CH640827A5 - Process for preparing novel peptides - Google Patents

Description

The present invention relates to a process for the preparation of new peptides which show a specific biological activity with respect to the inhibition of growth hormone, insulin and glucagon secretion. This invention particularly relates to novel peptides which can be prepared according to the invention and which are effective to selectively inhibit only the release of growth hormones by mucus-secreting glands or the release of glucagon or insulin by the pancreas.

A peptide that has a curbing effect on growth hormone secretion is described in US Patent No. 3,904,594 to Guillemin et al. characterized and described. This peptide was given the name "soma-tostatin". Somatostatin, also known as the "containment factor for somatotropin release", is a tetradecapeptide of the formula

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH.

Somatostatin, the linear form of somatostatin (di-hydrosomatostatin), and various acylated derivatives of somatostatin and dihydrosomatostatin have been described in the aforementioned U.S. patent application.

Somatostatin and many analogs of somatostatin show activity in inhibiting growth hormone (GH) secretion from in vitro grown, dispersed, previously mucus-releasing rat cells and have been shown to contain insulin and glucagon secretion in the rat in vivo. When using somatostatin, it has been considered highly desirable to selectively limit the secretion of growth hormones, insulin or GIu-cagon. Efforts have been made to develop analogues of somatostatin that show specific biological activity and only inhibit the secretion of growth hormones, insulin or glucagon. Although there have been publications showing differences in the amounts of somatostatin that are necessary to contain insulin compared to glucagon in the human and in vitro rat pancreas, somatostatin and some somatostatin analogues show similar efficacy in relation to that Inhibition of these two hormones.

The present invention relates to the discovery that certain amino acids in somatostatin and dihydrosomatostatin can be replaced by other amino acids to produce peptides which have a specific biological activity with a view to inhibiting the secretion of growth hormones, insulin or glucagon. In a conventional summary, the new peptides that can be produced according to the invention are in the form of a

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Amino acid unit is described, and the type of substitution, the location of the substitution and the fact whether the substitution takes place in somatostatin (SS) or dihydrosomotatstatin (DHSS) is given above. The nomenclature used to describe the new peptides that can be produced according to the invention is in accordance with conventional rules; in accordance with these rules, it represents the L-form of the desired amino acid, unless expressly stated otherwise.

The individual amino acids of the peptide are expediently numbered from left to right in the new peptides which can be prepared according to the invention.

The first method according to the invention for the production of new peptides with pharmaceutical utility of the formula II

H-Ala-Gly-X-Lys-X1-Phe-Phe-X2-Lys-Thr-Phe-Thr-X3-X4

(II)

and the non-toxic salts thereof, is characterized in that a peptide intermediate of the formula III

R-Ala-Gly-X- (R1) -Lys (R2) - (X1) -Phe-Phe- (X2) -Lys (R3) -Thr- (R4) -Ph.e-Thr (R5) - ( Xa) - (R6) - (X4) - (R7) - (X5) (III)

forms what

X is selected from Cys and D-Cys;

Xj is selected from Asn and Des-Asn;

X2 is selected from Trp and D-Trp;

X3 is selected from Ser and D-Ser; and X4 is selected from Cys and D-Cys,

with the proviso that at least one of the groups X3 and X4 is D-Ser or D-Cys;

R is selected from the class consisting of H and an alpha-amino protecting group;

R1 and R7 are selected from the group consisting of H and a protecting group for Cys selected from S-p-methoxybenzyl, S-p-methylbenzyl, S-acetamidomethyl, S-trityl and S-benzyl;

R2 and R3 are selected from the group consisting of

H and a side chain amino protecting group;

R4, R5 and R6 are selected from the group consisting of H and a hydroxyl protecting group selected from the group consisting of acetyl, benzoyl, tert-butyl, benzyl and benzyloxycarbonyl;

with the further proviso that at least one of the radicals R, R1, R2, R3, R4, R5, R6 and R7 has a different meaning than hydrogen; and

Xs is selected from the group consisting of hydroxy, esters, preferably methoxy, amides, hydrazides, -0-CH2-polystyrene resin carrier and 0-CH2-benzyl-poly-styrene resin carrier,

cleaves the protective group or groups from the intermediate compound of the formula III, and, if X5 denotes a resin support, cleaves the peptide from the resin, and optionally produces the non-toxic salts from the compounds obtained.

The second method according to the invention for the production of new peptides with pharmaceutical utility of formula I.

H-AIa-Gly-X-Lys-X1-Phe-Phe-X2-Lys-Thr-Phe-Thr-X3-I

X4 (I)

in which the substituents are defined further above, and the non-toxic salts thereof, is characterized in that peptides of the formula II are prepared in accordance with the first process according to the invention described above and the peptides are then oxidized in order to form a disulfide bond between the cysts mentioned. Produce residues, and optionally the compounds thus obtained, the non-toxic salts.

Examples of new peptides which can be prepared according to the invention and which have a specific biological activity with regard to the release of growth hormones, insulin and glucagon are: [D-Ser13] -SS; [D-Ser13] -DHSS; [D-Trp8] - [D-Ser13] -SS; [D-Trp8] - [D-Ser13] -DHSS; [D-Cys14] -SS; [D-Cys14] -DHSS; [D-Trp8] - [D-Cys14] SS and [D-Trp8] - [D-Cys14] DHSS.

Pharmaceutically acceptable acid addition salts of the peptides are also within the scope of the present invention. Such acid addition salts are, for example, the hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc.

Compounds are preferably prepared in which X is D-Cys, Xx is Asn, X2 is Trp, X3 is Ser and X4 is D-Cys; as well as compounds in which X has the meaning of D-Cys, Xi means Asn, X2 means Trp, X3 means D-Ser, and X4 means Cys.

The a-amino protecting groups denoted by R are those which are known in the art to be useful in the stepwise synthesis of the polypeptides. The class of a-amino protecting groups which R includes include, for example, the following: (1) protecting groups of the acyl radical type, such as formyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl , Chloroacetyl, acetyl, y-chlorobutyrul, etc .; (2) aromatic urethane type protecting groups such as benzyloxycarbonyl and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethane type protecting groups such as a-t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thiourethane type protecting groups such as phenylthiocarbonyl; (6) protecting groups of the alkyl radical type, such as triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such as trimethylsilane. A preferred a-amino protecting group defined with R is t-butyloxycarbonyl.

R1 and R7 both represent a protecting group for Cys or D-Cys, which are selected from the group of S-p-methoxy-benzyl, S-p-methylbenzyl, S-acetamidomethyl, S-trityl, S-benzyl, etc. A preferred protecting group is S-p-methoxybenzyl. R1 and / or R7 can represent hydrogen, which means that there is no protective group on the thiol group.

R2 and R3 both represent a protecting group for a side chain amino substituent of lysine or R2 and / or R3 represent hydrogen, which means that there is no protecting group on an amino substituent of a side chain. Examples of suitable protecting groups for a side chain amino group are benzyl, chlorobenzyloxycarbonyl, benzyloxycarbonyl, tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, etc. The selection of such an amino protecting group for a side chain is not critical, except that it is intended to be such that it is not removed during the synthesis during the removal of the a-amino groups. As a result, the> «amino protection

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group and the protecting group for the side chain amino function may not be the same;

R4, R5 and R6 are protecting groups for the hydroxyl group of Thr and Ser, and they are selected from the group of acetyl, benzoyl, t-butyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and benzyloxycarbonyl. A preferred protecting group is the benzyl radical. R4 and / or R5 and / or R6 can also represent hydrogen, which means that there is no protective group on the hydroxyl group.

Xv X2, X3 and X4 are defined as above. X5 is selected from the group of OH, esters, for example OCH3, amides, hydrazides,

-0-CH2 polystyrene resin support and

0-CH2-Benzylpolystyrene resin support selected.

The polymer is preferably a copolymer of styrene with about 0.5 to 2% divinylbenzene as a crosslinking agent, resulting in a polystyrene polymer which is completely insoluble in certain organic solvents. In formula III, at least one of the radicals R, R1, R2, R3, R4, R5, R6 and R7 represents a protective group.

When selecting a special side chain protecting group which is used in the synthesis of the peptides of the formula I or formula II, the following rules should preferably be listed:

(a) the protecting group must be stable enough to withstand the reagents and reaction conditions used to remove the "amino protecting group at each stage of the synthesis,

(b) the protecting group must retain its protective properties and must not be split off during the connection conditions, and

(c) The protecting group of the side chain must be removable upon completion of the synthesis, which contains the desired order of the amino acids under the reaction conditions, which should not change the peptide chain.

The peptides of formulas I and II can be prepared using solid phase synthesis. It is expedient to begin the synthesis at the C-end of the peptide, using an a-amino-protected amino acid. Such a starting material can be prepared by connecting a Cys having a protected a-amino group and a protected sulfur to a chloromethylated resin or a hydroxymethyl resin. The preparation of a hydroxymethyl resin was described by Bodanszky et al., Chem. Inc. (London) 38, 1957-98 (1966). A chloromethylated resin is commercially available from Bio Rad Laboratories, Richmond, California; the preparation of such a resin was described by Stewart et al., “Solid Phase Peptide Synthesis” (Free-man & Co., San Francisco 1969), chapter 1, pages 1-6. Cys with the protected a-amino group and the protected S is expediently bound to the chloromethylated resin by the method of Monahan and Gilon, Biopolymer 12, pages 2513-19, 1973. After the Cys with the protected a-amino group and the protected S have bonded to the resin support, the a-amino protecting group is removed, using, for example, trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HCl in dioxane. The protective group is preferably removed at a temperature between about 0 ° C. and room temperature.

Other standard cleavage reagents and conditions for removing specific a-amino protecting groups can also be used, as described in Schröder & Lubke, "The Peptides", 1 pages 72-75 (Academic Press 1965).

After removal of the a-amino protecting group from the Cys, the remaining amino acids with protected a-amino groups and side chains are preferably combined stepwise in the desired order in order to obtain a compound of the formula III or instead of having to separate each individual amino acid into the synthesis there may be some of these combined before adding to the solid reactor. The selection of a suitable coupling reagent is readily accessible to a person skilled in the art. NJSP-dicyclohexylcarbodiimide is a particularly suitable coupling reagent.

The activating reagents which are used in the synthesis of the solid phase peptides are preferably those which are well known in peptide chemistry. Examples of suitable activation reagents are: (1) carbodiimides, such as N, N-diisopropylcarbodiimide, N-ethyl Nx- (y-dimethylaminopropylcarbodiimide; (2) cyanamide, such as N, N-dibenzylcyanamide; (3) keteimines; (4) isoxazolium salts, such as N-ethyl-5-phenylisoxazolium-3l-sulfonate; (5) monocyclic nitrogen-containing heterocyclic amides of an aromatic nature which contain 1 to 4 nitrogen in the ring, for example imidazolide, pyrazolide, 1,2,4-triazolide, specific heterocyclic amides which are useful include N ^ N ^ carbonyldiimidazole, N ^ carbonyl-di-1,2,4-tri-azole; (6) alkoxylated acetylene, such as ethoxyacetylene; (7) reagents which are a mixed anhydride with the carboxyl Form a unit of the amino acid, such as ethyl chloroformate and isobutyl chloroformate and (8) nitrogen-containing heterocyclic compounds which have a hydroxyl group on a ring nitrogen, such as N-hydroxyphthalimide, N-hydroxysuccinimide, N-hydroxybenzotriazole, other activating reagents and their use b The assembly of the peptides has been described by Schröder & Lubke supra, in Chapter III and by Kapoor, J. Pharm. Sei., 59, lines 1-27 (1970).

Each protected amino acid or amino acid sequence is preferably introduced into the solid state reactor in approximately fourfold excess and the assembly is carried out in a medium consisting of dimethylformamide: methylene chloride (1: 1) or in dimethylformamide or methylene chloride alone. In cases where incomplete connection occurred, the procedure for assembly prior to removal of the a-amino protecting group, i.e. before connecting the next amino acid, usually repeated. The success of the assembly reaction at each stage of the synthesis is usually followed by the ninhydrin reaction, as described in E. Kaiser et al. Analyte. Biochem, 34, 595 (1970).

After the desired amino acid sequence of Formula III was synthesized, the peptide was separated from the resin support by treatment with a reagent such as liquid hydrogen fluoride, which not only separates the peptides from the resin but also all other side chain protecting groups R1 , R2, R3, R4, R5, R6 and R7 and the a-amino protective group R split off to directly obtain a peptide of formula II. Peptides of formula I are obtained by using peptides of formula II bpw. oxidized by known methods. Another synthesis can consist in separating the peptide which is bound to the resin support from the resin by alcoholysis, whereupon the C-terminal methyl ester obtained is converted into the acid by hydrolysis. Any side chain protecting group can then be removed as described above or by other methods such as catalytic reduction, e.g. Pd on BaS04 using conditions in which the Trp unit remains undetected. Becomes hydrogen fluoride

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used for the cleavage, Ani-sol is expediently used in the reaction vessel as a cleaning agent.

The solid phase synthesis method discussed above is known to those skilled in the art and has been essentially described in Merrifield J. Am. Soc., 85, S 2149 (1964).

The novel peptides which can be prepared according to the invention and which have specific effects with regard to the inhibition of the release of growth hormones, insulin and glucagon are regarded, for example, as being particularly important in connection with the treatment of diabetes. The traditional view of diabetes has been that symptoms can only be attributed to insufficient insulin production. As more extensive research was done, it became apparent that some somatostatin was effective against diabetes after weakening insulin secretion. It is known that while insulin is usually insufficient in diabetes, glycagon is usually in excess. It is now recognized that the presence of glucagon is at least as important a factor in diabetes as the lack of insulin.

The fact that insulin deficiency is usually accompanied by an excess of glucagon has been difficult to explore in the role of glucagon in diabetes. While adding additional amounts of a hormone such as insulin is easy, it has proven difficult to lower the glucagon content. The discovery of somatostatin has facilitated research into the role of glucagon in diabetes. Somatostatin inhibits both insulin and glucagon release. The role of somatostatin in the study of diabetes is detailed in an article that appeared in Science, Vol. 188, lines 920-923, May 30, 1975. However, there are several problems with the use of somatostatin in the treatment of diabetes. Somatostatin inhibits the release of insulin when glucagon is added. Thus, the need to produce a peptide with a specific insulin and glucagon release control effect has been recognized in connection with the treatment of diabetes. The new peptides which can be prepared according to the invention have such specific efficacies. Certain of the new peptides that can be produced according to the invention are particularly effective in inhibiting glucagon secretion, while they have a smaller influence on the restriction of insulin secretion. The following examples illustrate various properties of the present invention.

example 1

The new peptides are made by solid phase synthesis, generally in accordance with the method described in U.S. Patent No. 3,904,594. The synthesis was carried out in stages on chloromethylated resin. The resin was composed of fine beads (20-70 microns in diameter) made of synthetic resin, which was prepared by copolymerization of styrene with 1 to 2% divinylbenzene. The benzene rings in the resin were chloromethylated in a Friedel-Crafts reaction with chloromethyl methyl ether and tin (IV) chloride. The chlorine thus introduced represents a reactive benzyl chloride, suitable for subsequent reactions. The Friedel-Crafts reaction continues until the resin contains 0.5 to 2 moles of chlorine per gram of resin.

In the further description of peptide synthesis, the reagents used are first described in terms of their chemical names with their common abbreviations in parentheses. Then one will refer to the common abbreviations.

A peptide of the formula

Ala-Gly-Cys-Lys-des-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-

1 2 3 4 5 6 7 8 9 10 11 12

Ser-Cys-OH I

13 14

was synthesized using the solid phase method. Other peptides, which are described below, were produced by similar processes.

The Cys t-butyloxycarbonyl-S-paramethoxybenzyl (Boc-SpOMe-Bzl) derivative was bound to the resin using one of three known methods: (1) heating in ethanol in the presence of triethylamine; (2) the cesium salt of the amino acid protected with Boc is kept at 50 ° C in dimethylformamide (DMF); (3) The potassium salt of the Boc-protected amino acid is kept at 80 ° C in dimethyl sulfoxide (DMSO) for 2 hours. Only one milliequivalent of the protected Cys per milliequi-valent Cl on the resin was used.

Method (3) is described in detail below: 0.9 mA of potassium butoxide (KOtBut) per mAe of amino acid are added to a slurry of the resin and the dissolved protected Cys in DMSO. The reaction mixture is exposed to the air as little as possible so that no amber color is observed. Reaction at 80 ° C for 2 hours gives a suitable substituted resin for the synthesis of the peptides (about 0.2 Ae amino acid derivative per resin). After the protective group has been removed and neutralized, the peptide chain is built up on the resin. Deprotection, neutralization and addition of each amino acid is carried out in accordance with Table I. The Na-t-butyloxycarbonyl (Boc) derivative of each amino acid is used, with the exception that an a-amino protecting group can be used for the alanine 1 residue, provided that it is cleaved with HF (benzyloxycarbonyl, Z; Boc and others). After removing the first residue (ie, SpOMe. Bzl. Cys) according to List I (steps 3 to and with 8), the N Boc derivative of Ser is added together with the binding agent, which is di-cyclohexylcarbodiimide (DOC) ( Level 8 in the list I). The side chain from Ser is protected with benzyl ether (OBzl). The O-benzyl protecting group (OBzl) is also used to protect the threonine side chain. p-Nitrophenyl Ester (ONp) is used to activate the car-boxyl end of Asn. o-Nitrophenyl ester can also be used for this purpose. Formyl groups can be used to protect the indole N-H. Benzyloxycarbonyl1 (Z) or benzyloxycarbonyl-2Cl [Z (2-CL)] were used as a protective group for the Lys side chain.

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1. List for linking the amino acids, which are different from Asn, in a synthesis in solid phase (5-10 g resin)

step

Reagents and operations

Mixing lines (min.)

1

Wash with 80 ml CH2C12 (twice)

3rd

2nd

Wash with 30 ml of methanol (MeOH) (twice)

3rd

3rd

Wash with 80 ml CH2C12 (three times)

3rd

4th

70 ml of 50 percent trifluoroacetic acid (TFA), which contains 5% 1,2-ethanedithiol, in CH2C12 (twice)

10th

5

Wash with 80 ml CH2C12 (twice)

3rd

6

70 ml 12.5% triethylamine (Aet N) in CH2C12 (twice)

5

7

Wash with 40 ml MeOH (twice)

2nd

8th

Wash with 80 ml CH2C12 (three times)

3rd

9

BOC amino acid (10 moles) in 10 ml DMF (once) and 30 ml CH2C12 plus DCC (10 moles) in CH2C12 (2 m)

30 to 120

10th

Wash with 40 ml MeOH (twice)

3rd

11

12.5% Aet3N in CH2C12 (twice)

3rd

12

Wash with 30 ml MeOH (twice)

3rd

13

Wash with 80 ml CH2C12 (twice)

3rd

After level 13 an aliquot is needed for a ninhydrin test:

if the test is negative, go back to level 1 to connect the next amino acid; if the test is positive or slightly positive, you go back to levels 9 to 13. Panel I was used to add any of the amino acids of the peptide to the Cys except for Asn (if present). If the peptides produced according to the invention contained Asn, stages 1 to 8 are the same and list II is used for the rest of the compound reaction:

II. List for Boc-Asn-ONp or for an active ester, which combine in a synthesis in solid phase (5-10 g resin)

step

Reagents and operations

Mixing times (min.)

9

Wash with 60 ml DMF (three times)

3rd

10th

Boc-Asn-ONp (15 mol) in 20 ml DMF (once)

800

11

Wash with 30 ml MeOH (four times)

3rd

12

30 ml 12.5% Aet3Nin DMF (twice)

3rd

13

Wash with 30 ml MeOH (twice)

3rd

14

Wash with 80 ml CH2C12 (three times)

3rd

After level 14, an aliquot is needed for a ninhydrin test:

if the test is negative, go back to level 1 to connect the next amino acid; if the test is positive or slightly positive, go back to levels 9 to 14.

The cleavage of the peptides from the resin (5 gr) and the removal of the side chain protecting groups of the peptide was carried out in hydrofluoric acid (75 ml) in the presence of anisole (8 ml). After removing the hydrofluoric acid under high vacuum, the resin peptide is washed with ether.

The dried resin is immediately extracted with 25% acetic acid (150 ml) and diluted to 3000 ml with degassed H20 (N2). The pH of the solution was adjusted to 6.6-7.0 with NH4OH. The solution was titrated dropwise with potassium ferricyanide solution (1 g / 500 ml HzO) while stirring until a permanent yellow color could be observed. The solution was allowed to stand for 10 minutes; pH was adjusted to 5.0 with glacial acetic acid; Bio Rad AG 3-X4A resin (0.074-0.149 mm, chloride form, 10-15 g) was added to the slurry solution and stirred for 15 minutes. The solution was filtered through Celite and successively placed in two columns: a) Bio Rad AG 3-X4A resin chloride form (10 ml); b) Bio Rex-70 resin (100 ml), cationic form. The Celite and resin cake was thoroughly washed with water (500 ml), whereupon it was applied to columns a) and b) as a washing liquid. The peptide material was then eluted from a Bio-Rex-70 resin column with pyridine: acetic acid: water (30: 4: 66) or 50% acetic acid. The fractions were collected; only those containing peptide (ninhydride positive) were diluted with water and immediately freeze-dried. 1.2 g of an off-white material was obtained. This was applied to a Se-phadex G-25 F gel column (3 × 200 cm), brought into equilibrium and eluted with 2N acetic acid.

The elution samples were observed at 280 nm and showed a symmetrical main peak centered at 2 V0 (400 mg). It was then subjected to a countercurrent distribution (solvent system n-buta-nol: acetic acid: water, 4: 1: 5) with 10 ml of the lower phase per tube. 237 transfers were made; the main peak was found in tubes 48-69. The compound (250 mg) appeared homogeneous on the thin layer chromatogram.

The specific optical rotation was

[«] D23 = -38.2 ± 1

(c = 1 in 1% acetic acid). An amino acid analysis of this material showed the expected ratio for different amino acids.

Active esters can be used in a solid phase synthesis and it is also possible that the classic synthesis methods are used to produce the peptides according to the invention.

In vitro bioanalysis: The effects of the various new peptides that can be produced according to the invention were tested in vitro on the secretion of growth hormones by primary cultures of enzymatically dissociated, previously mucus-secreting rat cells according to the method of Vale et al., Endocrinology 91: pages 562-571 (1972). The experiment was carried out by treating mucus-secreting glands, which were taken from rats, in order to separate cells therefrom. The cells were placed in culture dishes after modification of Dulbecco's modified eagle medium. (Vale et al., Methods in Enzymology). The cell cultures which one at

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Maintained at 37 ° C for 4-5 days prior to use in the experiment are supplied with carbon dioxide and oxygen gas. After changing the media, the cell cultures are incubated for 4 hours and special somatostatin peptides are added. A radioimmunoassay analysis is used to determine the rate of growth hormone secretion, which is expressed in nanograms per bowl per hour.

An investigation with respect to the effects of somatostatin, dihydrosomatostatin (as controls) and the novel peptides which can be prepared according to the invention and which inhibit the release of glucagon and insulin was carried out as follows:

In vivo bioanalysis: Male Sprague-Dawley-CD rats weighing 180-200 g were used in all experiments which lived in cells with controlled temperature and humidity with 14 hours of light and 14 hours of night (light 0700-21100 ). The animals were fed a standard ration and tap water ad libitum. The experiments were carried out by the supplier between the times (2:00 pm and 4:00 pm) for at least 5 days after the rats' arrival. After ether anesthesia, the peptides or saline solutions were administered in a volume of 0.2 ml through the external throat throttle. The animals remained anesthetized until the blood was collected from the portal vein. The blood samples were placed in chilled tubes containing 10 mg EDTA and 50 vI 2m benzamidine per ml blood.

The plasma was stored at -20 ° C for insulin and gluca gon determinations. The level of insulin was determined according to the method of Herbert et al., J. Chem.

Endoer. Metab. 25: 1375, 1965 using porcine insulin antisera and insulin tracers provided with iodine (1251). Human insulin standard was obtained from Schwarz-Mann, Orangeburg, New York. Gluca-5 gon was determined by the method of Faloona and Unger, In Jaffe et al ed., Methods of Homon Radioimmunoassay, Academic Press, New York, 1974, page 317, using Glucagon Antisera 30K. Glucose was determined using the glucose oxidase method, xo using a “Beckman” glucose analyzer.

Growth hormone determinations were carried out on tissue culture media using the following reagents: NIAMDD rat growth hormone standard (GH-RP-1), NIAMID monkey anti-15th growth hormone (GH-Serum-3), and very well purified Rat growth hormone for iodination.

All experiments were carried out in a random block arrangement. The following analysis of variance between treatments was determined using the Dunnett and Duncan multi-range 20 tests. Efficacy values were calculated from four to six point bioanalyses.

Various new peptides were made in solid phase in accordance with the procedures described above. The composition of the peptides is shown in Table I below. Table I also shows the ratio of the effectiveness of the peptide in containing growth hormone, insulin and glucagon secretion, based on the inhibition of 30 glucagon. Table I also shows the percent effectiveness of the peptide in reducing hormones with somatostatin, which is considered 100% effective.

TABLE I

Somatostatin ratio of effectiveness [<x] D percentage effectiveness of

(Control) growth hormone: insulin: growth hormones, base 100

Glucagon 1: 1: 1

[D-Ser13] -SS

10th

10th

1

-34.2 °

10th

[D-Trp8] - [D-Ser13] -SS

18th

261

<1

-21.9 °

[D-Cys14] -SS

2.7

1

1

-19.5 °

270

[D-Trp8] -SS- [D-Cys14] -SS

0.7

0.13

1

-33.6 °

650

[D-Cys3] - [D-Ser13] -SS

7

<1

-38.5 °

[D-Cys3] - [D-Cys14] -SS

-11.9 °

v

Claims (8)

640827 1. Process for the preparation of new peptides with pharmaceutical utility of formula II H-AIa-Gly-X-Lys-XrPhe-Phe-X2-Lys-Thr-Phe-Thr-X3-X4 (II) and the non-toxic salts thereof, characterized in that a peptide intermediate of formula III R-Ala-Gly-X- (R1) -Lys (R2) - (X1) -Phe-Phe- (X2) -Lys (R3) -Thr- (R4) -Phe-Thr (R5) - (Xg) - (R6) - (X4) - (R7) - (X5) (III) forms what X is selected from Cys and D-Cys; Xj is selected from Asn and Des-Asn; X2 is selected from Trp and D-Trp; X3 is selected from Ser and D-Ser; and X4 is selected from Cys and D-Cys, with the proviso that at least one of the groups X3 and X4 represents D-Ser or D-Cys; R is selected from the class consisting of H and an alpha-amino protecting group; R1 and R7 are selected from the group consisting of H and a protecting group for Cys selected from S-p-methoxybenzyl, S-p-methylbenzyl, S-acetamidomethyl, S-trityl and S-benzyl; R2 and R3 are selected from the group consisting of H and a side chain amino protecting group; R4, R5 and R6 are selected from the group consisting of H and a hydroxyl protecting group selected from the group consisting of acetyl, benzoyl, tert-butyl, benzyl and benzyloxycarbonyl; with the further proviso that at least one of the radicals R, R1, R2, R3, R4, R5, R6 and R7 has a different meaning than hydrogen; and X5 is selected from the group consisting of hydroxy, esters, amides, hydrazides, -0-CH2-polystyrene resin carriers and 0-CH2-benzyl-polystyrene resin carriers, the protective group or the protective groups from the intermediate compound of the formula III cleaves off, and, if X5 represents a resin support, cleaves the peptide from the resin, and optionally produces the non-toxic salts from the compounds obtained. 2. Process for the preparation of new peptides with pharmaceutical utility of formula I. H-Ala-Gly-X-Lys-XrPhe-Phe-X2-Lys-Thr-Phe-Thr-X3-I X4 (I) wherein the substituents are defined in claim 1, and the non-toxic salts thereof, characterized in that peptides of the formula II are prepared in accordance with the process according to claim 1 and then the peptides are oxidized to form a disulfide bond between the Cys residues mentioned to produce, and optionally the compounds thus obtained, the non-toxic salts. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that X has the meaning of Cys, X1 means Asn, X2 means Trp, X3 means Ser and X4 means D-Cys. 4. The method according to claim 1, characterized in that X has the meaning of Cys, Xt means Asn, X2 means D-Trp, X3 means Ser and X4 means D-Cys. 5. The method according to claim 1, characterized in that X has the meaning of Cys, X1 means Asn, X2 means Trp, X3 means D-Ser, and X4 means Cys. 6. The method according to claim 1, characterized in that X has the meaning of Cys, X! means Asn, X2 means D-Trp, X3 means D-Ser, and X4 means Cys. • 7. The method according to claim 1, characterized in that X5 has the meaning of -0-CH2-polystyrene resin carrier. 8. The method according to claim 7, characterized in that R has the meaning of t-butyloxycarbonyl, R4, R5 and R6 mean benzyl, R2 and R3 mean 2-chloro-benzyloxycarbonyl and R1 and R7 mean S-p-methoxy-benzyl.