NO813460L - PROCEDURE FOR THE PREPARATION OF NEW PEPTIDES. - Google Patents

Description

The present invention relates to a method for the production of peptides with the general formula A-B-C, where A is selected from the group consisting of L-pyroglutamyl, D-pyroglutamyl and L-homo-pyroglutamyl, B is selected from the group consisting of L-histidyl, D-histidyl, L-3 1-methylhistidyl, L-phenylalanyl,

L-p-aminophenylalanyl, and L-p-(pyrazolyl-1)alanyl; and C is

selected from the group consisting of glycine and its lower alkyl esters, glycinamide and lower alkyl amides thereof, D-alanine and'L-3-(2-thienyl)alanine, with the proviso that C cannot be glycine or glycinamide when A is L- pyroglutamyl' and B is L-histidyl.

Peptides of this type are obtained according to the present invention by coupling an appropriately protected α-amino-protected amino acid with formula C, in the presence of a catalyst,

to a resin carrier selected from 'hydroxyethyl and chloromethylated resins, so that the corresponding compound of formula

(protected C)-O-Cr^-(resin support) is obtained, removing the α-amino-protected group from the latter compound and coupling the resulting compound with an appropriately protected α-amino-protected amino acid of formula B in the presence of a appropriate dialkyl or dicycloalkyl carbodiimide to obtain the corresponding protected peptide linked to the resin support through the anchoring group -O-CI-^-, removing the α-amino protecting group from the latter compound and coupling the resulting compound with. an appropriately protected

α-amino-protected amino acid of formula A in the presence of e-t suitable dialkyl- or dicycloalkyl-carbodiimide, to obtain the corresponding protected peptide attached to the resin carrier through the anchoring group - O-CH^-, removal of the protecting groups from the latter compound, cleavage of the resulting peptide from the resin support and isolation of the corresponding peptide where A, B and C are as indicated above.

The invention also includes the intermediate products used in the process and the use of the compounds A-B-C as anorexics, for the treatment of obesity and other pathological conditions where reduced nutrient or pre-absorption in mammals is appropriate, as well as the use of these compounds for the treatment of pathological conditions that occur in connection

. with overproduction of stomach acid and pancreatic fluid in the case of stomach and

duodenal ulcer or acute pancreatitis and in the treatment of certain critical conditions in the central nervous system such as disturbances of consciousness or coma due to brain damage.

The abbreviations for the amino acids and their residues and protecting groups used here are based on recommendations from the IUPAC-IUB Commission on Biochemical Nomenclature (vgl.Bio-chemistry, Vol. 11 (1972), p. 1726. Examples of such are : (Pyro)-Glu = 5-oxoproline or pyroglutamic acid; homo-(pyro)-Glu = homopyroglutamic acid; His = histidine; Nim-3-His 3'-methylhistidine; 'Phe = phenylalanine; p-NH2Phe = p-aminophenylalanine, (3-(pyrazolyl-1)Ala-3-(pyrazol-1-yl)-alanine; Gly = glycine;

Gly-ol .= 2-aminoethanol; and Thi =3-(2-thienyl)-alanine.

All amino acids have the natural L configuration unless otherwise stated. D-His denotes the D-histidine residue, D-(pyro)-Glu is the D-pyroglutamic acid residue and D-Ala is the D-alanine residue. The abbreviations Me and Et are used for methyl and ethyl respectively.

For protecting groups, the following abbreviations are used, for example: Boe = tert-butyloxycarbonyl; Z = benzyloxycarbonyl; Tos = tosyl;'Dnp = dinitroph enyl; Nlm = imidazole nitrogen in histidine; and Y = a suitable anchoring group bound to a solid resin carrier which is used in solid-phase synthesis, preferably The humoral control of appetite, especially via the hypothalamus, has been discussed for many years, conf. e.g. the review article by Schally et al., Am. J.'Med. Sei., Vol. 248 (1964), p. 79 and the literature cited there. Since then, it has been known that the hypothalamus is involved in regulating nutrient absorption. Electrical stimulation of the lateral hypothalamic area leads to hyperphagia, and destruction of the area to aphagia. In contrast, stimulation of the ventromedial nucleus results in reduced nutrient uptake, and destruction, leading to hyperphagia and obesity. The obesity syndrome produced through the administration of gold-thioglucose is partly a consequence of a hyperphagia associated with. hypothalamic lesions caused by this agent.. Theories about the neural control of hunger, appetite and satiety have been discussed by various authors. Different mechanisms have been proposed:

1) The thermostatic theory

• According to this theory, appetite is regulated by a special dynamic effect of nutrition and its influence on body temperature. 2) Chemostatic theories.

These theories postulate that appetite is regulated through intracellular or extracellular concentrations of glucose (glucostatic theory), lipids (lipostatic theory) and various

metabolites, such as serum amino acids.

3). Another theory group assumes that sensations that occur in

the digestive tract during eating and in the presence of food in the stomach and intestines, regulates appetite. To the contributing factors

The expansion of the stomach due to food or non-nutritive substances is part of the feeling of satiety occurring, or causing nutrient absorption to decrease. Stomach contractions can be linked to hunger, but these contractions

are, however, the same in animals, which are induced into a hyperphagic state through artificial hypothalamic lesions, as in animals, which are prevented from having access to food and water. These theories cannot explain all experimental findings. For example, they do not explain why diabetic animals can feel hunger. Schally et al., mentioned above, assume that the obese person who appears at animals with hypothalamic lesions, is rather transmitted through a humoral than a neurogenic mechanism and that the hypothalamus possibly develops a substance that participates in the central appetite control. Preliminary findings are reported which point to the neurohypophysial extract containing a substance which affects the dynamic phase of weight gain in mice treated with gold-thioglucose.

A new short review article by Schally et al., regarding the status of research with regard to the hypothalamic control of pre-absorption and obesity is also out, conf. Resent Progress in Hormone Research, Proceedings of the 1967 Laurentian Hormone Conference, Vol.24 (1968), p .497, especially pp. 570 to 571 and

the references mentioned therein. These references also belong

the work of Schally et al., Science, Vol. 157 (1967), p. 210,

where it is shown that administration of purified entrogastron from dog duodenum reduces nutrient absorption in mice that have fasted for 17 hours. This effect is greatest during the first 30 minutes, however, the cumulative reduction lasts for at least another 4 hours. Other -peptides as well as glucagon, secretin, glucose and bovine serum albumin, isolated from dog duodenum'

or -colon, have been shown to be ineffective. These authors hypothesize that the action can be traced back to a direct abolition of hunger-gastric contractions or that it is caused by the central nervous system by the release of hypothalamic substances, although positive evidence that hypothalamic neurofluids are.an-responsible for an indirect or direct appetite control,

. at that time could not be presented.

It appears that this ■ positive evidence has now been presented by Trygstad et al., Acta Endocrinologica, Vol. 89 (1978), p. 196. These authors isolated a number of peptides from the urine of patients suffering from congenital generalized lipodystrophy (hypothalamus syndrome). These peptides elicited corresponding metabolic effects. Anorexia nervosa is associated with hypothalamic disorders. In primary hypothalamic anorexia nervosa, the hypothalamic-pituitary axis is disrupted, leading to a low level of gonadotropin release, amenorrhea,

loss of the^ daily ACTH secretion rhythm, decreased thyrotropin secretion and an initially increasing and later decreasing somatotropin secretion.

Precipitates" from urine samples of 25 patients with anorexia nervosa were chromatographed on Sephadex G-25 gel packed columns. This resulted in a division into 4 different chromatographic patterns: One resembled the pattern of normal control subjects, one showed similarity to that of schizophrenia patients. In 5 patients with a hysteriform neurosis a third pattern arose, and in 10 girls with a- primary "hypothalamus"-

type of anorexia nervosa also showed typical chromatograms. Fractions with an influence on appetite in mice were only found in the last group: Through several chromatographic steps, 2 appetite-influencing peptides were isolated. When these peptides were injected into mice, one caused an increase in appetite, while the other caused a decrease in appetite. The peptides are enveloped in peptide carrier proteins and thus protected against enzymatic degradation, which makes isolation from urine possible.

The structure of the anorexigenic peptide was elucidated by Reichelt et al., Neuroscience, Vol. 3 (1978) p. 1207 in close collaboration with Trygstad. It concerns the tripeptide H-(pyro)-Glu-His-Gly-OH, which was confirmed by synthesis.

With a daily injection of 12 nMol of the appetite-suppressing peptide in mice over 20 days, the pre-uptake decreased within approx. 6 months from 5.7 to 3.0 g per day. Body weight dropped from an average of 35 g to a minimum of 24 g.

The tripeptide has no acute effect on the glucose level

.in the blood or on the insulin level in serum. It seems open-

mustache on receptors in appetite-controlling hypothalamic centers.

The appetite-stimulating peptide increased the daily intake by more than 10 g, whereby the body weight rose on average to 57 g. The structure of the appetite-stimulating peptide has not yet been determined.

Two similar factors showing an increased intake and a decreased pre-uptake ratio were also isolated from patients with genetic 'conditional' metabolic obesity.

According to the invention, it has now been determined that peptides of the general formula I

A-B-C

where A, B and C have the meaning stated above, are more active and have a longer duration of action with regard to reducing appetite and lowering nutrient absorption than is the case for the tripeptide pyroglutamyl-histidyl-glycine (H-(pyro)- Glu-His-Gly-OH) that was isolated by Trygstad et al. and Reichelt et al. Peptides of the general formula I and their pharmacologically acceptable salts, which are biologically equivalent to said peptides, are therefore valuable appetite suppressant compounds

for the treatment of obesity and associated pathological conditions where a reduction in nutrient intake is necessary. These substances also reduce gastric and pancreatic secretion. They are therefore also suitable for the treatment of pathological conditions where there is overproduction of stomach acid and/or pancreatic fluid. Moreover, they have specific effects on the central nervous system

so that they are also suitable for acute treatment of consciousness-

disturbances.

The compounds according to the invention have the advantage over the natural tripeptide that it is more active and has

more long-lasting effect. These advantages are of particular practical importance: Due to the lower minimum effective dose, a reduction in side effects as well as in the costs of manufacturing the compounds is achieved. Due to the longer duration of action, less frequent dosing is required.

Due to the fact that the peptides of the general formula I and their pharmacologically acceptable salts are biologically equivalent, all references heretofore, and hereinafter, to peptides of the general formula I are to be understood

which includes both the peptide itself and its salts.

The invention also encompasses protected intermediates

which is bound to a solid resin carrier and which is used for the preparation of compounds with the general formula I.

As protecting groups in the step-by-step solid-phase synthesis of the intermediate products, such groups can be used that can be removed by one or more chemical treatments without this having an impact on the desired end product of formula I. The protecting groups are preferably chosen so that they can be removed in a single step together with other protecting groups, for example simultaneously with the anchoring group Y defined above. A particularly suitable protecting group R 2 for the stepwise solid-phase synthesis of the aforementioned intermediates is tert-butyloxycarbonyl (Boe).

Preferred protecting groups R 3 for protection of the imidazole nitrogen in histidine are tosyl (Tos) or dinitro-

4

phenyl (Dnp). A suitable protecting group R for protecting phenylamino groups in p-aminophenylalanine is the benzyloxycarbonyl group (Z). A suitable anchoring group for connection with the resin carrier is the group Y defined above,

for example -^O-CH 2 . The protective groups mentioned are stable towards the reagents and under the reaction conditions used to remove the protective group on the terminal ami.no group and in the subsequent coupling reactions.

The term "lower alkyl" means straight-chain or branched alkyl residues with one to three carbon atoms.

The peptides of the general formula I are prepared by stepwise solid phase synthesis starting with the terminal carboxyl group. The procedure can be short. can be summarized as follows: A specific amino acid, chosen as C, optionally as B, when C means NHR with R = lower alkyl, with a suitable

■protecting group on the a-amino group and. optionally with other protecting groups, which are combined with the above-defined group Y with the resin carrier, are subjected to a treatment to remove the protecting group on the α-amino group and connect it with the particular amino acid, which is chosen for B or, for A, when C has the meaning N.HR i, with a suitable dialkyl or dicycloalkyl carbodiimide being chosen as coupling agent. The aforementioned procedure is repeated until the desired number of amino acids are connected to each other. The protective group on the terminal amino group is then removed as well as any other protective groups and the anchoring group Y. After removal of the solvent, the desired crude peptide is obtained. The crude product is purified chromatographically, for example by gel filtration, whereby the desired peptide is obtained in a pure state.

The starting materials for the production of the peptides are either common commercial products or can be easily produced using known methods. All amino acids used in the synthesis are commercially available with the exception of homo-(pyro)-glutamic acid, which can be prepared according to Greenstein and Winitz in "Chemistry of the Amino Acids", J. Wiley, New York, 1961, p. 2407 to 2462, and 3-(2-thienyl)-alanine which can be prepared according to the aforementioned reference page 2707.

Suitable solid resin carriers are chloromethylated and hydroxymethylated resins, of which the former are preferred. Regarding the production of hydroxymethyl resins, refer to

M. Bodansky and J.T. Sheehan, Chem. Ind., London, Vol.. 38

(1966), p. 1597. A chloromethylated resin is supplied by Bio Rad Laboratories, Richmond, California. When using the chloromethylated resin, an ester anchoring group is formed with the α-amino protected amino acid. C (or B, when C has it above for specified NHR 1 meaning), which may have additional protection groups.

One. appropriate procedure for conversion of. the connected, protected peptide to C-terminal (lower-alkyl)-amide, consists in cleavage of the protected peptide from the resin by treatment with a lower-alkylamine, e.g. D.H. Coy et al., Biochem. Biophys. Res. Commun., Vol. 57 (1974), p. 335, whereby the corresponding protected peptide (lower alkyl) amide is obtained. The protective groups are then removed by treatment with sodium and liquid ammonia or, preferably, by cleavage with hydrogen chloride, whereby the corresponding peptide according to the invention is obtained. Another method consists in carrying out the cleavage by transesterification with a lower alcohol, preferably methanol or ethanol, in the presence of triethylamine, whereby the corresponding ester is then obtained. The ester can then be converted to the corresponding (lower alkyl)-amide. The protecting groups can then be removed according to the method described above. If free carbonic acid is desired, the cleavage is preferably carried out with hydrogen fluoride in anisole. If the acid amide is desired, the cleavage is carried out with ammonia. In this regard, reference is made to J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman&Co., San Francisco, 1969,

pp. 4 0-4 9. •

Following a technique according to the invention, is connected

glycine with • protected α-amino group, preferably tert.-butyl-oxycarbonylglycine, with a chloromethylated resin by means of a catalyst, preferably cesium hydrogen carbonate or triethylamine. After coupling to the resin support, the α-amino protecting group is removed, for example with trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrogen chloride in dioxane. The protective group is removed at temperatures from approx.

0° to room temperature. Other standard cleavage reagents and conditions for the removal of special α-amino protecting groups can also be used, e.g. E. Schroder and K.. Lubke, "The Peptides", Vol. T, Academic Press, New York, 19.65, pp. 72 to 75.

After removal of the α-amino protecting group, the other amino acids with protected α-amino groups are connected step by step in the desired order to form the desired peptide. The protected amino acids are added in a 3-fold excess to the solid-phase reactor. The coupling reaction is carried out in methylene chloride or in mixtures of dimethylformamide and methylene chloride.

If the coupling proceeds incompletely, the coupling step is repeated before removing the α-amino protection and before connecting the next amino acid. The correct progress of the coupling reaction in the individual synthesis steps is checked using the ninhydrin reaction according to E. Kaiser et al., Analyt. Biochem., Vol. 34 (1970), p. 595.

After synthesizing the desired amino acid sequence, the protected peptide is removed from the resin carrier by treatment with a (lower alkyl) amine to form the protected peptide (lower alkyl) amide. When using dinitrophenyl or tosyl as a protecting group for the histidyl residue, these protecting groups are also removed during the treatment with the (lower alkyl )-amine. The peptide can also be separated from the resin by transesterification with a lower alkanol, preferably methanol•or ethanol, in the presence of triethylamine, after which the obtained ester is purified chromatographically on silica gel. The fraction obtained can be subjected to treatment with a (lower alkyl) amine to convert the lower alkyl ester, preferably the methyl or ethyl ester, to the carboxyl-terminal (lower alkyl) amide. It must be noted that any dinitrophenyl or tosyl groups on the histidyl residue are also cleaved off. The remaining protecting groups for the side chains. in the protected alkyl amide is then cleaved off according to the method described above, for example by treatment with sodium in liquid ammonia

.or with hydrogen fluoride. The cleavage of the desired peptide from the resin carrier can also be carried out with ammonia during formation

of the corresponding amide or with hydrogen fluoride and anisole to form the corresponding free acid. Peptides of the general formula I, where C means 2-aminohydroxyethyl! (Gly-ol), is obtained by cleavage of an A-B-dipeptide resin with 2-aminoethanol. In this method, any dinitrophenyl or tosyl groups on the histidyl residue are also cleaved off.

As already mentioned, the peptides of the general formula I can also be obtained in the form of salts with acids, for example acetic acid, lactic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid and toluenesulfonic acid, polymeric acids such as tannic acid or carboxymethyl cellulose, and inorganic acids such as hydrogen halides, for example hydrogen chloride, as well as sulfuric acid and phosphoric acid. Optionally, special acid salts can be transferred into salts of other acids, for example into non-toxic pharmacologically acceptable salts by treating them with a suitable ion exchange resin according to R. A. Boissonnas et al., Heiv. Chim. Acta., Vol. 43 (1960), p. 1349. Examples of such ion exchange resins are cellulose-based cation exchangers, such as carboxymethyl cellulose or chemically modified, cross-linked dextran cation exchangers, for example of the Sephadex-C type, and strongly basic anion exchange resins, for example resins listed in J. P. • Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley and Sons, Inc.,, New York and London, 1961,, Vol. 2,.p . 1456.

The peptides of formula I and their salts have valuable and long-lasting effects, namely an appetite suppressant effect,

an inhibitory effect on the secretion of gastric and pancreatic juices, as well as a CNS-activating effect.

The appetite-reducing and anorexigenic effect of the peptides of formula I can be determined through feeding experiments on mice, rats and dogs as described in the aforementioned references to Trygstad et al., and Reichelt et al. The inhibitory effect of the peptides on gastric and/or pancreatic secretion is determined on unanesthetized rats or preferably on unanesthetized dogs, with oesophageal, gastric and pancreatic fistulas or on dogs with Heidenhain pockets, etc. B.' P. Babkin, "Secretion Mechanism of Digestive Glands", 2nd ed., B. P. Hoeber, New York, 1950, as cited in "Physiology", Ed. E. Selkurt, Little, Brown and Company, Boston, 1963, pp. 542 to 544; L-. Olbe, Gastroenterology, Vol. 32 (1959) p. 460; Antin et al., Journal' of Comparative and Physiological Psychology, Vol. 89 (1975),

p. 784; and Lieblihg et al., ibid., Vol. 89 (1975), p. 955. The action of the peptides on the central nervous system, especially their characteristic spectrum of neurotropic effects, allows

determine through a number of CNS tests, incl. e.g. A.J.

Kastin, R.D. Olson, A.V. Schally, and D.H. Coy, Life

Science Vol. 25 (1975), pp. 4.01; A. J. Prange, G. R. Breese,

J. M. Cott, B. R. Martin, B. R. Cooper, I. C. Wilson and N. T. Plotnikoff, Life Sciences, Vol. 14 (1974), p. 447 and M. Brown and W. Vale, Endocrinology, Vol. 96 (1975), p. 1333.

Mice, rats and dogs are preferred for determining the appetite-suppressing or anorexic effect of drugs and for determining the inhibitory effect on gastric and/or pancreatic secretion, as excellent parallelism has been established in numerous cases between animal experiments and clinical investigations on people, for example during controlled peroral or intragastric nutrition of people with a liquid diet, etc. H. A. Jordan, Journal of Comparative and Physiological Psychology,' Vol. 68 (1969), p. 498; G. A. Bray and F. L. Greenway, Clinics in Endecrinology and Metabolism, Vol. 5

(1976), p. 455; and H. D. Janowitz, “Role of gastrointestinal

tract in regulation of food intake", cited in "Handbook of Physiology", Section 6, Alimentary Canal., Publisher C. -.F., Code and W. Heidel',' American Phy siological Society, Washington, T967 .

The effect of the peptides of formula I and their pharmacologically acceptable salts as appetite suppressants and anorexic agents can be demonstrated in rats. Rats are preferred as experimental animals for demonstrating the effect of drugs that inhibit nutrient absorption. Various authors have established the parallelism between the results on the rat and man, cf. e.g. G. A. Bray, in "The Obese Patient," Vol.. IX of "Major." Problems in Internal Medicine", W. B. Saunders Company, Philadalphia, London, Toronto, 1976. In Chapter 9 entitled "Drug Therapy for the Obese Patient" there is under the section "Pharmacology - Effects

on the Central Nervous System", on p. 364, table 9-4 provided data, which, among other things, show the reduction of pre-absorption in rats as a result of a number of anorexic drugs. In the section "C.linieal use of Anorectic Drugs" it is shown on page 369 in table 9-7 that the same drugs as in table 9-4, also

■is suitable for clinical use.

Due to their aforementioned properties, the peptides of the general formula I are suitable for treating mammals within

veterinary medicine and also in human medicine.

Because of their appetite-suppressing and anorexic properties, they are suitable for the treatment of obesity, including the preoperative reduction of body weight which is often necessary before major interventions in obese patients. Their properties also make them valuable. treatment of other pathological conditions, which require a reduction in nutrient absorption and body weight, for example certain forms of diabetes and circulatory diseases.

Due to their inhibitory effect on the secretion of gastric acid and pancreatic fluid, peptides of the general formula I are suitable for the treatment of pathological conditions with the occurrence of gastric and/or pancreatic hypersecretion of gastric acid or gastrin, pepsin or histamine, for example in case of duodenal ulcer or acute pancreatitis.

The effect of the peptides on the central nervous system shows a neurotropic action profile, which differs significantly from the profile of tricyclic antidepressants and related drugs. Because of their CNS-activating action is

the valuable means for the treatment of disturbances of consciousness or coma due to brain lesions, for example in the case of cerebral trauma or tumors, severe frostbite or in certain post-operative conditions and in disturbances of consciousness caused by certain vascular diseases or by over-dosing with drugs (e.g. .eg drug intoxication.ions) .

When using the peptides of the general formula I, or their pharmacologically acceptable salts, as appetite-suppressing or anorexic agents for the treatment of obesity or other pathological conditions that require a reduction in nutrient absorption, when used as a means to inhibit excess stomach or pancreas -secretion or when used as a CNS activating agent, the active agents can be given alone or together with pharmacologically acceptable carriers. The ratio between drug and carrier is determined by the solubility and chemical nature of the compound, dosage method and common biological practice. For example, an amount of medication that is sufficient to reduce appetite and/or nutrient absorption can be given orally in solid form together with carriers such as starch, sugar,

specific types of clay and the like.

In a similar way, corresponding amounts can be given orally in the form of solutions or suspensions. The compounds can also be injected parenterally. For peroral or parenteral administration, sterile solutions or suspensions are used

in a pharmacologically acceptable carrier, such as water, ethanol, propylene glycol, or polyethylene glycol containing other solutes or suspending agents, such as sufficient amounts of sodium chloride or glucose to obtain isotonic solutions, salts, of bile acids, gum arabic, gelatin, sorbitan monooleate , polysorbate 80 (oleate esters of sorbitol and copolymers of their anhydrides with ethylene oxide), such as Tween 80.

The dosage of peptides of the general formula I depends on the method of administration and on the particular compound chosen. Moreover, it depends on the patient. As a rule, the treatment is started with small doses significantly below the optimal dose of the compound. The dosage is then stepped up in small steps until the optimal effect - under the given conditions - is achieved. In general, the compound is given in amounts that reduce appetite and/or nutrient absorption, inhibit gastric and/or pancreatic secretion or activate the central nervous system without harmful or unfortunate side effects occurring. Such doses are usually in the range from approx. 1.0^ug to approx. -250^ug per kg. body weight per day, although these doses can be varied as mentioned. Particularly preferred is. dosages in the range from approx. 5 to approx. 100 µg/kg per day, preferably in the form of divided doses.

The individual pharmacological effects of the peptides of formula I are, in relation to. weight, more powerful than a number of drugs for the same purpose. Most clinically usable appetite suppressants also have a phenylethylamine-related structure and therefore exhibit the unwanted side effects associated with this. The peptides of formula I have no phenylethylamine structure and are therefore free from these side effects. In addition, they have a lower toxicity and thus guarantee safe use.

In the case of human medical use of the peptides of formula I, preferably in the form of salts with acids, the administration can take place systemically, either by intravenous, subcutaneous or intramuscular injection, or by sublingual or nasal administration in connection with pharmacologically acceptable carriers.

Pharmaceutical preparations for oral administration can also be prepared. Below, the active substances are mixed with suitable known pharmaceutical diluents and processed in a known manner into preparations such as tablets, capsules, suspensions, emulsions, solutions or dispersible powders.

Solid mixtures can be filled into capsules for oral administration. Such mixtures can contain the solid active substance in a mixture with solid materials with a buffering effect, for example in a mixture with colloidal aluminum hydroxide or calcium hydrogen phosphate, or together with an inert solid substance such as lactose.

The preparations in the form of tablets can be coated in a known manner or produced in the form of foaming or non-foaming preparations. In addition, inert diluents or carriers, such as magnesium carbonate or lactose, together with common blasting agents, such as corn starch and alginic acid, and lubricants,

such as magnesium stearate, is used.

For nasal administration in the form of drops or spray, becomes. the peptides of formula I added to a sterile, aqueous carrier which may also contain other dissolved substances such as buffer or preservative or sufficient quantities of pharmacologically acceptable salts or glucose to prepare isotonic solutions. The doses for intranasal administration range from 1.0 to 250 µg/kg/day, or preferably from about 5 to about 100^ug/kg/day.•

The peptides of the general formula I can also be given as nasal powders or inhalants. For these purposes, the peptides are given in finely divided form together with a pharmacologically acceptable solid carrier, e.g. finely divided polyethylene glycol (Carbowax 1 540.), finely divided lactose or very finely divided silicon dioxide

(Cab-O-Sil). Such agents can also contain other additives in finely divided form, such as preservatives, buffers or surfactants.

The peptides can also be given. [ in the form of long-acting agents with slow release of the drug or depot effect,. preferably, by intramuscular injection or implantation. Such preparation forms have the property that they release from approx. 5 to approx. 100 yug per kg of body weight per day.

It is often desirable that the peptides of formula I are given continuously over a longer period of time in the form of long-acting preparations with a depot effect. Such preparation forms can either contain a pharmacologically acceptable salt of the active substance that has low solubility in the body fluids, for example salts with pamoic acid, tannic acid or carboxymethyl cellulose or in the form of a water-soluble salt together with a protective agent that prevents rapid release. In the latter case, for example, the peptide of formula I can be converted into a viscous liquid with non-antigenic, partially hydrolyzed gelatin or absorbed into a pharmacologically acceptable solid carrier, for example zinc oxide, possibly together with protamine. The active substance can also be given in the form of a suspension in a pharmacologically acceptable liquid carrier. In addition, it is also possible to prepare gels or suspensions with a non-antigenic protective hydrocolloid, e.g. sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatin polygalacturonic acids, such as pectin, or certain mucopolysaccharides, together with aqueous or non-aqueous pharmacologically acceptable liquid carriers, preservatives or surfactants. Examples of such preparations can be found in common pharmacological handbooks such as Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975. Long-acting preparations with slow release of the active substance are also obtained by microencapsulation in a pharmacologically acceptable coating material, such as gelatin, polyvinyl alcohol or ethyl cellulose. For further examples of coating materials and methods of microencapsulation, see J. A. Herbig in "Encyclopedia. of Chemical Technology", Vol. 13, 2nd ed., Wiley, New York, 1967, pp. 436 to 456. Such preparations and suspensions of peptide salts with formula I, which are only slightly soluble in the body fluids, have the property that they release approx. 5 weeks to approx. 100 yug of the peptide per kg of body weight per day. They are preferably given by intramuscular injection. Another possibility consists in preparing some of the above-mentioned solid preparations, for example certain poorly water-soluble salts or dispersions or adsorbates of the salts with solid carriers, for example dispers ions, in a neutral hydrogel of a polymerized ethylene glycol methacrylate or similar cross-linked monomers, .(cf. US-PS 3 551 556), in the form of pellets which release the above stated amounts of drug. Such pellets can be implanted subcutaneously or intramuscularly.

Special peptides of formula I which are poorly soluble in water can be prepared in the form of suspensions in water or in an emulsion base. Water-based suspensions are produced using wetting agents, such as condensation products of polyethylene oxide and alkylphenols, fatty alcohols or fatty acids and suspending agents, such as hydrophilic colloids, e.g. polyvinylpyrrolidone. Emulsion-based suspensions are prepared by suspending peptides using a wetting agent and suspending agents, in the emulsion base using the above-mentioned emulsifying agents. Suspensions may also contain buffers and/or sweeteners, flavourings, colourings, preservatives and antioxidants.

As explained above, pharmaceuticals are prepared for peroral or parenteral administration of compounds of formula I so that 5 to 100 µg/kg/day are delivered, preferably in the form of divided doses. Each unit dose may contain 0.3 to 15 mg of active substance. For parenteral administration, the use of a water-soluble peptide or a water-soluble salt of a less soluble peptide, in aqueous, sterile solutions, is preferred. Examples of preservatives are p-hydroxybenzoic acid methyl and -propyl ester to which, together with other soluble components, sufficient amounts of sodium chloride are added or glucose to obtain isotonic solutions. Sparingly water-soluble peptides of formula I can also be administered intramuscularly in the form of solutions or suspensions in sterile, liquid, non-aqueous carriers, for example vegetable or animal oils. The aforementioned additional solution components or suspending agents may also be present in these.

In - the following, the method for producing peptides with the general formula I will be explained in more detail -.

The terminal carboxylic amino acid chosen for C or B, now that C has the aforementioned NHR 1 meaning, is protected in the α-amino group position, preferably with tert.-butoxycarbonyl

2. (R = Boe). The protected acid is then coupled with a hydroxymethyl resin or preferably a chloromethylated resin,

by means of a catalyst, preferably cesium hydrogen-

carbonate•or triethylamine. In connection with the coupling,

the protective group on the α-amino group is removed, e.g. using trifluoroacetic acid in methylene chloride or by means of one of the other methods mentioned above. The removal of the protecting group is preferably carried out at temperatures from 0°C to room temperature.

As a chloromethylated resin (1% cross-linking) it is preferably used. commercial product from the company BioRad Laboratories, Richmond, California.

The carboxyl-terminal amino acid which is bound to the resin carrier by means of the previously defined anchoring group, and which constitutes the residue C, or B when C stands for NHR, is transferred to the reaction chamber by an 'automatic peptide synthesizer which is programmed for the following washing cycle:' (a) methylene chloride., (b) 33% trifluoroacetic acid in methylene chloride (2 times),

(c) methylene chloride,

(d) ethanol,

(e) chloroform,

(f) 10% triethylamine in chloroform (2 times),

(g) chloroform and

(h) methylene chloride.

The washed resin is then stirred in methylene chloride with a correspondingly protected amino acid which makes up the residue B, or A

when C means NHR 1: Tert-butoxycarbonyl (Boe) is preferred as a protecting group for the α-amino function. By amino acids for. the residue B, the tosyl group (Tos) is preferred to protect the imidazolium nitrogen (N<im>) in hdstidine. Benzoylcarbonyl (Z) is preferred as a protecting group for the phenylamino group in p-aminophenylalanine. 3-Methylhistidine, 3-(pyrazolyl-1)-alanine and 3-(2-thienyl)-alanine need no protection, except for the α-amino group. A substantially equimolar amount of a coupling agent, such as dicyclohexylcarbodiimide or preferably diisopropylcarbodiimide, is added. The mixture is stirred in

two hours at room temperature (22-25°C). The amino acid resin is then washed with methylene chloride (3 times). The protecting group for the attached amino acid is removed with 33% trifluoroacetic

acid in methylene chloride. In connection with this, steps (c) to (h) of the above-mentioned wash cycle are carried out.

The above steps are repeated with a corresponding protected amino acid chosen for residue A when C stands for an amino acid. The protected peptide resin thus obtained is washed three times with methylene chloride and then three times with methanol and dried under reduced pressure. In this way, a practically theoretical yield is achieved. (96% or more) .

To prepare a peptide of formula I, where C means NHR 1 or an amino acid lower alkyl amide, the protected peptide resin is suspended at 0°C in the respective lower alkyl amine and stirred for several hours. Excess alkylamine is then evaporated at room temperature. The cleaved peptide is washed out of the resin with dimethylformamide. The protected peptide is then precipitated by the addition of acetic acid ethyl ester and filtered. In this way, the protected peptide is obtained, where C means NHR 1 or an amino acid lower alkyl amide.

For the preparation of protected peptides, where C means an amino acid amide or an amino acid lower alkyl ester, is carried out

.the above-described method with ammonia or the corresponding alcohol + triethylamine instead of lower-alkylamine. The protection groups are removed by treating the material with hydrogen fluoride and anisole at 0°C for 15 to 60 minutes, preferably approx. 30 minutes. Hydrogen fluoride and anisole are removed respectively under reduced pressure and by washing with ether. For the preparation of peptides of formula I, where C means a free amino acid, the corresponding protected peptide resin is treated in the aforementioned manner with hydrogen fluoride and anisole. In this way, the protective groups and the bond to the resin carrier are removed at the same time. The desired peptide of formula I is thereby obtained, where C stands for a free amino acid. For the production of peptides of formula I, where C means 2-aminohydroxyethyl (Gly-ol), the A-B-dipeptide resin is treated with 2-aminoethanol, whereby the desired peptide of formula I, where C means Gly-ol is obtained directly . Any dinitrophenyl or . the tosyl protecting groups on the histidyl residue are simultaneously removed.

In what follows, biological investigations of the peptides according to the invention are reproduced.

The peptides' reduction of appetite and nutrient uptake is confirmed through feeding experiments on mice and rats, essentially according to the methods mentioned by Trygstad et al., and Reichelt et al. At regular intervals, the daily preconsumption is determined with an accuracy of 0.1 g and the body weight with an accuracy of 1 g. Other data, such as oxygen consumption, body temperature, glucose and/or insulin levels in plasma and quantitative aminograms are recorded to specific times.

The effect of the peptides on gastric and/or pancreatic secretion is determined in rats, or preferably dogs, provided with esophageal, gastric, and pancreatic fistulas according to the aforementioned authors, Babkin, Liebling et al., or Olbe. A leather lining of dogs led to a marked increase

of the acid secretion, which amounted to 82% of what can be maximally achieved by the administration of 320 µg/kg/hour histamine (13.55 + 1.8 meq./15 min).' At the same time, a significant increase in the serum gastrin level occurred from a baseline value of 32 + 5 pg/ml to 73 + 10 pg/ml. can be achieved by administration of 0.5 µg/kg/hour cerulein (650 + 86 mg/15 min). The peptides with formula I reduce the maximum acid secretion by approx. 34%, without a significant change in the serum gastrin level, and the pancreatic protein secretion by approx. 38%, by intravenous infusion of doses of 25 -' -50 µg/kg/hour. 30 minutes before and under the leather lining.

In dogs with stomach fistulas. and Heidenhain pouches, gastric administration of a liver extract led to a marked increase in acid secretion. It included approx. 90% of the maximum value that can be achieved through histamine administration to dogs with stomach fistulas, and approx. 35% of the maximum value.for■

dog with Heidenhain pockets, while the serum gastrin level rose very significantly to 76 + 12 pg/ml which is more than double the baseline value of 29 + 5 pg/ml. By adding the peptides of formula I during 1 hour under the

plateau through intravenous infusion of doses of 50 µg/kg/hr, the induced acid secretion was reduced by 35% in dogs with gastric fistulas and by 41% in dogs with Heidenhain-. pockets'. without significant changes in serum

the gastrin mirror. Peptides with the general formula I therefore also inhibit the feeding-related increase in insulin and gastrin.

In dogs with .pancreatic fistulae stimulated normal

lining the pancreatic HCO^ to 80% of the maximum value that can be obtained by the supply of 3 KU/kg/hour secretin, and the protein excretion to 92% of the maximum value that can be obtained with 0.5 µg/kg/hour cerulein. The peptides of formula I, given in the above-mentioned manner, during the second hour after feeding, decreased HCO 3 secretion by 35% and protein (enzyme) secretion by 46% without causing vomiting or other side effects.

These results clearly show that peptides of the general formula I inhibit the brain, stomach and intestinal channels of gastric and pancreatic secretion, presumably through suppression of the neuro-hormonal mechanism responsible for these secretions. The brain phase refers to the neural, psychic or appetitive phase of gastric secretion, the inhibition of which shows a suppression of the appetite and the anorexic effects of the peptides of formula I Example 1 D- pyroglutamyl- L- histidyl-( tosyl)- glycyl- O- CRy-resin,

D- H-( pyro)- Glu- His( Nim- R3)- Gly- Q- CH2- resin, R3 = Tos.

2.70 g (1.00 mmol glycine) of the Boe glycine resin of formula

Boe - Gly 0 - CH2- resin

placed in the reaction compartment of an automatic peptide synthesizer (Beckmann Model 990) which is programmed for the following washing cycle:

(a) methylene chloride

(b) 33% trifluoroacetic acid in methylene chloride (2 times,

1 or 25 minutes),

(c) methylene chloride,

(d) ethanol,

(e) chloroform, (f) 10% triethylamine in chloroform (2 times 2 minutes), (g) chloroform and (h) methylene chloride.

The washed resin is then stirred with 3.0 mmol of tert-butyloxycarbonyl-tosyl-histidine in methylene chloride and 3.0 mmol of diisopropylcarbodiimide is added. The mixture is stirred at room temperature for 1 hour. The peptide resin is subjected to steps (a) to (h) of the above washing cycle. 3.0 mmol of D-pyroglutamic acid is then added. same way, by which ' the title compound is obtained.

The finished tripeptide is washed 3 times with methylene chloride

and then 3 times with methanol. After drying, 2.89 g of material is obtained.

The glycine resin in this example was prepared from commercially available chloromethylated resin (1% cross-link, Bio Rad Laboratories, Richmond, California).

Example 2

D- pyroglutamyl- L- histidyl-glyein.

2.89 g of the protected tripeptide obtained in Example 1. is suspended during 45 minutes at 0°C in 40 ml of hydrogen fluoride and 10 ml of anisole. The hydrogen fluoride is then evaporated with a stream of dry nitrogen. The anisole is removed by washing

with ether.

The crude peptide is purified by gel filtration on a column packed with Sephadex G 15 (chemically modified, cross-linked dextran, "fine") measuring 2.5 x 95 cm. The corresponding fractions (320 to 340 ml) are lyophilized. This gives 320 mg of D-H-(pyro)-Glu-His-Gly-OH in the form of a colourless, flaky powder.

In thin-layer chromatography on silica gel, the product • behaves homogeneously in 4 different solvents. Below this, 20-30 µg are deposited each time in the form of spots. Development takes place with chlorine gas and subsequent spraying with starch reagent. The following R^ values are obtained: 1-butanol : acetic acid : water (4:1:5, upper phase), 0.08; Acetic acid ethyl ester : pyridine : acetic acid : water (5:5:1:3) 0.28;. 1-butanol : acetic acid : water : acetic acid ethyl ester (1:1:1:1) 0.18; 2-propanol : 1 m acetic acid (2:1)0.<2>0.

By amino acid analysis, the following values are obtained:

Glu 1.03, Gly 1.00, His 0.99.

Example 3

3 parts of D-pyroglutamyl-L-histidyl-glycine are dissolved in 1000 parts

distilled, pyrogen-free water. To prepare an isotonic solution, a sufficient amount of sodium chloride is added. The mixture is sterilized by autoclaving and filled into ampoules. This results in an infusion solution for therapeutic purposes.

Example 4

A mixture of 12 parts of D-pyroglutamyl-L-histidyl-glycine and 500 parts of white magnesium carbonate and 20 parts of magnesium stearate is pressed into coarse pieces. The pieces are granulated, sieved through an 8 mesh sieve (2.38 mm) and pressed. The tablets obtained in this way are suitable for oral administration.

Example. 5

A mixture of 12 parts D-pyroglutamyl-L-histidyl-glycine and 500 parts white magnesium carbonate. granulate in a sufficient amount of methanol while mixing in a solution of 2 parts sodium dioctyl sulphosuccihat. The granulate is sieved through a 12 mesh sieve (1.68 mm) and dried at 50 to 55°C. The granulate

then passed once more through a 12" mesh sieve. After adding 8 parts magnesium stearate, the mixture is pressed to

tablets for oral administration.

Example 6

A mixture of 24 parts D-pyroglutamyl-L-histidyl-glycine, .475 parts lactose, 94 parts corn starch and .3 parts magnesium stearate is pressed together into coarse pieces. The pieces are crushed into a granulate which is passed through an 8 mesh sieve (2.38 mm). The granulate is then coated with a sufficient amount of a solution of 50 parts shellac and 3 parts castor oil in 800 parts ethanol. 3 parts of magnesium stearate are then added. The granulate is pressed into tablets for oral administration.

Example 7

24 parts of D-pyroglutamyl-L-histidyl-glycine are mixed with 576 parts

lactose in a ball mill. The mixture is filled into gelatin capsules. A preparation for oral administration is obtained.

Claims (11)

1.. Process for the production of peptides with the general formula formula A-B-C, where A is selected from the group consisting of L-pyroglutamyl, D-pyroglutamyl and L-homo-pyroglutamyl, B is selected from the group consisting of L-histidyl, D- histidyl, L-3 <1-> methylhistidyl, L-phenylalanyl, L-£-aminophenyl-alanyl, and L-3-(pyrazolyl-1)alanyl; and C is selected from the group consisting of glycine and its lower alkyl esters, glycinamide and lower alkyl amides thereof, D-alanine and L-3-(2-thienyl)alanine, with the proviso that C cannot be glycine or glycinamide when A is L-pyroglutamyl and B is L-histidyl, characterized by the coupling of an appropriately protected α-amino-protected amino acid of formula C, in the presence of a catalyst, to a resin carrier selected from hydroxyethyl and chloromethylated resins, so that the corresponding compound of formula (protected C)-0-CH2 -(resin carrier) is obtained, removal of the α-amino protected group from the latter compound and coupling the resulting compound with an appropriately protected α-amino protected amino acid of formula B in the presence of a suitable dialkyl or dicycloalkyl carbodiimide to obtain the corresponding protected peptide attached to the resin support through the anchoring group -O-CH -, removal of the α-amino protecting group from the latter compound and coupling of the resulting compound with an appropriately protected α-amino-protected amino acid of formula A in the presence of a suitable dialkyl or dicycloalkyl carbodiimide, to obtain the corresponding protected peptide attached to the resin support through the anchoring group -O-CH,,- , removing the protecting groups from the latter compound, cleaving the resulting peptide from the resin support and isolating the corresponding peptide where A, B and C are as indicated above. 2. Method as stated in claim 1, characterized in that the catalyst is cesium bicarbonate or triethylamine. 3. Method as stated in claim 1, characterized in that the peptide of formula A-B-C is treated with a pharmaceutically acceptable acid and the corresponding pharmaceutically acceptable salt of the peptide is isolated. 4. Method as stated in claim ^ characterized in that the peptide is cleaved from the resin carrier with hydrogen fluoride in anisole and the peptide is isolated as the free carboxylic acid. 5. Method as stated in claim 1, characterized in that the peptide is cleaved from the resin carrier with ammonia and the peptide is isolated as the acid amide. 6. Method, as stated in claim 1, characterized in that the peptide is cleaved from the resin carrier with a lower, alkylamine and the peptide is isolated as C^terminal-(lower-alkyl)-amide 7. Process as stated in claim 1, characterized by treatment of Boc-Gly-O-CPI^ - (the resin carrier) with trifluoroacetic acid followed by triethylamine, coupling of the resulting compound with t-butyloxycarbonyl-tosyl-histidine in the presence of diisopropylcarbodiimide and treatment of the resulting compound with trifluoroacetic acid followed by triethylamine, coupling of the resulting compound with D-pyroglutamic acid in the presence of diisopropylcarbodiimide, and isolation of D-pyroglutamyl-L-histidyl (tosyl)-glysyl-O-CI-^- (resin carrier), which is then treated with hydrogen fluoride in anisole, after which D-pyroglutamyl-L-histidyl-glycine is isolated. 8. Process for the production of a peptide of formula A-B-C, where A is selected from the group consisting of L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl, B is selected from the group consisting of L-histidyl, D-histidyl, L- 3'-methylhistidyl, L-f enylalanyl, L-p_-aminof enylalanyl and L-3-(pyrazolyl-1)alanyl, and C is NHR 1 where R <1> is lower alkyl, characterized by cT^ linkage~of a suitable " protected' α-amino-protected amino acid of formula B in the presence of a catalyst to a resin support selected from hydroxyl and chloromethylated resins to obtain the corresponding compound of formula (protected B)-O-CH 2 -(resin support), removal of the α-amino protecting group from the latter compound and coupling the resulting compound with a suitably protected α-amino-protected amino acid of formula A in the presence of a suitable dialkyl or dicycloalkyl carbodiimide, to obtain the corresponding protected peptide bound to the resin carrier by means of the anchoring group -0-CH2 -, remove g of the protecting groups, cleavage of the resulting peptide from the resin carrier by treatment with a lower alkylamine of formula P^NR 1 , where R is lower alkyl, and isolation of the corresponding peptide of formula A-B-C where A and B. are as indicated above and C is an NHR, where 1 R is lower alkyl. 9. Process for producing a peptide of formula A-B-C where A is selected from the group consisting of L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl, B is selected from the group consisting of L-histidyl, D-histidyl. L-3'-methylhistidyl, L-phenylalanyl, L-p_-aminophenylalanyl and L-3-(pyrazolyl-1)alanyl and C is 2-amino-1-hydroxyethyl, characterized by the coupling of an appropriately abbreviated α-amino -protected amino acid of formula B .in the presence of a catalyst, to a resin support selected from hydroxymethyl and chloromethylated resins to obtain the corresponding compound of formula (protected B)-O-CH2 -(resin support), removal of the α-amiho protecting group from the latter compound and coupling the resulting compound with a suitably protected α-amino-protected amino acid of formula A in the presence of a suitable dialkyl or dicycloalkylcarbodiimide to obtain the corresponding protected peptide bound to the resin carrier through the anchoring group -O-CI^-, removal of the protecting groups, cleavage of the resulting peptide from the resin support by treatment with 2-aminoethanol and isolation of the corresponding peptide of formula A-B-C where A and B are as indicated above and C is 2-amino -1-hydroxyethyl1. 10. Method as stated in claims 8 or 9, characterized in that the catalyst is cesium bicarbonate or triethylamine. 11. Procedure as specified in claims 8 or 9, ■ characterized in that the peptide of formula A-B-C is treated with a pharmaceutically acceptable acid and the corresponding pharmaceutically acceptable salt of the peptide is isolated.