DE2361135A1 - Pyroglutamyl seryl glycine amide - from cattle hypophysis fragments, for stimulating growth hormone release - Google Patents

Abstract

The cpd. of formula (I) is new (I) stimulates the release of growth hormone from the hypophysis so is useful in diagnosis and treatment of hypothalamus disorders, and also stimulates analogous processes in cases of disorders of non-endocrinal origin. It is used in place of anabolic steroids and has no local irritation, histamine-like, histamine-sensitising or pyrogenic side effects. A dose, equivalent to 20 times the maximum human dose given s.c. to mice, caused no toxic symptoms. Suitable dosage forms are tablets contg 1-10 mg or solns. contg. 10-500 ug/ml. (I) is prepd. from comminuted fragments of (cattle) hypothalami by treating with cold (-10 to -15 degrees C) acetone and aq. acetic acid or HCl; lyophilising cleaning and recovering the active matl. by adsorption onto a cellulose cation exchanger.

Description

Institute eks • perimental endocrinology

i chimii gormonov Akademii Medicinskich Nauk SSSR

Moscow / USSR '■

P 52 823

'' ■ "-, - ■ 7 · Dec. 1973 ' ^: .'." L / Br

PYROGLÜTAMYIr-SERYL-G'LYZINAMID,. PROCEDURE FOR ITS MANUFACTURING AND USE

The present invention relates to a new substance, the pyroglutamyl-seryl-glyzinaxaid Process for its manufacture and use.

According to the present invention, said substance has the following Formula on:,

H ₅ G CH ₀ GH ₀ OH

² I "<I ²

O = G - ^ CH - CO - NH - CH - CO - MT - CH ^ - CO N . . . ., ■■ - '■. H ■■■ "■ '■'

The proposed new compound represents an amorphous one Powder whiter. Color with no taste or odor present in

50 9-808 / 1 132

Water, is readily soluble in ethyl and methyl alcohol.

The pyroglutamyl-seryl-glyzinamid was found in the hypothalamus of bull and besitzt.die ability to stimulate the release of growth hormone from the pituitary. Therefore, it can be used as a drug for the diagnosis and treatment of diseases caused by disorders of the function of the hypothalamic-pituitary system and insufficient secretion of growth hormone, as well as for the stimulation of analogous processes in the organism in the treatment of non-endocrine diseases that are associated with the Disturbance of trophic processes (dystrophic phenomena, sequelae of infectious diseases, burns, etc.).

For diagnostic purposes it is currently used to assess the "reserve" of growth hormone in the pituitary gland the so-called "argine, insulin or glucose tolerant tests". A disadvantage of these tests is that they work with a 30 minute infusion of the substances mentioned in larger doses (g / kg body weight), which often leads to negative side effects.

Known preparations for the treatment of diseases related to the lack of growth hormone in the body are preparations based on anabolic steroid hormones and human growth hormone. The anabolic steroid hormones do not eliminate the organic cause of the disease, but cause better utilization of the in

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growth hormone present in the organism. Also call anabolic steroid hormones have a number of undesirable effects like androgenization, premature sex Development of boys and other hormonal erects. The 7 series. Turn of growth hormone is largely limited by that it is species-specific, which is why in medicine only that inaccessible human growth hormone can be used. Also, in some cases, the usage leads such a preparation leads to strong immunological reactions or to a lack of effect.

The process according to the invention for the preparation of pyroglutamyl-seryl-glycinamide consists in being hypothalamic Fragments are crushed, these one after the other with acetone cooled to a temperature of minus 10 to minus 15 ° C and aqueous solution of acetic or hydrochloric acid treated, the acidic solution obtained lyophilized, the lyophile Powder in a solvent with a pH of 4 to 5 -. · ■ dissolves the "solution obtained" on organic molecular sieves separating the end product from the fraction obtained to obtain a fraction containing the end product on a cellulose cation exchanger under subsequent Eluants adsorbed, the eluate obtained from a chromatography on cation exchangers and then on organic molecular sieves to obtain one containing the end product

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Subjects fraction and subsequent separation from the said fraction of the end product.

The process for preparing the is expediently carried out proposed connection using / as ion exchanger (»on Carboxymethyl cellulose ^ by.

With the help of chromatography and electrophoresis Experiments carried out have shown that the substance obtained is an individually pure peptide. Due to the Determination of the acidic composition, the IT-terminal Amino group and the step-wise implementation of the reactions for cleavage of the peptide according to the Edman method information obtained and a comparison with the known literature on the structure of the secretion of the thyrotropic hormone and melanocytostimulating hormone and the gonadotropin regulating factors, the structure of the separated peptide was determined which is proved to be pyroglutamyl-seryl-glycinamide.

7 / ie above · erwäiint - _f the proposed new compound has the ability to stimulate the release of growth hormone. And that is why, according to the invention, it is the one

Active ingredient ^ drug for diagnosis and treatment

the
of diseases, ((caused ^ by disturbance of the function of the hypothalamus-pituitary system and insufficient secretion of the growth hormone <> sir &

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The medicinal product is expediently used to diagnose

■ the e. ■

nostics and treatment of diseases, <conditional> by the Disruption of the functioning of the hypothalamic-pituitary system and

</ y are

insufficient secretion of growth urine, in the form of powder of its active ingredient and in the form of a solution which contains 10 to 500 / Ug active ingredient in 1 ml pharmaceutical solution ^J ; ^:

agent that is a physiological solution.

It is expedient to use a preparation which contains the active ingredient in an amount of 1 to 10 mg in combination with ■ contains a pharmaceutical filler for tablets, which is a mixture of lactose and starch.

In experiments carried out on animals in vitro and In vivo it was found that the proposed preparation the release of growth hormone is stimulated »In the system in vitro during incubation with isolated halves of the adenohypophyses of the rats, the preparation shows a stimulating effect Effect in picogram boxes. The released into the medium Growth hormone was determined with the help of electrophoresis in polyacrylamide gel proven. The in vitro tests testify to the strong specific effect of the preparation. It acts directly on the pituitary gland.

The examination of the biological activity of the preparation under the conditions in vivo ₉ ie its ability to

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Growth hormone levels in the blood were increased Rhesus monkeys carried out. The growth hormone content of the blood was determined by the radioimmunological method, .eis it was found that in the blood of the rhesus monkeys (11 males), with the help of injections of morphine and nembutal brought up to the state of deep anesthesia, 30 minutes after after intravenous injection of the preparation, the growth hormone content is 3 to 4 times higher than the initial content increases.

A single intravenous injection of the drug ^ ⁱⁿ animals from three species (mice, rats, Kanienchen) in doses exceeding the recommended one-time maximum human dose (500 ^ g per man) by 5 to 20 times, showed no toxic effect .

Multiple subcutaneous injections of the preparation in white mice at a dose that is the maximum recommended Dose for humans exceeded by 20 times, also showed no toxic effect.

The preparation showed no local irritation, no histamine-like and mstamine-sensitizing effect and no pyrogenic properties.

A single intravenous injection of the preparation in rhesus monkeys in a dose of 250 to 500 A'g has no effect Changes in pulse rate and blood pressure size.

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The "ability" of the preparation, the release of the waxes Tumor hormones in humans. to stimulate, according to the radioimmunological Method determined on 22 healthy men and 2 women. - ■

With intravenous injection of a sterile solution of the drug in a dose of 10, 25, 100, 250 and 500 a g. Active substance in 1 ml of physiological solution was observed after only 15 minutes an increase in the content of growth hormone in the blood, which reached its maximum after 30 to 60 minutes. The individual fluctuations in the maximum increase in the growth hormone content in the blood are 140 to 1700%. The preparation also shows activity in sublingual or peroral application in the form of active ingredient powder in doses of 1 to 100 mg or in combination with fillers for tablets. The active ingredient content of the tablets is 1 to 10 mg.

The tablets are used for sublingual application the following composition in question: ■

Pyroglutamyl-seryl-glyzinamide - 0.001 g-

Lactose. -0.061 g _ ··

Sugar - 0.027 g?

Starch -'0.01 g _;

Magnesium stearate - 0.001 g

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For oral application, the tablets have the the following composition:

Pyroglutamyl-seryl-glycinamide - 0.001 g; Lactose - 0.059 Si
Strength - 0.039S
Magnesium stearate - 0.001 g

. , It is also possible to use the preparation in the form of suppositories that contain 10 to 500 μg / g of active ingredient.

Since the proposed preparation in doses of micrograms the By stimulating the release of growth hormone from the pituitary gland into the blood, it can cause the disease eliminate, which are connected with the lack of growth hormone in the organism.

In contrast to the growth hormone itself, the preparation should not be species-specific. Separated from the Hypothalamus from the bull, it is effective in testing Rats, monkeys and humans. In addition, it does not call any general generate reactions ..

The preparation, which contains pyroglutamyl-seryl-glycinamide, is harmless, which was determined by determining the "acute" and "chronic" toxicity. A sterile solution of the preparation for intravenous injection in bottles containing 10 or 500 g of the proposed peptide in 1 ml of pharmaceutical solvent is usable for a period of at least up to 9 months.

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Can be used for at least 12 months.

The process for the preparation of pyroglutamyl-serylglyzinamide will be carried out as follows.

Freshly frozen hypot hai ami see fragments are comminuted and homogenized with acetone cooled to a temperature of minus 10 to minus 15 ° C. The acetone is sucked off and. the obtained dry powder of the tissue twice 'at a temperature of plus 4 ⁰ G for 3 hours under
Stirring treated with aqueous 1N hydrochloric acid solution or preferably with aqueous 2N acetic acid solution. The solution obtained is lyophilized. The dry powder is dissolved in a minimal volume of the solvent with a. pH-vVert from 4 to 5, from which the end product is based
Cation exchanger is adsorbed, for example
Buffer solutions, a solution of acetic or hydrochloric acid.
The solution is passed through a column filled with organic molecular sieves, for example Sephadex G-25 (Sweden,
Pharmacia) or Biogel P-4 is filled. The active fraction is removed and mixed in a container with activated cellulose cation exchanger. -

The end product adsorbed on this cation exchanger is eluted with a solution of sodium chloride and lyophilized, then dissolved in water, desalinated by passing the solution through a column with organic molecular sieves,

509 808/1 132.

- ίο -

for example Sephadex G-15 or Biogel P-2 is passed. For further purification: the end product is applied to chromatography (Columns with cation exchangers, for example with! carboxymethyl cellulose or synthetic resin ion exchanger Dowex 5QxQ (Serva Development Laboratory Heidelberg) with subsequent lyophilization of those containing the end product Solution. The dry powder dissolves in minimal

this volume of the solvent and passes through columns with organic molecular sieves, for example Sephadex G-10 in aqueous solutions or in systems of organic solvents, for example n-butanol / acetic acid / water in a ratio of 4: 1: 1;: n-butanol / Pyridine / acetic acid / water in a ratio of. 15ϊ1Οϊ3ϊ12, or uses thin-layer chromatography in the above-mentioned solvent systems.

The proposed solution is separated from the solution obtained Peptide by lyophilization or evaporation of the solution in a vacuum.

For a better understanding of the present invention, the following concrete examples are given below the process for the production of pyroglutamyl-seryl-glyzinamide illustrate.

Example 1.

5000 g freshly frozen hypothalamic fragments from .Are homogenized in to a temperature of minus

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Cooled 15 ° C em, Az et on and obtained an acetone powder of tissue. The dry powder was extracted twice with three times the volume (weight / volume) of 2m acetic acid for 3 hours at a temperature of ⁴⁰ G with stirring. The acetic acid extract was separated by centrifugation and lyophilized, then dissolved in a minimum of 0.005 J & -

Ammonium acetate buffers at a pH of 4.5 and this. - ■ - ..

passed through a column with Sephadex G-25. (Sweden, pharmacist, Upssala) in the same buffer. The active fraction with Ef = 0.63 to 0.53 was mixed in a glass with carboxymethyl cellulose, suspended in the above buffer. The active ingredient, adsorbed on a cat ion exchange, it was eluted with 0.1 M NaCl with a pH of 6.5 and lyophilized, then dissolved the dry powder in 0.005 M ammonium acetate buffer with a pH of 4.5 and free of the salts by passing the solution through a column of Sephadex G-15 in the same buffer. Those containing the active ingredient Fractions were subjected to chromatographic separation on a column of carboxymethyl cellulose among the Conditions of gradual renewal. The active ingredient was adsorbed on a column of 0.005 M buffer with a pH value of 4.5, eluted with 0.1 M NaCl and lyophilized · Das, dry preparation was dissolved in water and the solution was desalted by passing it through a column of Sephadex G-15 fine headed. The active ingredient was lyophilized, then dissolved

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in Y / ater, passed the solution through a column of Sephadex G-10 fine, removed the fraction which eluted with Rf = 0.7 and lyophilized it. In the obtained preparation were with the help of the automatic amino acid microanalyzer Technicon only detected three amino acids, namely • Serine (86 z / m), glutamic acid (0.89 / Tm) and glycine (0.96 #m). The Dansil chloride method showed that the N-terminal amino acid of the resulting peptide is blocked.

The activity of the obtained peptide was as follows The pituitary gland of male rats weighing 140 to 180 g After removing the posterior flap, it was separated in half and one half was placed in the control sample. the other in the test sample. Each sample contained 12 adenohypophyseal halves. After a previous three hour Incubation of the adenohypophyses in the Erebs-Ringer solution with a pH value of 7.4, the solution was replaced by a fresh solution and the test sample was given the test sample Peptide and incubated the samples for 1 hour. The amount of growth hormone released into the incubation— medium spontaneously (in the control sample) and under the action of the peptide (in the test sample), one determined after the physico-chemical method using electrophoresis in the polyacrylamide gel.

The 2 mg tissue equivalent incubation medium brought

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on gel sticks. After electrophoresis, they were stained the gel with 0.01% solution of amide black «The colored Strips corresponding to the growth hormone are cut out from the gel and the dye is eluted with a mixture of methanol and In-HaOK in a ratio of 1: 1 »up the amount of growth hormone was determined by the size of the ab- ■ sorption closed at 570 m // m.

Growth hormone in the medium

optical obligation at 570

1. Control sample 0.060 100

2. Trial sample.
(contains 0.0001 z / g

Peptide) - 'J. \ 0.150, 250

Example '2.

8400 g freshly frozen hypothalamic fragments, dated; Hind was homogenized to a temperature of minus Acetone cooled at 15 ° C, separated the residue by filtration and dried in the air. The dry powder extracted one twice with three times the volume of 2m acetic acid at a temperature of 4 C with stirring. The acetic acid extract was separated by centrifugation and lyophilized

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sized, then dissolved 0.005 & ammonium acetate buffer, with a

this
pH value of 4.5 and passed through a column with biogel P-4 (Sweden Pharmacia) in the same buffer by removing the fraction with Ef = 0.6 and mixing this solution with the carboxymethyl cellulose suspended in the same buffer. The active ingredient adsorbed on a cation exchanger was eluted with 0.1 Im-KaGl with a pH of 6.5 and lyophilized. The dry powder was dissolved in water and the solution desalted by passing it through a column of biogel P-2. The fractions containing the active ingredient were passed through a column with the synthetic resin ion exchanger Dowex 50x8 (Serva Development Laboratory Heidelberg) and

lyophilized. The dry material was dissolved in water on this.

and subjected to further purification by chromatography on a column with Sephadex G-10 in the system n-butanol / acetic acid / water with a ratio of 4: 1: 1 and then in the system n-butanol / pyridine / acetic acid / water with a ratio of 15-10: 3:12. The solution was evaporated in vacuo. In the dry matter, the Dansil chloride method on polyamide plates were three Amino acids detected, namely serine, glutamic acid and (xlycine. Free IT-end amino acid was not detected. The peptide obtained by this method showed in vitro an activity in the system described above that of the Activity of the peptide © obtained in Example 1 is identical.

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Claims (1)

P 52 823 ' _ ₁₅ - 7 Dec. 1973 . ■ VBr ¹ -viii! .. r 1. Pyroglutainyl-seryl-glycinamide, d ad.ur ch g e denotes, that it has the following formula: H ₀ C-CH ₀ CH ₀ OH ² II ² j 2 O = C-CH-CO-. NH - CH - CO - NH - CH ₀ - CO - NH ₀ \ / ^{2 2} N 2. Process for the production of Pyroglutamyl-seryl-> _ glycinamide according to. Claim 1, characterized in that hyp ot hai ami see fragments are crushed, these one after the other to a temperature of minus 10 "to minus 15 ° C cooled acetone and aqueous Treated acetic or hydrochloric acid solution, lyophilized the acidic solution obtained, the lyophilic powder dissolved in the solvent with a pH of 4 to 5, the obtained Solution on organic molecular sieves to give a fraction containing the final product separates the final product from the fraction obtained on a cellulose cation exchanger adsorbed, then eluted, the eluate obtained a chromatography on cation exchangers and then on subjected to organic molecular sieves to obtain a fraction which contains das.Endprodukt, and then separating the end product from said fraction. 509808/1 132 • 3 · Expires after. Claim 2, characterized in that that one uses carboxymethyl cellulose as a cation exchanger. · 4. Preparation for the diagnosis and treatment of disorders of the function of the hypothalamic-pituitary system and insufficient secretion of growth hormone related diseases, characterized by that it contains pyroglutamyl-seryl-glycinamide. 5. Preparation according to claim 4, characterized in that that it consists of pyroglutamyl-seryl-glycinamide as an active ingredient in combination with a pharmaceutical Carrying fabric consists of 6. Preparation according to claim 4, characterized in that that there are 10 to 500 // - g of active ingredient in Contains 1 ml of pharmaceutical solvent which is a physiological solution. 7. Preparation according to claim 4, characterized in that that it contains 1 to 10 mg of active ingredient in combination with a pharmaceutical filler for tablets contains, which is a mixture of lactose and starch. ·. 5 09 80 87 1132