount of circulating GH at levels usually associated with normal growth.

Usually a dose of 0.05 mcg to 10 mcg per kilo-gram of body weight per day is employed although the aforementioned variations will occur. However, a dosage in the range of from about 0.1 mcg to 10 mcg per kilogram of body weight per day is most desir-ably employed in order to achieve effective re-sults.
Accordingly, a method of treatment for growth-related disorders due to growth hormone deficiency in an animal is provided which comprises administer-ing to the animal an effective amount of the peptide of formula 1, or its therapeutically acceptable salt, to stimulate the endogenous production of growth hormone to levels associated with normal growth.

When the peptides of formula 1, or their thera-peutically acceptable salts, are employed to stimu-late growth activity in livestock, e.g. to increase milk production in dairy herds or to improve and increase meat production, the dosage will generally be greater than that used to obtain normal levels of 1 3~ OB 77 GH in growth hormone deficiency states. In live-stock, the systemic dosage generally ranges from 0.1 mcg to 20 mcg per kilogram of body weight per day, preferably 0.5 to 20 mcg per kilogram of body weight per day. The dosage is preferably administered sub-cutaneously.

Thus, there also is provided a method of accel-erating the growth rate of non-human animals and/or increasing milk production in female species thereof which comprises administering to the animal an effective amount of the peptide of formula 1, or a therapeutically acceptable salt thereof, to stimu-late the production of GH at a level greater than that associated with normal growth.
It is sometimes desirable to administer the peptides of this invention continuously over pro-longed periods of time in long-acting, slow-relase, or depot dosage forms. Such dosage forms may either contain a pharmaceutically acceptable salt of the respective peptide having a low degree of solubility in body fluids, for example one of those salts described above, or they may contain the peptide in the form of a water-soluble salt together with a protective carrier which prevents rapid release and decomposition of the peptide. Examples of such formulations are found in standard pharmaceutical texts, e.g., in "Remington's Pharmaceutical Sci-ences", cited above. Long-acting, slow-release 3o preparations of the peptides of this invention may also be obtained by microencapsulation in a pharma-ceutically acceptable coating material, for example gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coating materials and of the processes used for microencapsulation are described by J.A. Herbig in "Encyclopedia of Chemical Technol~
ogy". Vol. 13, 2nd ed., Wiley, New York, 1967, pp.
436-456. Such formulations, as well as suspensions of salts of the peptide which are only sparingly soluble in body fluids, are designed to release from about 0.1 mcg to 20 mcg of the peptide per kilogram body weight per day, and are preferably administered by intramuscular injection.
The following examples illustrate further this invention. Abbreviations used in the examples in-clude BOC: t-butyloxycarbonyl; TFA: trifluoro-acetic acid; DCC: N,N'-dicyclohexylcarbodiimide;
HOBT: l-hydroxybenzotriazole monohydrate; HF:
hydrofluoric acid; and HPLC: high performance liquid chromatography. Solution percentages are calculated on a volume/volume basis unless stated otherwise. The following terms are trademarks:
Pharmacia, Vydac and Waters.

Finally, with reference to formula 1, the symbols Tyr, des-NH2-Tyr and His for R1 include the terminal hydrogen. In other words, for example, Tyr- has the same meaning as H-Tyr- with reference to Rl 13llO077 Example 1 Preparation of (Serl7,Leu18)hGRF(1-29)NH2 having the formula:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 The title compound was synthesized by the solid-phase technique of B. Merrifield, J. Amer.
Chem. Soc., 85, 2149 (1963). The synthesis of the fully protected linear peptide having the correct sequence of amino acids was conducted on a benzhy-drylamine resin. The following protocol was used:
a) BOC-deprotection: 45% TFA in chloroform (2 times, firstly for 5 minutes then for 25 minutes);
b) wash: chloroform (3 times for 2 minutes each);
c) neutralization: 5% diisopropylethylamine in methylene chloride (2 times for 3 minutes each); d) amino acid coupling: symmetrical anhydride or active ester mediated methods using a three fold molar excess of the preformed active ester or sym-metrial anhydride and a reaction time of 3 to 5 hours, and e) wash: methylene chloride (3 times for 2 minutes each). With reference to the amino acid coupling, each Asp and Ile residue was double coupled by the symmetrical anhydride method. The 134~077 Arg residue was double coupled in methylene chlor-ide/dimethylf'ormamide after activation of the cor-responding amino-acid with DCC-HOBT. The Gln and Asn residues were double coupled in dimethylforma-mide via their corresponding 2-nitrophenyl esters.

The BOC group gave N~ protection for all amino-acids. Side chain protection was as follows:
2-chlorobenzyloxycarbonyl for tyrosine, benzyl for aspartic acid, benzyl for threonine, benzyl for serine, tosyl for arginine and 2-chlorobenzyloxy-carbonyl for lysine. After each coupling, a resin sample was removed during synthesis for a ninydrin test. On completion of the synthesis, the protected peptide- resin was removed from the reaction vessel and dried in vacuo over phosphorus pentoxide and then sodium hydroxide.

The protected peptide resin was mixed with dl-methionine (10:1, w/w) and the mixture was treat-ed at 0~ C for one hour with an excess of anhy-drous HF and anisole (9:1, v/v). After rapid re-moval of the HF in vacuo, the resulting residue was triturated thrice with diethyl ether. The suspended material, a mixture of peptide and resin, was col-lected by filtration and dried in vacuo. The dried solid was extracted with TFA. The TFA extract was concentrated in a rotary evaporator at 30~ C. The residue was triturated with diethyl ether and the resulting white solid, the crude peptide, was col-lected by filtration.

~ 3~1Q~77 Purification of the white solid ~rude product was effected by reversed-phase HPLC on a Pharmacia octadecasilyl- silica (ODS) column (2.5 x 40 cm, C-18, Vydac, 30 ~ particle size) using a gradient of 0.06% TFA in H20 and 0.06% TFA in MeOH. The purity of collected fractions were monitored using analytical reverse phase HPLC 'Waters). Pure fractions were combined to give the title compound.
Reverse HPLC in two different buffer systems and amino acid analysis confirmed t~,at the desired peptide ;~as been obtained in a pure form.

Example 2 By following the procedure ~f example 1, but using ~OC-Ala instead of BOC-Gly for introducing the 15th amino acid residue (determineà from the carboxy terminus), (Alal5 Serl7, r eul 8 )hGRF(l-29)NH2 having the formula:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-~ln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Iie-Met-Ser-Arg-NH2, is obtained.

3o -'~

~ ~q~(J77 Example 3 By following the procedure of example 1, but using N~-BOC-Nim-tosylhistidine instead of N~-BCC-04-(2,6-dichlorobenzyl)tyrosine for introducing the 29th amino acid residue, (Hisl, Alal5,Serl7,Leul8)hGRF(1-29)NH2 having the formula:

H-His-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 is obtained.

Other examples of peptides within the scope of this invention include (AcTyr ,Ser 7,Leu )bGRF-1-40)0H, (AcTyrl,serl7,Leu18)hGRF(l_29)NH2, (serl7~Leul8~Ile27)hGRF(l-29)NH2~ (AcTyr ,Ala ,-Serl7,Leul8,Nle27)hGRF(1-29)NH2, ~Serl7,Leul8,-Lys24)hGRF(1-29)NH2, ~Serl7,LeU18,ser24)hGRF(l_ NH2, (Ser 7,Leu 8,Glu24)hGRF(1-29)NH (AcTyrl Ala 5,Ser 7,Leu 8)hGRF(1-29)NH , (Leu 5,Serl7,-Leu 8)hGRF(1-29)NH2 and (AcTyr~,Leul5,Serl7,Leul8)-hGRF(1-29)NH2.

Claims (17)

1. A human growth hormone-releasing factor (hGRF) or a bovine growth hormone-releasing factor (bGRF) represented by formula 1 R1-R2-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-R3-Leu-R4-Gln-Ser-Leu-Ala-Arg-R5-Leu-Leu-R6-Asp-Ile-R7-Y-Z

in which R1 is Tyr, (lower alkanoyl)-Tyr, des-NH2-Tyr, His or (lower alkanoyl)-His, R2 is Ala or D-Ala, R3 is Val or Ile, R4 is Gly, Ala, Leu, Ile, Sar, Val or Aib, R5 is Lys or Glu, R6 is Gln, Lys, Ser or Glu, R7 is Met, Nle, Nva or Ile, Y is des Y or the amino acid sequence R8-Arg-Gln-Gln-Gly-Glu-R9-Asn-Gln-Glu-R10-Gly-Ala-R11-R12-Arg-Leu wherein R8 is Ser or Asn, R9 is Ser or Arg, R10 is Arg or Gln, R11 is Arg or Lys and R12 is Ala or Val, or Y is a fragment selected from the group consisting of: Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu, Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala, Ser-Arg and Asn-Arg, and Z is hydroxy, amino, lower alkylamino or Gly-OH; or a therapeutically acceptable salt thereof.

2. A peptide of formula 1 of claim 1 wherein R1, R2, R3, R4, R5, R6 and R7 are as defined in claim 1, Y is des-Y, or an amino acid sequence selected from the group of Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu, Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala, Ser-Arg and Asn-Arg, and Z is hydroxy, amino or Gly-OH; or a therapeutically acceptable salt thereof.

3. A peptide of formula 1 of claim 1 wherein R1, R3 and R6 are as defined in claim 1, R2 is Ala, R4 is Gly, Ala, Leu, Ile or Val, R5 is Lys, R7 is Met or Nle, Y is Ser-Arg or Asn-Arg, and Z
is amino or Gly-OH; or a therapeutically acceptable salt thereof.

4. A peptide of formula 1 of claim 1 wherein R1 is Tyr or AcTyr, R2 is Ala, R3 is Val, R4 is Gly or Ala, R5 is Lys, R6 is Gln, Lys, Ser or Glu, R7 is Met or Nle, Y is Ser-Arg and Z is NH2; or a therapeutically acceptable salt thereof.

5. (Ser17, Leu18)hGRF(1-29)NH2 of claim 1.

6. [Ala15, Ser17, Leu18]hGRF(1-29)NH2 of claim 1.

7. [His1, Ala15, Ser17, Leu18]hGRF(1-29)NH2 of claim 1.

8. [AcTyr1, Ser17, Leu18]bGRF(1-40)OH of claim 1.

9. [AcTyr1, Ser15, Leu18]hGRF(1-29)NH2 of claim 1.

10. [Ser17, Leu18, Ile27]hGRF(1-29)NH2 of claim 1.

11. [AcTyr1, Ala15, Ser17, Leu18, Nle27]hGRF(1-29)NH2 of claim 1.

12. [Ser17, Leu18, Lys24]hGRF(1-29)NH of claim 1.

13. [Ser17, Leu18, Ser24]hGRF(1-29)NH of claim 1.

14 [Ser17, Leu18, Glu24]hGRF(1-29)NH of claim 1.

15. A peptide of claim 1 selected from the group of [AcTyr1, Ala15, Ser17, Leu18]hGRF(1-29)NH2, [Leu15, Ser17, Leu18]hGRF(1-29)NH2, and [AcTyr1, Leu15, Ser17, Leu18]hGRF(1-29)NH2.

16. A pharmaceutical composition which comprises a peptide of claim 1, or a therapeutically acceptable salt thereof, and a pharmaceutically or veterinarily acceptable carrier.

17. A use of the peptide of claim 1, or a therapeutically acceptable salt thereof, for stimulating the release of growth hormone in an animal.