WO1983001251A1

WO1983001251A1 - Polypeptide, preparation method thereof, utilization thereof and pharmaceutical products containing it

Info

Publication number: WO1983001251A1
Application number: PCT/DE1981/000171
Authority: WO
Inventors: Arzneimittel Gmbh Ferring
Original assignee: HÖRIG, Joachim
Priority date: 1981-10-09
Filing date: 1981-10-09
Publication date: 1983-04-14
Also published as: EP0089950A1

Abstract

Polypeptides having the formula I and the salts thereof <IMAGE> wherein the symbols X, Y and Z are as follows: X: D-Pro, D-p-Cl-Phe; Y: Phe, Ile; Z: Met, Nle. The invention also relates to a method for the preparation of said compounds. The invention also includes the utilization of said polypeptides or the pharmaceutically acceptable salts thereof for the relief of pain and hypertension, as well as pharmaceutical products containing one or more of said polypeptides having the formula I or the pharmaceutically acceptable salts thereof and agents and/or conventional pharmaceutical adjuvants.

Description

Polypeptide, process for its preparation, its use and pharmaceutical preparations containing it

The present invention relates to a novel polypeptide which has pronounced antagonistic properties against substance P, a process for its preparation, its use in combating diseases, and pharmaceutical preparations containing it.

Euler and Gaddum ((1931), J. Physiol. (London) 72, 74-87) were the first to describe a biological activity from tissue extracts that had a contraction-stimulating effect on the jejunum of the guinea pig and a blood vessel-expanding effect in rabbits. They called the activity "Substance P", but were unable to isolate the responsible compound in its pure form. This only happened later, when Leeman and Hammerschlag 1967 (Endocrinology 81, 803-810) found the salivary effect of a hypothalamic fraction. As a result of this simple detection system, the working group was able to maintain the effective principle in a homogeneous form (Chang & Leeman 1979, J. Biol. Chem. 245, 4784-4790). It was a peptide with the following structure:

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂

This peptide was soon identified as substance P, since it had the same properties as the fractions partially cleaned by Euler and Gaddum. Since the peptide was now synthetically accessible (the first synthesis was described by Tregear et al. (1974, Nature (London) 232, 87-89)), a specific radioimmunoassay for the peptide could also be developed. This allowed the distribution of the substance in different tissues to be determined and thus stimulated Substance P research considerably.

Substance P has been detected in a variety of different tissues and cellular structures. Since this was done in most cases by radioimmunological methods, the literature speaks of ISP (immunoreactive substance P). Organs in which ISP occurs are: mammalian intestine, salivary gland, trachea, pancreas, kidney, bladder, prostate, substantia nigra, hypothalamus, pineal gland, dorsal horn.

ISP is apparently located in nerves that belong to the autonomic nervous system and primary sensory nerve fibers (e.g. fibers that are responsible for the transmission of pain). ISP was found in the myenteric plexus (Auerbach plexus) and in scattered nerve fibers between the smooth muscle cells of the small intestine. ISP also occurs in endocrine cells of the intestinal mucosa.

A major advantage in researching the biological functions of substance P would be an inhibitor that specifically blocks the action of the peptide. Recently, specific and competitive inhibitors have been successfully synthesized. These are substances in which the amino acids in positions 2, 7 and 9 are replaced by other amino acids. D-Pro ² , D-Phe ⁷ , D-Trp ⁹ -SP (Folkers et al. (1981) Acta Physiol. Scand. 111, 505-506; Rosell et al. (1981) Acta Physiol. Scand. 111, 381 -382) inhibits the contraction of the guinea pig ileum by SP, that by SP-induced saliva secretion and by antidromic nerve irritation and by SP-induced vasodilation.

Nature, Vol. 293, page 222 reports on a synthetic peptide which has an antagonistic effect against substance P. Formula II, given later, is given for this polypeptide. According to this literature reference, the peptide tested there also has pronounced agonistic properties which preclude the peptide from being used as a medicinal product in humans and animals. A method for producing the peptide allegedly used there or physical and chemical data are not specified. On the basis of the knowledge gained according to the invention, it must be assumed that the substance reported in the literature reference Nature does not have the formula given there or represents a Geraisch from unknown substances.

The object of the present invention is to find new polypeptides which have a practically exclusive antagonistic effect against substance P and which are suitable as medicaments for humans and animals for combating certain diseases.

The present invention accordingly relates to a Polypeptide of the following general formula I and its salts

Arg-X-lys-Pro-Gln-Gln-D-Trp-YD-Trρ-Leu-Z-NH ₂ (I) where the symbols x, y and Z have the following meanings:

X: D-Pro, D-p-Cl-Phe

Y: Phe, Ile

Z: Met, never.

In particular, the subject of the present invention is the peptide of the formula

D-Pro ² , D-Trp ⁷ ' ⁹ substance P (II) and their salts.

The process for the preparation of the polypeptides mentioned is characterized in that, in a manner known per se, the C-terminal amino acid and then the other amino acids are gradually coupled to a carrier resin by a solid resin method and the carrier resin is split off.

The invention furthermore relates to the use of the polypeptides mentioned or their pharmaceutically acceptable salts for combating, in particular chronic, painful conditions and high blood pressure disorders.

The invention furthermore relates to pharmaceutical preparations which contain one or more of the abovementioned peptides or their pharmaceutically acceptable salts together with customary pharmaceutical carriers and / or auxiliaries.

The peptides according to the invention have an extremely pronounced antagonistic property with no discernible agonistic effect on substance P. The invention is further illustrated by the following examples.

With methionine

Leu Leucih

Trp tryptophan

Phe phenylalanine

Gin glutamine

Per proline

Lys Lys in

Arg arginine

DCC dicyclohexylcarbodiimide

Ac ₂ O acetic anhydride

Py pyridine

HONp p-nitrophenol

Np nitrophenyl

Boc t-butyloxycarbonyl

TFA trifluoroacetic acid

DMF dimethylformaid

BHA benzhydrylamine

Ile isoleucine

Never norleucine

Unless otherwise stated, it is the L configuration.

example 1

(1) materials

Boc-Arg-Tos, Boc-Gln-ONp, Boc-Leu, D-Pro, Pro, D-Trp, Phe and benzhydrylamine resin are commercially available.

The t-butyloxycarbonyl protecting group (Boc) was introduced at D-Pro, Pro, Phe, D-Trp using 2- (t-butyloxycarbonyloxy imino) -2-2 phenylacetonitrile (Boc-ON). (M. Itoh, D. Hagiwara, T. Kamiya, Tetrahedron Letters 4393 (1975)). (2) Synthesis method (general description) The peptide was produced by the solid phase method according to Merrifield (RB Merrifield (1963), J. Amer. Chem. Soc. 85, 2149-2154). As a solid phase, this was first described by Monahan et al. (Biochem. Biophys. Res. Com. 48, 1100-1105) (1972) used benzhydrylamine resin.

In the solid phase synthesis of peptides, the terminal amino acid is first coupled to the resin. In the case of the benzhydrylamine resin, which carries the amino group as a functional group, this is done with the help of dicyclohexyl carbodiimide (DCC), which prevents the formation of an acid amide or. Free amine and free carboxyl group peptide bond.

Apart from the carboxyl group, the amino acid component must not carry any other groups which are reactive during the coupling. The t-butyloxycarbonyl group (Boc) is generally used to block the alpha-NH ₂ group. The entire peptide is then gradually built up on the first C-terminal amino acid coupled to the resin.

a. Since the commercially available benzhydrylamine resin is supplied in the hydrochloride form, it must first be converted to the free amine form. This is done with the help of triethylamine. Cl-H ₃ N ⁺ -BHA + Et ₃ NH ₂ N-BHA + Et ₃ NH ⁺ Cl-

b. The coupling can then be carried out with the first araino acid.

Boc-AS ₁ -OH + H ₂ N-BHA

Boc-As ₁ -NH-BHA

c. The temporary protecting group introduced with the first amino acid is now removed with methylene chloride / tri fluoroacetic acid. In order to trap the t-butyl cations formed during the reaction, which can undergo side reactions with nucleophilic centers of the growing peptide chain, 10% by volume of anisole is added to the reaction mixture.

Boc-As ₁ -NH-BH

TFA-H ⁺ ₂ As ₁ -NH-BHA + CO ₂ + CH ₂ C (CH ₃ ) ₂

d. After cleavage of the Boc protective group by tri fluoroacetic acid, the trifluoroacetyl salt of the bound amino acid is now available. In order to enable the next coupling, the ammonium group must be converted into the form of the free amine. This in turn happens with triethylamine. 3

TFA-H ₂ ⁺ As ₁ -NH-BHA

HAs ₁ NH-BHA + Et ₃ NH ⁺ Cl-

e. The free amine is then acidified with the next Boc-amino acid as in point b. coupled.

HAs ₁ -NH-BHA + Boc-As ₂ -OH

ΕOC-AS ₂ -AS ₁ -NH-BHA

f. Acetylation After coupling the Boc-protected amino acid with The free amino groups of the resin or the peptide chain are examined with the ninhydrin test according to Kaiser (Kaiser, E., Coloscott, RL, Bossinger, CD, Cook, PI (1970) Anal. Biochem. 34, 595-598) on a small resin sample, whether free amino groups are still detectable (blue color). If this is the case, the resin must either be subjected to a new coupling (double coupling) or acetylated in order to prevent the formation of peptides which lack an amino acid. These peptides would be very difficult to separate from the original product during final cleaning.

HAs ₁ -NH-BHA + Ac ₂ O → AcAs ₁ -NH-BHA + AcOH

G. Coupling of active esters to the growing peptide chain. If asparagine or glutamine are to be introduced into the amino acid sequence to be synthesized, dicyclohexylcarbodiimide is not suitable as a coupling reagent, since it causes nitrile formation on the carboxamide side function of the two amino acids. An alternative coupling method is the active ester method, in which the p-nitrophenyl ester of the relevant Boc-amino acid is cleaved aminolytically from the amine component to form the peptide bond. In the reaction, a catalytic amount of 1-hydroxybenzotriazole is added to accelerate the reaction rate,

Boc-As ₂ -ONp + H ₂ N-As ₁ -... → Boc-As ₂ -As ₁ -... + HONp

(3) Detailed description of the individual reaction steps The following are the individual steps involved with the resin in one and the same manual peptide synthesis apparatus were carried out in detail. The amounts are given for 5 g of BHA resin.

a. Neutralization (compare 2 a or 2 d)

Solvent / reagent Volume stirring time Type of operation (ml) (min)

CH ₂ C1 ₂ 40 3 wash

Et ₃ N / CH ₂ Cl ₂ = 9/1 50 3 neutralization

Et ₃ N / CH ₂ Cl ₂ = 9/1 50 10 neutralization

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

2-propanol (repeat 3 times) 40 3 wash

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

b. Coupling of the amino acid (formation of the peptide bond) (see point 2 b)

Solvent / reagent Volume stirring time Type of operation (ml) (min)

CH ₂ CV 40 3 wash

10 mmol Boc amino acid in

20ml CH ₂ C1 ₂ + 10 mmol DCC about 40 10 coupling in 20 ml CH ₂ C1 ₂

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash 2-Proρanol (repeat 3 times) 40 3 wash DMF (repeat 3 times) 40 3 wash

CH ₂ C1 ₂ 40 3 wash c. Boc protecting group is split off (see point 2 c)

Solvent / reagent Volume stirring time Type of operation (ml) (min)

CH ₂ C1 ₂ 40 3 wash

CH ₂ C1 ₂ / CF ₃ COOH / anisole = 50 3 deprotection 45/45/10

CH ₂ C1 ₂ / CF ₃ COOH / anisole = 50 20 deprotection 45/45/10

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

2-propanol (repeat 3 times) 40 3 wash

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

d. Acetylation of the resin (see point 2 f)

Solvent / reagent Volume stirring time Type of operation (ml) (min)

CH ₂ C1 ₂ 40 3 wash

CH ₂ C1 ₂ / Ac ₂ 0 / pyridine = 50 180 coupling 80/10/10

CH ₂ C1 ₂ / Ac ₂ O / pyridine = 50 3 wash 80/10/10

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

2-propanol (repeat 3 times) 40 3 wash

CH ₂ C1 ₂ (repeat 3 times) 40 3 wash

b. Cleavage of the peptide from the resin ^:

After the coupling steps had been completed, the peptide-resin was removed from the reaction vessel and dried under high vacuum for 24 hours. The resin (9.7 g) was treated in a Kel-F apparatus with 20 ml distilled anisole and 60 ml double distilled hydrogen fluoride for 1 hour at room temperature. The hydrogen fluoride was then distilled off in vacuo and the residue was dried in vacuo. This was then extracted with 3 x 100 ml of ethyl acetate in order to remove residual anisole. The crude peptide was dissolved with 3 × 100 ml of 5% acetic acid from the resin, the suspension was filtered and the filtrate was lyophilized. The dry, slightly yellowish residue weighed 3.48 g.

c.1. Column chromatography (gel filtration)

1.0 g of the raw material was dissolved in 25 ml of 5% acetic acid and applied to a column of Sephadex G-25 fine (5x85 cm) which had been equilibrated with 5% acetic acid. The flow rate was 24 ml / h, the fraction size 10ml. Fractions 157-175 contained the desired product and were lyophilized together. After drying, 260 mg of product were obtained.

d.2 column chromatography (distribution chromatography

Before applying the substance, the column (Sephadex G-25 fine, 2 x 71 cm) was prepared in the following way: First, the column material with the lower phase of the solvent system BuOH-Py-H ₂ O-AcOH = 7/3/10 / 0.1 equilibrated. The mobile phase was then exchanged for the upper phase of the same system by pumping 2 l of this phase through the column at a flow rate of 24 ml / h. On the column prepared in this way, 260 mg of peptide from the first column chromatography, dissolved in 20 ml of the upper phase, were applied and chromatographed at a flow rate of 24 ml / h. Fractions of 10 ml were collected. Fractions 67-83 contained the pure peptide. They were combined, diluted with the same volume of water, frozen and lyophilized. Yield: 180mg

This product was uniform in the analytical systems described below.

5. Analytics

HPLC: column: μ-Bondapak C 18 (Waters), 2.4 x 300mm mobile phase: 32% CH ₃ CN, 68% K-phosphate buffer

0.1 M, pH 3.0 flow rate: 1.5 ml / min detection: 210 nm In this system the substance had a retention time of 8.14 min. It delivered a single symmetrical peak without a shoulder with an integral of 98.1 area percent.

Thin layer chromatography:

Thin layer chromatography was performed on Merck HPTLC Plates (silica gel 60) performed; Running distance: 15 cm. The substance was uniform in the following fluid systems:

CHCl ₃ conc. NH ₃ -CH ₃ OH = 60/20/45 R _f : 0.4

EtOAc-Py-AcOH-H ₂ O = 5/5/1/3 R _f : 0.78 n-BuOH-EtOAc-AcOH-H ₂ O = 2/2/1/1 R _f : 0.02 n- BuOH-Py-AcOH-H ₂ O = 30/3/6/24 R _f : 0.59

2-PrOH-IN AcOH = 2/1 R _f : 0.03

Amino acid analysis:

Before analysis, the substance (1 mg) was dissolved in 1 ml of 4 M

Methanesulfonic acid containing 0.2% tryptamine 24

Hydrolyzed for hours in a sealed bomb tube at 120 ° C. The amino acid analysis was based on a

Biotronik-LC-2000 amino acid analyzer performed.

The following were found for the individual amino acids

Values:

Glu: 2.08; Pro: 2.00; Met: 1.05; Leu: 0.99; Phe: 1.01;

Arg: 1.06; Trp:; 1, 9.

The peptide content of the material was calculated for that

Triacetate, 86.7%.

Optical rotation:

The measurement was carried out on a polarimeter from Perkin-Elmer. For the measurement, 10 mg of substance in 1 ml

MeOH dissolved and measured at a layer thickness of 10 cm

(T: 25 °).

Result: ^α D ^{25 °} : -40.1 °

The other polypeptides according to the inventions can be prepared analogously. The polypeptides according to the invention can be converted in a conventional manner into salts with organic or inorganic acids, which can be used for pharmaceutical use and as intermediates for cleaning. The salts can be converted into the free polypeptides. Pharmaceutical preparations naturally only contain the polypeptides or their pharmaceutically acceptable salts. Examples of salts with organic acids are the acetates, fumarates, succinates, propionates, etc. Examples of salts with inorganic acids are chlorides, phosphates, sulfates, etc.

The results of the experiments on the antagonistic or agonistic activity of the compounds according to the invention are described below.

The guinea pig ileum, whose smooth muscles are stimulated to contract by this neurotransmitter (similar to acetylcholine), is considered a test model for Substance P, material and method

Guinea pig ileum was cut out after killing the animals, largely freed of mesenteries by pulling on the caudal end and freed of digestive products with Tyrode solution. Various ca.5 cm long sections were cut out from caudal to cranial and stored in Tyrode solution. The Tyrode solution was gassed with carbogen (95% O ₂ , 5%. CO ₂ ). The Tyrode solution contained the following substances per liter:

8 g NaCl; 2 g KCl; 1.3 g CaCl ₂ .2H ₂ O; 2.1 g MgCl ₂ .6H ₂ O; 10 g NaHCO ₃ ; 0.58 g NaH ₂ PO ₄ . lH ₂ O; 11 g D (+) glucose. The Tyrode solution was heated to 38 ° C. in an organ bath. Each preparation retained its full contractility for 4 to 7 hours. Gut sections of 2-3 cm were tied on both sides and hung in the organ bath. The contractions of the preparation were recorded under isotonic measuring conditions using a lever on a kymograph drum. A felt-tip pen, which was attached as a page pen, was used for writing. The load arm was 225 mm, the power arm 20 mm long. The preload was approx. 0.3 g, the paper feed 5 mm / min. The organ bath had a capacity of 10 ml. As a rule, the test substances were added in volumes of 100 μl and were immediately distributed in the organ bath due to the gassing. All added test substances were dissolved in Tyrode solution or with Tyrode solution from one strain! Diluted solution.

Results

Maximum contractions of the ileum sections were noted

Acetylcholine in one. Final concentration from 10 ^-7 to

10 ^-6 M and achieved with Substance P 2 x 10 ^-8 to 10 ^-7 M.

A dose-response relationship was shown for both substances (FIG. 1). The reaction was expressed as a% of maximum contraction (e.g., acetylcholine 10 ^-6 M or substance

P 10 ^-7 M) specified, D-Pro ² , D-Trp ^7-9 -SP was used approx. 1 min before the application of SP, measuring points are individual values for acetylcholine, for SP and D-Pro ² , D-Trp ^{7 -9} -SP

Mean values from 2 to 6 measurements,

Most intestinal preparations reacted to substance PK concentrations between 2 x 10 ^-9 M and 10 ^-7 M with a Contraction, D-Pro ² , D-Trp ^7-9- Substance P acted in one

Concentration of 10 ^-5 M as an antagonist of substance P,

An approximately 50% inhibition with D-Pro ² , D-Trp ^7-9 -SP (10 ^-5 M) was in the range between 7.5 x 10 ^-9 and 2 x 10 ^-8 M

Substance P found.

The antagonist was added to the organ bath about 1 min before the agonist. Fig. 2 is the original record of the response of a guinea pig ileum to irritation with Substance P (2 x 10 ^-9 M, 5 x 10 ^-9 M and 10 ^-8 M) and the proof of the antagonistic effect of

D-Pro ² , D-Trp ^7-9 -SP (10 ^-5 M) when applied before

Agonists, SP.

It should be noted that no application is agonistic

Activity of D-Pro ² , D-Trp ^7-9 -SP appears. Administration of D-Pro ² , D-Trp ^7-9 -SP simultaneously or after administration of Substance P also caused a decrease in the Substance P response or relaxation of the already contracted bowel preparation. Figure 3 is the original record of a guinea pig ileum's response to irritation with Substance P (10 ^-8 M). Comparison of the inhibitory effects of D-Pro ² , D-Trp ^7-9 -SP when applied before the substance

P administration and after reaching the excitation maximum. An agonistic activity of D-Pro ² , D-Trp ^7-9 -SP could not be found in the range between 10 ^-9 and 5 x 10 ^-5 M.

Claims

Claims:

1. Polypeptide of formula I and its salts

Arg-X-Lys-Pro-Gln-Gln-D-Trp-YD-Trp-Leu-Z-NH ₂ (I) where the symbols X, Y and Z have the following meanings: X: D-Pro, Dp- Cl-Phe Y: Phe, Ile Z: Met, Never.

2. Polypeptide according to claim 1 of the formula

D-Pro ² , D-Trp ⁷ ' ⁹ -Subs dance P (II) and their salts,

3. polypeptide of formula I according to claim 1, except D-Pro ² , D-Trp ^7.9 substance P, and their salts.

4. Process for the preparation of the polypeptides of the formula I and their salts Arg-X-Lys-Pro-Gln-Gln-D-Trp-YD-Trp-Leu-Z-NH ₂ (I) in which the symbols X, Y and Z have the following meanings:

X: D-Pro, D-p-Cl-Phe

Y: Phe, Ile

Z: Met, never. characterized in that one is known per se

How to couple the C-terminal amino acid and then the other amino acids step by step to a carrier resin and that Splits off carrier resin,

5. Use of the polypeptides and their pharmaceutically acceptable salts according to claim 1 to 4 in the control of, in particular chronic. Pain and hypertension.

6. Pharmaceutical preparations, characterized in that they contain one or more polypeptides and their pharmaceutically acceptable salts according to claim 1 to 4 together with conventional pharmaceutical carriers and / or excipients.