HU183636B - Process for preparing new peptides with anorexigenic activity - Google Patents

Abstract

The present invention relates to novel peptides having the formula ABC, wherein AL is pyroglutamyl or D-pyroglutamyl, B is L-histidyl, D-histidyl or L-3'-methyl-histidyl, and C is glycine. , glycm-NHR (where R is hydrogen or C1-C3 alkyl) or a derivative of D-alanine or a group of formula -NHR1, and in the latter R1 is alkyl having 1 to 3 carbon atoms, provided that A L -pyroglu tamyl and B are L-histidyl, then C is other than a derivative derived from glycine or glycine-NHR. These peptides can be prepared by methods well known in the art of peptide chemistry. The peptides of the present invention have anorexigenic activity, but also reduce gastric and pancreatic secretions and also stimulate the central nervous system. -1-

Description

The present invention relates to the production of novel anorexigenic peptides. The novel peptides obtainable by the process of the invention are the

They are characterized by the general formula A-B-C (I). In the formula

L-pyroglutamyl (L-H- / Pyro / -Glu) or D-pyrogluamamyl (D-H- / Pyro / -Glu) represents

B is L-histidyl (L-His), D-histidyl (D-His) or L-3'-methyl histidyl (L-N * '- 3-MeHis), and

C is derived from glycine, glycine monoalkilamidból (Gly-NHR where R is hydrogen or C 1-3 alkyl) or D-alanine (D-Ala) group or NHR ¹ group of formula, and in the latter R ¹ 1- It represents an alkyl group containing 3 carbon atoms, with the proviso that when A represents a L-pyroghitamyl group and B represents an L-histidyl group, then C represents a group other than that derived from glycine or glycine monoalkylamide.

The compounds of formula (I) may be used as active ingredients in pharmaceutical compositions having aneroxygenic activity. Such formulations may be used to reduce fat requirements in lactating animals or to treat other pathological conditions that are intended to reduce feed or nutrient uptake. The said pharmaceutical compositions may also be used to treat pathological conditions associated with excessive secretion of gastric acid and pancreatic fluid. Such pathological conditions include gastric and duodenal ulcer and acute pancreatitis. However, said pharmaceutical compositions are useful in the treatment of certain critical states of the central nervous system, such as cerebral palsy or coma.

Abbreviations used for amino acids, derivative groups thereof, and protecting groups are based on the recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry, 11, 1726 (1972)). Examples include:

Phe = phenylalanine (pyro) -Glu = 5-oxo-proline or pyroglutamic acid homo- (pyro) -Glu = homopyroglutamic acid His = histidine

Ν '™ - 3 - His = 3' - Methylhistidine p-NH ₂ Phe = α- (4-aminophenyl) -alanine fi- (pyrazolyl-1) -Ala = 6- (1-pyrazolyl) -alanine Gly = glycine

Gly-ol = 2-aminoethanol and Thi = phi- (2-thienyl) -alanine.

All amino acids are of natural or L-configuration unless otherwise specified. D-His aD-histidine residue, D- (Pyro) -Glu a D-pyroglutamine residue and D-Ala a D-alanine residue. The abbreviations "Me" and "Et" refer to the methyl and ethyl groups respectively. Examples of protecting groups are the following abbreviations: Boc = tert-butoxycarbonyl, Z = benzyloxycarbonyl, Tos = tosyl, Dnp = dinitrophenyl, N ^1m = nitrogen of the imidazole moiety of histidine and Y = a suitable anchor, a solid support , a group commonly used in solid phase peptide synthesis, such as a -O-CH ₂ group attached to a carrier resin.

There has been a long-standing debate in the profession about how humor is influenced by appetite, especially through the pituitary gland. In connection with this discussion, Schally et al., Am. J. Med. 79 (1964) refers to the review article and the publications cited in this article. Thus, since the publication of this article, it is known that the hypothalamus is involved in the regulation of food intake. Electrical stimulation of the lateral hypothalamus leads to excessive appetite and its destruction leads to loss of appetite. At the same time, ventromedial stimulation of the hypothalamus leads to reduced food intake, but its destruction leads to increased fat requirements and excessive appetite.

The fat demand syndrome that can be induced by the administration of gold thioglucose is in part due to the excessive appetite associated with the hypothalamic injury caused by the said compositions. Theories on the control of hunger, appetite and satiety in the nervous system are discussed by many authors and refer to a variety of mechanisms. These mechanisms include:

1) Thermostatic theory

According to this theory, appetite is controlled by a specific dynamic effect of food and its influence on body temperature.

2) Chemostatic theories

These theories assume that appetite is controlled by intracellular or extracellular concentrations of glucose (glucostatic theory), lipids (lipostatic theory), or other metabolites (such as serum amino acids).

3) Other

A further group of existing theories suggest that appetite is controlled by gastrointestinal sensations that occur in the stomach and intestine during food intake and in the presence of food. Enlargement of the stomach by dietary or non-nutritive substances is also one of the factors involved in the development of satiety or a reduction in food intake. Gastric contractions, i.e., gastric contractions, may be related to the feeling of hunger, although these contractions are the same in animals that have been artificially suppressed or loss of appetite by hypothalamic injury, and have been deprived of food and nutrition.

The theories described above are not suitable for explaining all experimental experiences. This category of questions also includes the fact that diabetic animals also have a feeling of hunger. Schally et al., In their earlier article, assume that fat requirements in animals with hypothalamic injury are due to a humoral rather than a neural mechanism and that the hypothalamus is thought to be a substance involved in the regulation of appetite in the central nervous system. The foregoing authors disclose experimental results suggesting that the neurohypophyseal extract contains a substance that influences the dynamic phase of weight gain in mice treated with gold thioglucose.

Another review article by Schally et al. (Recent Progress in Hormones Research; Proceed-21

183,636 of the 1967 Laurentian Hormones Conference, 24,497 (1968); in particular pages 570 and 571, and the references cited therein, have reported on the state of the research on the regulation of food intake and fat requirements by the hypothalamus. Of the publications cited in their review article, special mention is made by Schally et al., Science, 15, 7, 210 (1967), according to which administration of isolated and purified enterogastrone from canine intestine reduces the food intake of mice that had been fasted for 17 hours. This effect is greatest in the first 30 minutes, but the cumulative decline lasts for at least 4 hours. However, other peptides isolated from canine duodenum or colon as well as glucagon, secretin, glucose and bovine auxin albumin are ineffective. These authors hypothesize that the said appetite suppressant effect is based on the direct elimination of gastric contractions due to hunger or the effect of substances released from the hypothalamus via the central nervous system, although at that time it was not possible to obtain positive evidence for hypothalamic neurofluids can be used to control appetite directly or indirectly.

This positive evidence appears to be provided by Trygstad et al., Acta Endocrinologica, 89, 196 (1978). These authors have isolated a number of peptides from the urine of patients with congenital generalized lipodystrophy (hypothalamic syndrome). These peptides induce appropriate metabolic changes. Anorexia nervosa is associated with hypothalamic disorders. In primary nervous hypothalamic anorexia, the hypothalamic-pituitary axis is disturbed, leading to inadequate gonadotropin secretion, loss of menstrual bleeding, loss of daily ACTH secretion, decreased thyrotropin secretion and subsequent increase in somatotropin secretion.

Precipitates from urine specimens from 25 patients with anorexia nervosa were subjected to chromatography on Sephadex G-25 gel-loaded columns. The chromatograms obtained were classified into four different chromatographic samples.

One sample was similar to that of normal controls; showed similarity with another schizophrenic patient; a third sample was seen in five patients suffering from hysterical-type neurosis; and finally, the fourth sample was seen in 10 girls suffering from a so-called primary hypothalamic-type anorexia of nervous origin. In this latter group, it was possible to find fractions that were able to influence the appetite of mice. Several peptide-influencing peptides were isolated by multiple chromatographic steps. When these peptides were injected into mice, one induced an increased appetite and the other a decreased appetite. These peptides were encapsulated with carrier proteins that protected them from enzymatic degradation and thus allowed their isolation from urine.

The structure of the appetite suppressant peptide, in close collaboration with Trygstad, was established by Reichelt et al., Neuroscience, 3, 1207 (1978). This peptide is the H- (pyro) -Glu-His-Gly-OH tripeptide. This was also proved by synthesis.

By injecting 12 nanomoles of this peptide daily for 20 days, food intake decreased from 5.7 g to 3.0 g / day for about 6 months. Weight decreased from an average of 35 g to just 24 g.

Tripeptide has no acute effect on blood glucose and serum insulin levels. Obviously, it acts on the receptors of hypothalamic centers that control appetite.

The aforementioned appetite suppressant peptide, on the other hand, increased daily food intake to above 10 g, resulting in an increase in average body weight to 57 g. The structure of the appetite stimulating peptide cannot be revealed.

Patients with metabolic fat requirements of genetic origin have succeeded in isolating two similar factors, one that increases and decreases food intake.

We have found that the tripeptides of formula I are more potent and sustained in their appetite and food intake reduction activity than the previously mentioned tripeptide isolated by Trygstad et al., Reichelt et al. ) Glu-His-Gly-OH. Biologically equivalent peptides of the formula (1) and their pharmaceutically acceptable salts are thus valuable appetite suppressants in the case of excessive fat requirements and associated pathological conditions in which a reduction in food intake is essential. The active compounds of the present invention also reduce the amount of gastric acid and / or pancreatic secretion. They are therefore also suitable for the treatment of pathological conditions associated with said over-excretion. In addition, the compounds of the present invention also have certain effects on the central nervous system, and are thus useful in the acute treatment of a disturbed consciousness.

The compounds of the present invention, as mentioned above, have the advantageous properties of being more active and longer lasting than the natural tripeptide. These latter two advantages are of particular practical importance, since lower minimal effective doses reduce side effects and manufacturing costs, while less frequent administration requires less frequent administration.

As will be described below, the compounds of formula (I) are prepared by stepwise solid phase synthesis starting with a carboxy carboxyl group.

Suitable protecting groups for intermediates used in the solid phase synthesis of the steps include those which can be removed by one or more chemical treatments without affecting the final product of formula (I). Preferably, these protecting groups are selected such that they can be removed in one step together with other protecting groups, such as the Y-protecting group mentioned above. A particularly useful protecting group R ² is tert-butoxycarbonyl (Boc).

Preferred R ³ protecting groups for the imidazole ester of histidine imidazole are tosyl (Tos) and dinitrophenyl (Dnp). The preferred R ⁴ protecting group for the phenylamino group of α- (4-aminophenyl) alanine is

183,636 zyloxycarbonyl groups (Z). Especially suitable attachment moiety Y to connect to the resin support the previously mentioned _O-CH2 group. Said protecting groups are stable to the reagents and reaction conditions used to deprotect the terminal amino group and to carry out the subsequent coupling reaction.

The alkyl chain of the above-mentioned C 1-3 alkyl group may be straight or branched.

As indicated, the compounds of formula (I) may be prepared by solid phase synthesis in steps starting from the terminal carboxyl group. This synthesis can be briefly summarized as follows: First, a treatment for the deprotection of the α-amino group is carried out using a B or C, when C is -NHR ¹ , or R ^{1 is a C} 1-3 alkyl group, an appropriately selected amino acid (which has a suitable protecting group on its α-amino group and optionally other protecting groups and is attached to the resin carrier by means of the Y-bonding group as defined above) and then a selected amino acid according to B or A, using a suitable dialkyl or dicycloalkylcarbodiimide. This procedure is repeated until the desired number of amino acids are linked. The terminal amino protecting group, as well as any other protecting groups and the Y-protecting group, may be removed. After removal of the solvent, the crude peptide to be prepared is obtained. The crude product is purified by chromatography, for example, gel filtration to give the desired peptide in pure form.

The starting materials used in the preparation of the compounds of formula (I) are commercially available compounds or may be readily prepared in known manner. All amino acids used in the preparation of the compounds of formula I are commercially available, except for homo-pyrroglutamic acid (the preparation of which is described in Greenstein and Winitz, pages 2407-2462, "Chemistry of the Amino Acids", J. Chem. Published in New York in 1961 by publisher Wiley) and β- (2-thienyl) -alanine (the preparation of which is described on page 2707 of the now-mentioned köay).

Suitable solid carrier resins include chloromethylated or hydroxymethylated resins. Of these, the first mentioned are more preferred. The preparation of hydroxymethyl resins is described in Bodansky, M. and Sheenan, J.T. Chem. Ind. (London), 38, 1597 (1966). Chloromethylated resin is marketed by the US company Bio Rad Laboratories (Richmond, CA). When using a chloromethylated amino resin, an ester protecting group is formed with the C-amino acid protected at its α-amino group and optionally bearing another protecting group (or B when C is a -NHR ¹ group as previously defined). A preferred process for converting a fixed and protected peptide into a C-terminal alkylamide is to cleave the protected peptide from the resin by treatment with an alkylamine (the alkyl moiety of which contains 1 to 3 carbon atoms). See Coy, DH et al., Biochem. Biophys. Gap. Commun., 57, 335 (1974)] which results in the formation of the corresponding protected peptide alkylamide. The protecting groups of the latter compound are then removed by treatment with sodium and liquid ammonia, or preferably cleavage with hydrochloric acid, to give the corresponding peptide to be prepared. Another possibility is to carry out the cleavage by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine to give the corresponding ester. This ester can then be converted to the corresponding alkyl amide. Finally, the protecting groups can then be removed as described above. If it is desirable to produce the free carboxylic acid, the cleavage is preferably carried out with anisole hydrobromide. If the acid amide is to be produced, the cleavage is performed with ammonia. In this regard, reference may be made, inter alia, to Stewart, JM and Young, JD, "Solid Phase Peptide Synthesis". 40-49 of his book. pages (published in 1969 by WH Freeman and Co., San Francisco Publisher).

According to a preferred embodiment of the process according to the invention, the glycine protected on its α-amyro group, preferably tert-butoxycarbonylglycine, is coupled to a chloromethylated resin with the aid of a catalyst, preferably cesium bicarbonate or triethylamine. Following coupling, the α-amino protecting group is removed, for example, using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane.

Deprotection is carried out at temperatures between 0 ° C and room temperature. Other reagents or reaction conditions may be used to remove a particular α-amino protecting group (see Sehröder, E., and Lubke, K., pp. 72-75 in "The Peptides," 1965, edited by the New York Academic Press). appeared). After removal of the α-amino protecting group, the other amino acids protected at the α-amino group are sequentially linked in the desired order to give the desired peptide. In each case, the protected amino acids were added in triplicate to the reactor for solid phase synthesis. The coupling reactions are carried out in methylene chloride or in a mixture of dimethylformamide and methylene chloride. In cases where the coupling is not complete, the coupling step is repeated before removing the α-amino protecting group and coupling the following amino acids to the solid phase. The successful completion of the coupling reaction in each synthesis step is described in Kaiser, B. et al., Analyt. Biochem., 34, 595 (1970), as determined by the ninhydrin reaction. Following the successful synthesis of the desired amino acid sequence, the protected peptide is removed from the resin support as described above by treatment with one of the alkyl anrines containing 1 to 3 carbon atoms in the alkyl moiety to form the corresponding peptide alkyl amide. When used as a protecting group for bistidine-dough, these protecting groups are cleaved by treatment with alkylamine. The peptide may also be removed from the resin support by transesterification with a lower alkanol such as methanol or ethanol in the presence of triethylamine to give the resulting

183,636 volumes were purified by chromatography on silica gel. The product thus obtained is then treated from the alkyl ester to give a corresponding alkylamine which can be treated with a suitable alkylamine to form a carboxy-terminal alkylamide. Again, it should be noted that the dinitrophenyl cutosyl group optionally present in the histidyl moiety is cleaved by this reaction. The remaining side chain protecting groups of the resulting protected alkyl amide are then removed by treatment with one of the previously mentioned methods, for example, in liquid ammonia with sodium metal or hydrogen fluoride. Removal of the desired peptide from the resin support may be accomplished by treatment with ammonium hydroxide to give the corresponding amide or with hydrogen fluoride and anisole to form the corresponding free acid. The peptides of formula (I) wherein C is 2-amino hydroxyethyl (Gly-ol) can be prepared by cleavage of an A-B dipeptide resin with 2-aminoethanol. Here too must be taken into account. it is understood that the dinitrophenyl or tosyl optionally present in the histidyl moiety is cleaved during this treatment.

As mentioned above, the peptides of formula (I) are valuable and long-lasting therapeutically effective in that they reduce appetite, inhibit gastric and pancreatic secretion and activate the central nervous system.

The appetite suppressant and anorexigenic activity of the compounds of formula (I) in mice, rats or dogs may be determined by feeding experiments similar to those previously described by Trygstad et al. And Reichelt et al. The inhibitory effect of the peptides of the formula I on gastric and pancreatic secretion in non-stunned rats or, preferably, in non-stunned dogs with esophageal, gastric and pancreatic fibrils or, in the case of the dog, by the Heidenhain can be determined by: Babkin, Β. P. "Secretion Mechanism of Digestive Glands" (2nd edition; published in 1950 by B. P. Hoeber New York Publisher) - quoted in ".Physiology". book on pages 542 to 544 (the book was published in 1963 in the

Published by E. Selkurt, publisher of Little, Brown and Company, Boston); Olbe, L: Gastrpenterology, 32, 460 (1959); Antin et al., Journal of Comparative and Physiological Psychology, 89, 784 (1975) and Liebling et al., Ibid, 89, 955 (1975). The activity of the compounds of formula (I) on the central nervous system, in particular their characteristic spectrum of activity on neurotropic agents, can be investigated in a series of central nervous system-related pharmacological tests. Examples of these tests are Kastin, A.J., Olson, R.D., Schally, A.V. and Coy, D.H., Life Sciences, 25, 401 (1975); Prange, A.J., Breese, G.R., Cott, J.M., Martin, B.R., Cooper, B.R., Wilson, I.C. and Piotnikoff, N.P., Life Sciences, 14, 447 (1974); and Brown, M. and Vale, W., Endocrinology, 96, 1333 (1975).

In order to determine the appetite suppressant or anorexigenic effect of mice, rats and dogs and the inhibitory effect on gastric and / or pancreatic secretion, it is advantageous for animals to have excellent parallelism between animal studies and clinical trials in humans, e.g. or in people undergoing intragastric nutrition (reference is made to Jordan, HA Journal of Comparative and Physiological Psychology, 68, 498 (1969) by Bray, GA and Greenway, FL: Clinics in Endocrinology and Metabolism, 5, 455). 1976) and Janowitz, HD, "The Role of the Gastrointestinal Tract in the Regulation of Food Intake". Article "Handbook of Physiology" (1967, edited by Code, C.F. and Heidel, W., edited by the American Physiological Society in Washington), 6, "The Alimentary Canal". Chapter].

The appetite suppressant and anorexigenic activity of the peptides of formula (I) can be tested in rats. Rats are favorably used as experimental animals for demonstrating the effects of therapeutic agents that inhibit food intake. Numerous authors have found a parallel between rats and humans; See, in this respect, Bray, G.A.'s findings in "The Obese Patient". [This Issue, Vol. 9 of the Major Problems in Inter Medicine, published in 1976 by the W. B. Saunders Company, Philadelphia, London, Toronto]. 9 of this volume, "DrugTherapy for the Obese Patient". In his chapter, "Phaimacology - Effects on the Central Nervous System," on page 364, pages 9-4. Table 6 provides data demonstrating, inter alia, a reduction in food uptake in rats due to a range of aneroxigenic pharmaceutical agents. The Ginical Use of Ánorectic Drugs. 9 to 7 on page 369. Table 9-4 shows that Table 9-4. The therapeutically active agents mentioned in Table II are also suitable for clinical use.

Thus, based on the above properties, the peptides of formula (I) are useful in veterinary treatment of lactating animals and in human medicine.

In view of their appetite suppressant and anorexigenic properties, the peptides of formula I are useful in the treatment of excess fat requirements, including preoperative weight loss, which is often required prior to major surgery in obese patients. Due to their properties, the peptides of the formula I can be used for the treatment of other pathological conditions requiring the reduction of food intake and body weight, such as certain forms of diabetes and circulatory disorders.

In view of their inhibitory effect on the secretion of gastric juice and pancreatic secretion, the peptides of formula I are useful in the treatment of pathological conditions associated with gastric and / or pancreatic hypersecretion of gastric and / or gastrin, pepsin or histarin, such as ulcerative or duodenal ulcer.

The activity of the compounds of formula (I) on the central nervous system exhibits a neurotrophic activity profile that is significantly different from that of tricyclic depression inhibitors and other conventional agents having such activity. By virtue of their central nervous system activating activity, the peptides of formula I are useful in the treatment of cerebral injuries or coma, such as cerebral injuries or tumors, cases of severe freezing or certain postoperative states. They are also effective

183,636, such as certain circulatory disorders or excessive drug consumption (such as intoxication by medication).

In order to provide the above listed therapeutic effects, the peptides of formula (I) may be used, after conversion to a pharmaceutical composition, with carriers and / or excipients conventionally used in veterinary and human medicine, or alone. The quantitative and qualitative relationships between the active ingredient and the carrier or excipient will be determined by the particularity and chemical nature of the active ingredient used, the mode of administration and standard biological practice. For example, an amount of an active ingredient which may be used to reduce appetite and / or nutrition can be orally administered in solid form with carriers such as sugar, starch or certain types of clay. Similarly, suitable amounts of the active ingredient may be administered orally in the form of solutions or suspensions. The compounds may also be administered parenterally by injection. For oral or parenteral administration, sterile solutions or suspensions in a pharmaceutically acceptable liquid carrier such as water, ethanol, propylene glycol or polyethylene glycol may be employed. Said liquid carriers, or the solutions or suspensions themselves, contain other solutes or suspensions, such as sodium chloride or glucose, for example, to prepare isotonic solutions, as well as salts of bile acids, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 and sorbitol. ethylene oxide copolymerized anhydride) or Tween 80.

The dosage of the peptides of formula (I) will depend on the route of administration and the nature of the active compound employed, and the subject to be treated. Generally, treatment is started with a small dose that is significantly lower than the optimal dose. Finally, the dose is increased in small increments until the optimum effect is achieved under the circumstances. Generally, the compounds of formula (I) are administered at doses that produce a reduction in appetite and / or food intake, inhibition of gastric and / or pancreatic secretion, or activation of the central nervous system, without adverse or adverse side effects. Such dosages will generally vary from 1.0 to 250 Mg per kilogram of body weight per day, although as mentioned above, these dosages may be varied. Particularly preferred is a dose range of 5 to 100 µg / kg body weight per day, preferably by multiple administration.

With respect to the same weight, some of the therapeutic effects of the peptides of formula I are greater than those of a number of drugs used for the same purposes. In addition, most of the clinically used appetite suppressants are phenethylamine related and thus exhibit undesirable side effects associated with said structure. The peptides of formula (I) have a structure different from that of Phentylamine and are thus free of these side effects. In addition, they are of low toxicity and thus safe to use.

When used in human medicine, the peptides of formula (0) may be systemically administered by intravenous, subcutaneous or intramuscular injection, or by sublingual or nasal administration, together with carriers and / or excipients commonly used in the pharmaceutical art.

Pharmaceutical compositions can be prepared for oral administration. For this purpose, the active ingredient is mixed with suitable pharmaceutical diluents known in the art and then formulated, for example, in the form of tablets, capsules, suspensions, emulsions, solutions or dispersible powders, in a manner known per se.

The solid compositions may be filled into capsules for oral administration. Formulations suitable for filling capsules contain the active ingredient in admixture with solid excipients which act as buffering agents. Such a buffering agent is colloidal aluminum hydroxide or calcium hydrogen phosphate. For the same purposes, inert carriers such as lactose may be used.

Tablet formulations may be coated in the conventional manner or may be provided in foaming or non-foaming form. For this purpose, inert diluents or carriers, such as magnesium carbonate or lactose, may be used together with conventional disintegrants, such as corn starch and alginic acid, or lubricants such as magnesium stearate.

For the preparation of droplets or spray formulations suitable for nasal administration, the peptides of formula (I) are preferably used in a sterile aqueous vehicle. The carrier may contain other solutes, such as buffering or preserving agents, and pharmaceutically acceptable sodium chloride or glucose in an amount sufficient to form an isotonic solution. For intranasal administration, the dosage range is from 1.0 to 250 mg / kg body weight per day, preferably from 5 to 100 mg / kg.

The peptides of formula (I) may also be formulated as a nasal powder or as an insufflating agent. For this purpose, a peptide of formula (I) is used in fine comminution with a pharmaceutically acceptable solid carrier such as finely divided polyethylene glycol (Carbowax 1540), finely divided lactose or very finely divided silica (Cab-O-Sil). Such formulations may also contain other finely divided additives such as preservatives, buffering agents or surfactants.

The peptides of formula (I) may be delivered to the body in the form of slow release or so-called depot preparations, preferably by intramuscular injection or by implantation, as described below. Such formulations are formulated so as to deliver between 5 and 100 mg of active ingredient per kilogram of body weight per day.

Thus, it is often desirable to deliver the peptides of formula (I) continuously over an extended period of time. In this case, slow release or depot formulations are used. Such formulations or a pharmaceutically acceptable salt of the active compound which is poorly soluble in body fluids, such as pamoasawal (2,2-dihydroxy-1,1'-dinaphthylmethane-3,3'-dicarboxylic acid), tannic acid or carboxymethyl or a water-soluble salt of a peptide of formula (I) together with a protective agent which prevents the rapid release of the active ingredient. The last one

In 183,636, for example, a peptide of formula (I) can be converted to a viscous liquid with a partially hydrolyzed gelatin, or adsorbed onto a pharmaceutically acceptable solid support, such as optionally protamine-zinc hydroxide. The active ingredient may also be administered in the form of a suspension in a pharmaceutically acceptable liquid carrier. Gels or suspensions may also be prepared with non-antigenic protective colloids such as sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturonic acids (such as pectin) or certain monopolysaccharides, carriers, preservatives or surfactants. Examples of such formulations can be found in standard drug manufacturing manuals such as Remington's Pharmaceutical Sciences. published in 1975 by the American publisher Mack Publishing Co. (Easton, Pennsylvania). Slow release sustained release formulations may be prepared by microencapsulation with a pharmaceutically acceptable coating material such as gelatin, pdivinyl alcohol or ethyl cellulose. For further examples of coatings useful for this purpose and for microencapsulation methods, see Herbig, J.A. "Encyclopedia of Chemical Technology". (Published in 1967 by John Wiley and Sons New York Publisher), Volume 2, Volume 13, pp. 436-456. pages refer to what is written. Formulations of this type and suspensions of the poorly soluble salts of the peptides of the formula (I) in body fluids are formulated so as to release 5 to 100 µg of active ingredient per kilogram of body weight per day. Such compositions are preferably administered by intramuscular injection. Another possibility is to provide pellets that deliver the aforementioned amounts of active ingredient or that can be implanted subcutaneously or intramuscularly. For the preparation of such pellets, some of the previously mentioned solid formulations, such as certain water-insoluble salts or dispersions or adsorbates of salts with solid carriers, such as ethylene glycol methacrylate or similar cross-linked monomer polymer dispersions in neutral hydrogel, see U.S. Pat. patent).

Certain water-insoluble peptides of Formula I may be formulated as suspensions in an aqueous medium or an emulsion medium. Aqueous suspensions are prepared by the use of wetting agents such as polyethylene oxide and condensation products of alkylphenols, fatty alcohols or fatty acids, and suspending agents such as hydrophilic colloids such as polyvinylpyrrolidone. Emulsion-based suspensions are prepared by suspending the peptide in an emulsion using a wetting agent and a suspending agent, using the aforementioned wetting and suspending agents. The suspensions may additionally contain buffering agents and / or sweeteners, flavoring agents, coloring agents, preservatives and antioxidants.

As mentioned above, the pharmaceutical formulations of the peptides of the formula I are formulated for oral or parenteral administration in such a way that they are capable of delivering 5 to 100 Mg of active ingredient per kilogram of body weight per day, preferably in divided doses. Dosage units may contain 0.3 to 15 mg of active ingredient. For parenteral administration, it is preferred to use a water-soluble peptide of formula (I) or a water-soluble salt of a peptide (I) which is slightly soluble in water in the form of a sterile aqueous solution. Examples of preservatives include methyl 4-hydroxybenzoic acid and propyl ester, which may be added to such solutions together with other soluble components, such as sodium chloride or glucose, to make up an isotonic solution. The poorly water-soluble peptides of Formula I may be administered intramuscularly in the form of solutions or suspensions in sterile, liquid, non-aqueous vehicles such as oils of vegetable or animal origin. These formulations may optionally contain the aforementioned additional solution components or suspending agents.

The process for the preparation of the peptides of formula (I) according to the invention will now be described in more detail.

The carboxyl-terminated amino acid at the C or B position, when C represents a -NHR ¹ group as defined above, is protected at the tx-amino group, preferably with a tert -butyloxycarbonyl group (R ² = Boc). The protected acid is then coupled with a hydroxymethyl resin or, preferably, a chloromethylated resin using a catalyst, preferably cesium bicarbonate or triethylamine. Following the coupling reaction, the protecting group of the o-amine group is removed, for example, by treatment with trifluoroacetic acid in methylene chloride, as mentioned above, or by another method, also previously mentioned. The deprotection is preferably carried out at temperatures between 0 ° C and room temperature.

Chloromethylated resin (1% crosslinking) is preferably a commercially available product from Bio Rad Laboratories, Richmond, CA.

The carboxyl-terminated amino acid at the C or B site, when C is attached to the NHR ¹ group, and attached to the resin carrier as defined above, is then introduced into the reaction vessel of an automatic peptide synthesizer. The unit is programmed for the following washing cycle:

(a) methylene chloride,

(b) 33% solution of trifluoroacetic acid in methylene chloride (twice),

(c) methylene chloride,

d) ethanol,

(e) chloroform,

f) 10% triethylamine in chloroform (twice),

(g) chloroform; and

h) methylene chloride.

The washed resin is then mixed with a suitably protected amino acid in group B or A, when C is -NHR ¹ . The t-butoxycarbonyl (Boc) group is preferably used as a protecting group for the α-amino group. For the B-residual amino acids, the imidazole nitrogen (N ^ta ) of histidine is preferably protected by a tosyl group (Tos). In the case of 4-aminophenylalanine, the protecting group of the phenylamino group is preferably benzyloxycarbonyl (Z). 3-methyl histidine, β- (1-pyrazolyl) alanine and β- (2-thio)

183,636 enyl) -alanine do not require a protecting group except the a-amino group. A substantially equimolar amount of a condensing agent such as dicyclohexylcarbodiimide or preferably diisopropylcarbodiimide is added to the reaction mixture. The reaction mixture was stirred for 2 hours at room temperature (22-25 ° C), and then the amino-linked resin was washed three times with methylene chloride. Subsequently, the coupled amino acid is deprotected by double treatment with 33% methylene chloride in trifluoroacetic acid. Thereafter, steps c) through h) of the aforementioned wash cycle are performed.

The operations described in the previous paragraph are then repeated with a suitably protected amino acid corresponding to group A when C is an amino acid. The protected peptide resin thus obtained was washed three times with methylene chloride and then three times with methanol and then dried under reduced pressure. In practice, the theoretical yield (96% or more) can be achieved.

To prepare a peptide of formula I wherein C is -NHR ¹ or an amino acid monoalkyl amide, the protected peptide resin is suspended at 0 ° C in a suitable alkylamine containing 1 to 3 carbon atoms in its alkyl moiety and stirred for several hours. The excess alkylamine is then distilled off at room temperature while the cleaved peptide is washed from the resin with dimethylformamide. The protected peptide is then precipitated by addition of ethyl acetate and filtered. Thus, a protected peptide is obtained in which C is -NHR ¹ or an amino-monoalkylamide moiety.

The peptide prepared as described above is deprotected by treatment with hydrogen fluoride and anisole at 0 ° C for 15-60 minutes, preferably 30 minutes. Hydrogen fluoride was removed under reduced pressure and the anisole was washed with diethyl ether. The corresponding protected peptide resin is treated with a mixture of hydrogen fluoride and anisole as described above to produce peptides of formula (I) wherein C is a free amino acid. This treatment involves simultaneous cleavage of the substrate and deprotection.

In the following, specific results of biological assays with peptides of formula (I) are described.

The effect of the peptides of formula (I) on food intake and appetite and the appetite control effect in mice and rats were investigated essentially by methods previously described by Tiygstad et al., Reichelt et al. At regular intervals, daily food intake is determined to an accuracy of 0.1 g and body weight to 1 g. Other data such as oxygen consumption, body temperature, blood glucose / or insulin mirrors, and quantitative aminograms are also determined at certain times.

The effect of peptides of formula (I) on gastric and / or pancreatic secretion in rats or, preferably, in dogs with oesophageal, gastric and pancreatic fistulas was determined by the methods previously described by Babkin, Ölbe or Liebling et al. In dogs, apparent nutrition specifically increases acid secretion, which corresponds to 82% of maximum secretion. Maximum secretion is achieved by administration of 320 pg / kg / hr / 13.55 ± 1.8 milliequivalents / 15 min / histamine. At the same time, serum gastrin levels increase significantly, to 73 ± 10 pg / ml from a 32 ± 5 pg / ml baseline. However, apparent nutrition significantly increases pancreatic protein excretion, which corresponds to 71% of the maximum excretion. Maximum excretion is achieved by administration of 0.5 mg / kg / h / 650 ± 86 mg / ½ min / caerulein. Peptides of Formula I were administered at a dose of 25-50 µg / kg / hr by intravenous infusion 30 minutes prior to apparent feeding and at approximately 34% maximum apparent acid excretion (without substantially altering serum gastrin levels) and maximum pancreatic protein excretion is reduced by about 38%.

Gastric feeding of liver extract in dogs with gastritis and Heidenhain bag leads to a marked increase in acid secretion, which is 90% in maximal whereas, at the same time, serum gastrin levels are significantly increased from the initial value of 29 ± 5 pg / ml to more than twice 76 ± 12 pg / ml. Intravenous infusion of the peptide (I) at a dose of 50 mg / kg / hr to the test animal showed that in dogs fed gastric distillation, the acid excretion induced by liver extraction was reduced by 35% in fed dogs and 41 % without significant changes in serum gastrin levels. The peptides of formula (I) also inhibit nutritionally induced increases in insulin and gastrin levels.

In dogs with pancreatic fistula, one normal diet is 80% of the maximum secreted secretion of HCO3 by the pancreas and 93% of the secreted secretion of CaCO by 0.5 mg / kg / h price increases. The peptides of formula (I), administered as described above, reduce HCO3 secretion by 35% and protein (enzyme) secretion by 46% in the second hour of feeding without causing nausea or other side effects.

The results described above clearly show that the peptides of formula (I) inhibit the brain, gastrointestinal and intestinal secretions of the gastrointestinal tract, presumably by suppressing the neuro-hormonal mechanisms responsible for these choices. The brain phase is the nervous, physical, or appetite phase of gastric secretion which is inhibited by the appetite suppressant and anorexigenic activity of the peptides of formula (I).

The invention is further illustrated by the following embodiments.

Example 1

Prioglutamil D-L-histidyl (tosyl) glycyl-OCH2-resin (DH- (pyroJ-Glu-His) Nún.R ³ (CH ₂ -Gly-resin; ^R3 = Tos)

A Boc-Gly-O-CH ₂ resin (2.70 g, containing 1.00 mmol glycine) was added to a reaction vessel of an automatic peptide synthesizer (Model 990 from Beckmann). The machine is programmed with the following washing program:

183,636 (a) methylene chloride, (b) 33% solution of trifluoroacetic acid in methylene chloride (double treatment; 1 and 25 minutes, respectively), (c) methylene chloride, (d) ethanol, (e) ) chloroform, (f) 10% solution of triethylamine in chloroform (double treatment; both for 2 minutes each), (g) chloroform, and (h) methylene chloride.

The washed resin was then stirred with 3.0 mmol of tert-butoxycarbonyl tosyl histidine in methylene chloride, and 3.0 mmol of diisopropylcarbodiimide was added to the reaction mixture. After stirring at room temperature for 1 hour, the resulting peptide resin was subjected to steps (a) to (h) of the washing program described above. Next, 3.0 mmol of D-pyroglutamic acid is coupled to the peptide resin to give the title product.

The final tripeptide resin was washed three times with methylene chloride and three times with methanol. After drying, 2.89 g are obtained.

The glycine resin used as a starting material is from a commercially available chloromethylated resin [1% sterilized; manufactured by Bio Rad Laboratories (Richmond, CA).

Example 2

D-Pyroglutamyl-L-histidyl-glycine

The protected tripeptide resin obtained in Example 1 (2.89 g) was suspended at 0 ° C for 45 minutes in a mixture of 40 ml of hydrogen fluoride and 10 ml of anisole, and the hydrogen fluoride was evaporated by passing anhydrous nitrogen gas. The anisole is removed by washing with diethyl ether.

The crude peptide was purified by gel filtration over a 2.5 x 95 cm column packed with Sephadex G 15 ("fine"; chemically modified, cross-linked dextran). The fractions containing pure product (320 to 340 mL of azelate) are lyophilized. 320 mg of D-H- / pyro / -Glu-His-Gly-0H are obtained in the form of a colorless, fluffy powder.

The product was homogeneous by thin layer chromatography on silica gel plates with four different solvents. Samples of 20-30 Mg are always applied in the form of a spot. It is developed by treatment with chlorine gas followed by spraying with starch reagent. The following R _f values are obtained.

1: butanol: acetic acid: water 4: 1: 5 v / v = 0.08;

ethyl acetate: pyridine: acetic acid: water 5: 5: 1: 3 v / v = 0.28;

1: 1: 1: 1: 1 v / v 1-butanol: acetic acid: water: ethyl acetate = 0.18; and

2-Propanol: 1 M Acetic Acid 2: 1 = 0.20.

Amino acid analysis gives the following values: Glu = 1.03, Gly = 1.00 and His = 0.99.

EXAMPLE 3 D-Pyroglutamyl-L-histidylglycine, by weight, is dissolved in 1000 parts of distilled, antifreeze-free water, and the resulting solution is added with enough sodium chloride to form an isotonic solution. The solution is then sterilized in an autoclave and filled into ampoules. An infusion solution suitable for medical use is obtained.

Example 4 A mixture of parts by weight of D-pyroglutamyl-L-histidyl glycine, 500 parts by weight of white magnesium carbonate and 20 parts by weight of magnesium stearate is pressed into coarse pieces. The pieces were granulated and passed through a sieve having a mesh size of 2.38 mm and pressed. The tablets thus obtained are suitable for oral administration.

Example 5 A mixture of part by weight of D-pyroglutamyl-L-histidylglycine and 500 parts by weight of white magnesium carbonate was granulated with 2 parts of sodium dioctylsulfosuccinate in sufficient methanol and passed through a 1.68 mm sieve and 5055 ° C. dried. The granulate is then passed through a screen of said mesh size once more. After the addition of 8 parts by weight of magnesium stearate, the mixture is compressed into tablets. They are suitable for oral administration.

Example 6 A mixture of parts by weight of D-pyroglutamyl-L-histidylglycine, 476 parts by weight of lactose, 94 parts by weight of corn starch and 3 parts by weight of magnesium stearate was crushed into granules and passed through a 2.38 mm sieve. The granulate is then coated with a sufficient amount of a solution of 15 parts by weight of shellac and 3 parts by weight of castor oil in 800 parts by weight of ethanol. After the addition of 3 parts by weight of magnesium stearate, the granulate is compressed into tablets. They are suitable for oral administration.

Example 7

In a ball mill, 576 parts of lactose are mixed with 24 parts by weight of D-pyroglutamyl-L-histidylglycine and the resulting mixture is filled into gelatin capsules. Thus, capsules for oral administration are obtained.

Example 8

L-Pyroglutamyl - L-3'-Methyl-Histidyl-glycyl-O-CH3-resin

In the same manner as in Example 1 but using t-butoxycarbonyl-tosyl-histidine instead of t-butoxycarbonyl-3'-methyl-histidine and L-pyroglutamic acid instead of D-pyroglutamic acid, 2.90 g of the title product are obtained. .

Example 9

L-Pyroglutamyl-L-3'-methyl-histidyl-glycine

In the same manner as in Example 2, but using 2.90 g of the product prepared in Example 8 as starting material, the eluate is SiO2. The fractions from 1 to 320 ml were collected to give the title compound as a colorless fluffy powder (251 mg).

Thin layer chromatography on silica gel plates using four different solvent systems shows the product to be homogeneous. Samples of 20 to 30 Mg are always spotted during the test. It is developed by treatment with chlorine gas and then by spraying with starch reagent. The following Rf values are obtained.

183,636

1: butanol: acetic acid: water 4: 1: 5 v / v = 0.04;

ethyl acetate: pyridine: acetic acid: water 5: 5: 1: 3 v / v = 0.15;

1: 1: 1: 1: 1 v / v 1-butanol: acetic acid: water: ethyl acetate = 0.10;

2-propanol: 1 M acetic acid 2; 1 volume = 0.04.

Amino acid analysis gives the following values: Glu = 1.05, Gly = 1.00 and 3-MeHis = 0.96.

Example 10

L-Pyroglutamyl-L-histidyl (tosyl) -D-alanyl-O-CH ₂ resin

The procedure described in Example 1 was followed except that Boc-Gly-O-CH ₂ resin was commercially available from chloromethylated resin [1% crosslinked by Bio Rad Labs Richmond, California, USA]. ] Boc-D-Ala-O-CH ₂ Resin (2.78 g, corresponding to 1 mmol of D-alanine), 3.0 mmol of L-pyroglutamic acid in place of D-pyroglutamic acid and 3.0 mmol of tert-butoxycarbonyl- tosyl histidine is used. 3.37 g of the title compound are obtained.

Example 11

L-Pyroglutamil - L-Histidyl-D - Alanine

The procedure of Example 2 was followed, except that 3.37 g of the product of Example 10 were used as starting material and a fraction of 260 to 320 ml of eluate was isolated. This fraction was further purified by applying it to a 1.5x145 cm silica gel column and eluting with 1: 1: 1: 1 by volume of 1-butanol, acetic acid, water and ethyl acetate. Fractions between 380 and 560 ml of eluate were separated and lyophilized to give the title compound as a colorless powder (390 mg).

Thin layer chromatography on silica gel plates with four different solvents shows the product to be homogeneous. Samples of 20-30 Mg are spotted in the assay. It is developed by treatment with chlorine gas followed by spraying with starch reagent. The following Rf values are obtained.

1: butanol: acetic acid: water 4: 1: 5 v / v = 0.10;

ethyl acetate: pyridine: acetic acid: water 5: 5: 1: 3 v / v = 0.30;

1: 1: 1: 1 v / v 1-butanol: acetic acid: water: ethyl acetate = 0.21;

2-propanol: 2: 1 v / v acetic acid = 0.25.

Amino acid analysis gives the following values: Glu = 1.04, Ala = 1.00 and His = 0.95.

Example 12

D - Pyroglutamyl - L - 3 - Methylhistidyl - D - Alanyl - O - CH ₂ Resin

The procedure is as in Example 1, but 1.39 g (0.5 mmol of D-alanine) of the Boc-D-Ala-O-CH ₂ resin prepared as in Example 10, 5 mmol of tert-butoxycarbonyl-3'-methyl histidine and 1.5 mmol of D-pyroglutamic acid were used. 1.64 g of the expected product are obtained.

- Example 13

D-Pyroglutamyl-L-3'-methyl-histidyl-D-alanine

In the same manner as in Example 2, 1.64 g of the product of Example 12 was treated with a mixture of 20 ml of hydrogen fluoride and 5 ml of anisole to give 300 mg of the title compound as a colorless fluffy powder.

The product was found to be homogeneous with the following Rf values in thin layer chromatography as described in Example 2:

1-butanol: acetic acid: water 4: 1; 5 phase v / v = 0.05;

ethyl acetate: pyridine: acetic acid: water 5: 5: 1: 3 v / v = 0.15;

1: 1: 1: 1: 1: 1 butanol: acetic acid: water: ethyl acetate = 0.10;

2-propanol: 2: 1 v / v acetic acid = 0.05.

Amino acid analysis gives the following values: Glu = 1.07, Ala = 1.00 and 3-MeHis = 1.01.

Example 14

L-Pyroglutamyl-L-histidyl (tosyl) - 0 - CH ₂ - resin

The procedure described in Example 1, but substituting Boc-His (N ^im- Tos) -O-CH ₂ - from a commercially available chloromethylated resin (quality as in Example 10) was used instead of Boc-Gly-O-CH ₂ of 3.00 grams of resin (equivalent to 1.0 milliliters of histidine), followed by washing steps a) to h), followed by the addition of 3.0 millimoles of L-pyroglutamic acid to 3.0 milliliters of diisopropylcarbodiimide . The dipeptide resin was then washed three times with methylene chloride and three times with methanol and dried to give 3.1 g of the title compound.

Example 15

L-Pir oglutamil - L-histidyl ethyl amide

The product (3.1 g) obtained as described in Example 14 was suspended in 0 ml of dimethylformamide at 0 ° C and 3 ml of ethylamine were added to the suspension. The reaction mixture was stirred at 0 ° C for 18 hours, then the solvent and excess ethylamine were removed under reduced pressure. The residue was extracted with 2M acetic acid and filtered to remove the resin. The peptide-containing filtrate was purified by gel filtration over a 2.5 x 95 cm column of Sephadex G-15 (fine-grained, chemically modified cross-linked dextran) column. The product-containing fractions were lyophilized to give 100 mg of the title compound as a colorless fluffy powder.

The product was homogeneous by thin layer chromatography as described in Example 2, with the following Rf values obtained with the four solvent systems mentioned therein:

1-butanol: acetic acid: water, 4: 1: 5, first phase = 0.18;

ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3), first phase = 0.56;

1-butanol: acetic acid: water: 1: 1: 1: 1 ethyl acetate = 0.55;

2: 1 2-propanol / 1 M acetic acid = 0.29. Amino acid analysis gives the following values: Glu = 1.05, His = 1.00 and EtNH ₂ = 1.03.

-101

183,636 matogran examinations basic] with four solvent systems

Example 16

L - Pyroglu tamil - L-Histidyl (tosyl) - Glycyl - O -CH ₂ - Gyta

In the same manner as in Example 1, 3.0 mmol of L-pyroglutamic acid was used instead of D-pyroglutamic acid. 2.90 g of the expected product are obtained.

Example 17

L-Pyroglutamyl-L-histidyl-glycine ethylamide

The product (2.90 g) obtained in Example 11 was suspended in dimethylformamide (20 ml) at 0 ° C, then ethylamine (3 ml) was added to the suspension, and the resulting mixture was stirred at 0 ° C for 18 hours. The solvent and excess ethylamine were then removed under reduced pressure and the residue was extracted with 2M acetic acid and filtered to remove the resin. The peptide-containing filtrate was then purified as in Example 2 to give, after evaporation, the title compound as a colorless fluffy powder (138 mg).

The product is homogeneous for the thin film chronicle described in Example 2, and the following Rp values are obtained:

1: butanol: acetic acid: water (4: 1: 5) = 0.15;

ethyl acetate: pyridine: acetic acid: water 5: 5; 1: 3 v / v = 0.54;

1: 1: 1: 1: 1 v / v 1-butanol: acetic acid: water: ethyl acetate = 0.53;

2-Propanol: 1M Acetic Acid 2: 1 v / v = 0.26.

Amino acid analysis gives the following results: Glu = 1.02, Gly = 1.00, His = 0.97 and EtNH ₂ = 0.98.

Example 18

L- Pyroglu Tamil- D - Histdiol (Tosyl) - Glycyl - O - CH ₂ - Resin

In the same manner as in Example 1, but using 3.0 mmol of tert-butoxycarbonyl tosyl histidine and 3.0 mmol of L-pyroglutamic acid instead of t-butoxycarbonyl tosyl-D-histidine and 3.0 mmol g of product dm is obtained.

Example 19

L-pyroglutamyl-D-histidylglycine

Following the procedure described in Example 2, but treating 2.91 g of the product obtained in Example 18 with 20 ml of hydrogen fluoride and 5 ml of anisole, 125 mg of the title compound are obtained as a colorless fluffy powder. The product was found to be homogeneous in the thin-layer chromatography assays mentioned in Example 2, yielding the following Rp values with the four solvent systems mentioned therein:

1: butanol: acetic acid: water 4: 1: 5 v / v = 0.14;

ethyl acetate: pyridine: acetic acid: water 5: 5: 1: 3 v / v = 0.56;

1: 1: 1: 1: 1 Butanol: Acetic acid: water: ethyl acetate = 0.51;

2-propanol: 2: 1 v / v acetic acid = 0.25.

Amino acid analysis gives the following results: Glu = 1.01, Gly = 1.00 and His = 0.97.

Claims (5)

Patent claims A process for the preparation of peptides of formula A-B-C 5 The L-pyroglutamyl or D-pyroglutamyl group has been reported, ' B is L-histidyl, D-histidyl or L-3'-methyl histidyl, and C is gjicin, glycine-NHR (where R is hydrogen or alkyl having 1 to 3 carbon atoms or a group derived from D-alanine or -NHR ¹ and in the latter R ^{1 is} alkyl having ^{1 to} 3 carbon atoms, with the proviso to ha 15 A represents a L-pyroglutamyl group and B a L-histidyl group, then C represents a group other than a derivative derived from gticin or glycine-NHR, characterized in that a) For the preparation of peptides of the formula ABC, wherein C is selected from glycine, glycine-NHR or D-alanine20, wherein A and B are as defined herein, in the presence of a catalyst in the presence of a catalyst, an amino acid of formula C -ethylated or chloro-25 methylated resin, the (-amino protecting group of the resulting (protected C 1 -O-CH ₂ - / resin carrier) compound is removed and the resulting compound is coupled with an amino acid of formula B suitably protected at its α-amino group. dicildoalkií-karbodfimid In the presence of 3Q, the aamino protecting group of the thus-protected protected dipeptide attached to the resin carrier via an -O-CH ₂ - protecting group is cleaved, coupled with an α-amino protected amino acid of formula A in the presence of a dialkyl or dicycloalkylcarbodi deprotecting the resulting protected tripeptide attached to the resin carrier via the -O-CH ₂ attachment group, cleaving the resulting tripeptide from the resin carrier and isolating it, or b) C is -NHR ¹ - a In Formula 40, R ¹ is as defined above for the preparation of peptides of formula AB-C wherein A and B are as defined herein, in the presence of a hydroxyethylated or chloro-protected amino acid in the presence of a catalyst suitably protected at its α-amino group. is coupled to a methylated resin, the α-amino protecting group of the resulting compound (protected B / -O-CH ₂ - / resin carrier) is removed, and the resulting compound is coupled with an azo-amino protected amino acid in the presence of a dialkyl50 or dicycloalkylcarbodiimide. The o (. amino group) of the protected peptide thus attached to the resin support via the -O-CH ₂ - attachment group is removed and the resulting dipeptide is cleaved from the resin support with an alkyl 55 amine of the formula H ₂ NHR ^] - wherein R 1 is given - by treatment and the resulting pe silicon is separated. 2. A process according to claim 1, wherein the process is a) or b) 60 triethylamine was used. 3. The process of claim 1, wherein the peptide is cleaved from the resin carrier with a mixture of hydrogen fluoride and anisole and the peptides are isolated as the free carboxylic acid. 65 el. -111 183,636 4. The process of claim 1, wherein the peptide is cleaved from the resin support with an alkylamine having from 1 to 3 carbon atoms in the alkyl moiety, and the peptide is isolated as a C-terminal alkyl amide. 5 A process for the preparation of a pharmaceutical composition comprising the steps of: A peptide of formula A-B-C obtained by the process of claim 1 Denotes L-pyroglutamyl or D-pyroglutamyl, B is L-histidyl, D-histidine or L-3'-methyl histidyl, and C is selected from the group consisting of glycine, glycine-NHR (where R is hydrogen or C 1-3 alkyl) or D-alanine, or -NHR ¹ , and in the latter R ^{1 is C} 1-3 alkyl, with the proviso that when A represents a L-pyrogluamylamyl group and B represents an L-histidyl group, then C represents a group other than a derivative derived from glycine or glycine NHR, mixed with a pharmaceutical carrier and / or excipient.