JPS6037090B2 - Polypeptide and a pharmaceutical composition containing this polypeptide as a main component that suppresses the secretion of acidic gastric juice - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polypeptide having the property of suppressing the secretion of acidic gastric juice, and a pharmaceutical composition containing this polypeptide as a main component.

It has long been known that extracts of human urine inhibit the secretion of acidic gastric juices in warm-blooded animals, and that the two active ingredients can be separated in their pure form in West German Patent Application No. 2 and 3.
No. 59,564.

These two pure components are known as 8-urogastrone and y-urogastrone, respectively. The present invention is based on the discovery that oxygen degradation of 3- or y-urogastrone results in novel polypeptides and that the novel polypeptides and their reduction products are effective inhibitors of acidic gastric secretion. The object of the present invention is a white, water-soluble acidic polypeptide consisting of a single polypeptide chain with three internal disulfide linkages, having aspartic acid at the N-terminus in the dansylation, and having a molecular weight of about 550.
0 and the following physical properties: R-paper chromatography with n-butanol:acetic acid:pyridine:water (30:6:24:20): single spot RFO. 62, paper electrophoresis in acetic acid/formic acid at pH 2.1: Comets with a tip mobility of 1.67 for do-DNP-lysine, pH 8.9
Acrylamide gel electrophoresis in 0.1 molar Tris/HCl buffer: 0.7 to bromophenol
It has a single spot that moves towards the anode with a mobility of 7, and the following amino acid ratios when analyzing the hydrolyzate: aspartic acid (Asp) 7, serine (Ser) 3, glutamic acid (GIu) 4, proline (Pro)1, glycine (GIy)4, alanine (AIa)2, valine (Val
) 3, cysteine (Cys) 6, methionine (Met)
1, isoleucine (lie) 2, leucine (ju) 3,
Tyrosine (Tyr) 5, histidine (His) 2, lysine (L)S) 1 and arginine (~g) 2 were obtained, and the biological titer described below was within the range of 0.2 to lr/kg. The objective is to obtain a polypeptide or a reduction product in which a cystine residue is reduced to a cysteine residue.

The polypeptide in the present invention is produced by first incubating 81 or y-urogastrone with lysine-specific aminoendobeptidase to produce a polypeptide having 47 amino acid residues, and then producing a polypeptide having 47 amino acid residues. can be obtained by incubation with carboxybeptidase A, which cleaves one leucine residue from the C-terminus.

Therefore, the polypeptides of the present invention include ship p7, Ser3,
01u 4, Pro 1, GIy 4, AIa 2, V
a13, Cys 6, Met l, lie 2, Leu
4, TM5, has 47 amino acid residues in the ratio of His 2, L$1, Arg 2, forming a single polypeptide chain with 3 internal disulfide bonds, and has the following properties:
n-butanol:acetic acid:pyridine:water (30:6:24
:20): single spot with a mobility of 0.90 for phenylalanine, pH 2
．． Paper electrophoresis method with acetic acid/formic acid in 1: "Comets" with a tip mobility of 1.58 for go-DNP-lysine
Acrylamide gel haze in 0.1 molar Tris/HCl buffer at pH 8.9: a single spot migrating in the opposite direction with a mobility of 0.81 relative to bromophenol. , 0.2 to 1 Buddha's evening/k9 as described below
Polypeptides exhibiting biological potency within the range of carboxybef in aqueous media.

The desired polypeptide is isolated from the reaction mixture by exposure to the action of Tidase A, and then, if reduced products are desired, the polypeptide thus obtained is treated with reducing agents customary in peptide chemistry to reduce disulfide linkages, For example, it can be obtained by reduction with a thiol, such as mercaptoethanol or dithiothreitol. Carboxybef starting polypeptide.

Exposure to Tidase A can be carried out simply with the enzyme dissolved in the aqueous medium or with the enzyme bound to a support which may be soluble or insoluble in the aqueous medium. The rate and extent of reaction of the starting polypeptide with the enzyme depends on the enzyme/substrate ratio, the pH and temperature of the medium, the concentration of the substrate and the incubation time.

Generally, of course, the time required to cleave the substrate is reduced by increasing the concentration of the substrate or the enzyme/substrate ratio. Similarly, changes in the pH or temperature of the medium will affect the separation time. The progress of the reaction can be monitored by testing the reaction medium with an amino acid autoanalyzer to determine the amount of cleaved leucine or by screening the sample with 1-dimethyl-aminonaphthalene-5-sulfonyl chloride. ration (ship nsyiation) [Ha
roey) ``Biochemical Journal'' (Bioc
hemJ. ), 1970, No. 119, p. 805] and can be tracked by roughly estimating the amount of N-dansyl-L-leucine produced. In this way, the effect obtained by arbitrarily changing the reaction conditions can be measured. Although reactions can occur over a wide range of enzyme/substrate ratios, a minimum enzyme/substrate ratio of 1 part enzyme to 100 parts mature substrate is preferred.

The reaction is carried out at a medium pH value of 5-10 and a temperature of 1000-45°C.
occurs in Preferred conditions for obtaining the desired cleavage of the starting polypeptide in a relatively short period of time, e.g. 1 hour, are:
starting polypeptide concentration of 9/' or more of 1, pH 7-9,
The temperature is 2,500 to 40 qo and the enzyme/substrate ratio is from 10 parts of substrate to 1 part of enzyme to 1 part of substrate to 1 part of enzyme. Although the polypeptides of the present invention can be isolated from the reaction mixture by any conventional method for isolating polypeptides, it is particularly preferred to use gel chromatography. The aqueous medium remaining after cleavage of the leucine from the starting polypeptide is passed through a column of cross-linked dextran gel or polyacrylamide gel, and the column is loaded with components that will volatilize during freeze-drying under high vacuum, such as acetic acid with a pH of 7.2. By elution with a buffer consisting of an aqueous ammonium solution, the desired product can be obtained, which is separated by freeze-drying of the port brood fluid. If a reduced product is desired, the polypeptide of the present invention may be prepared at a pH of 7 to 1.
Preferably, the reduction is carried out at 0.0 in an aqueous buffer and in the exclusion of enzymes and light.

The products can again be separated using gel chromatography. As described above, the starting polypeptide can be obtained from 81 or y-urogastrone by isothermal maintenance of 81 or y-urogastrone with a lysine-specific aminoendobeptidase. Particularly suitable enzymes of this type are 'Naratake' (Armaria
It is an AM protease obtained from the mature fruiting body of Cormorant ellea. Gastron concentration 1 to 9/skin and above, pH 6 to 9, temperature 20oC to 4
0o○ and enzyme/substrate ratio of 1:8 to 1:100. When the salt isotherm is complete, the desired biologically active polypeptide is separated by gel chromatography using cross-linked dextran gels or polyacrylamide gels. As stated above, the polypeptides and reduced products thereof according to the present invention are useful inhibitors of acidic gastric secretion in bleeding animals.

This property is evidenced by the effect of suppressing the secretion of acidic gastric juice in dogs with Idenheim's microgastric glands whose gastric secretion was stimulated with histamine. This test is used to determine the biological potency of the product, which has a small Idenheim stomach and whose gastric acid secretion is stimulated to 60-80% of the maximum standard of secretion by infusion of histamine. It is defined as the amount of product that inhibits gastric acid secretion by 50-70% when administered by intravenous injection to dogs. Although some variation in the biological potency of a particular sample is seen and produced in different dogs or when measured on different occasions, the results are best expressed as a dosage range. The biological effect of suppressing the secretion of acidic gastric juices is useful in the treatment of duodenal hyperplasia, and no significant toxic effects were observed under the test conditions. The polypeptide according to the present invention has a strong hot spring characteristic in the standard test. When used to suppress acidic gastric secretion in hyperemic animals, a wide range of dosages e.g. 0.05-1
0/k9 can be used, depending on the suppression situation and degree desired.

The dosage can be administered by injection, especially intravenous or subcutaneous injection, or by intravenous infusion. The effects of a single intravenous injection last approximately 11 hours, and the effects of the infusion last approximately 11 hours after completion of the injection. Therefore, keeping acidity at a low level requires repeated dosing or injections of devo formulations in which the active ingredient is released gradually over a more delayed period. For human use, a typical single dose administered by injection or infusion is 5 to 500 French yen per person. The polypeptide or reduction product thereof according to the invention can be administered in the form of a pharmaceutical composition, and therefore, according to the invention, the polypeptide or reduction product thereof as described above and a pharmaceutically acceptable diluent or A pharmaceutical composition comprising excipients is obtained.

Preferred compositions are those suitable for parenteral administration, such as sterile injectable solutions or suspensions, and sterile injectable devo or slow release formulations. Injectable solutions or suspensions may contain from 0.5 to 500 mg/di, more dilute solutions are administered by injection, and in injectable depot or slow release formulations. may contain up to 2 to 9 parts of active ingredient per dose. Particularly preferred sterile injectable solutions may be provided with isotropic saline or isoperitoneal dextrose, optionally buffered to a pH of 5 to 9, and may contain 1 to 100 ph. The sterile injectable compositions described above may be prepared and stored as such, or immediately prior to use, the sterile component may be enclosed in a known quantity of the sterile component, e.g. It can also be prepared by adding a sterile medium, such as water, in the evening/day.

The known amount of sterile ingredients may include sufficient sterile dextrose or sodium chloride to provide an isoperitoneal solution after dilution with a sterile medium. The invention will now be explained in more detail on the basis of non-limiting examples, in which a special chromatography material according to the manufacturer's registered trademark can be obtained as follows: Acrylamide gel: Biogel (Bio Saddle 1) P6, Bio
Manufactured by RadLa's es, Richmond, Ca.
Porous cross-linked dextran gel: Sephadex G-25, Pha
Dish made by acia Fine Chemicals, Upps
ala, Sweden, carboxybeptidase A;
Power Ruboxybeptidase A-DFP, Woringt
onBiochemica I Corporation, Freehold, N.C. J, based in USA.

For each method, including the use of a chromatography column, the acid solution was purified using Ultrorac LKB7.
000 fraction collector 1 (LKB1nstru
Manufactured by menGLW, Croydon, Smrey, U.S.
K. These fractions were collected in fractions consisting of a certain number of drops using a U. The peptide materials were analytically tested by measuring V absorption. The material was then collected into fractions and the material was analyzed for biological activity as follows: Dogs had isolated denervation stores and histamine was injected subcutaneously to increase the maximal ratio to about 60-80. stimulated secretion of gastric juice up to % [usually 0.0% per hour].
Injection of 600 ml of solution of histamine (as base) in Z.48 is sufficient]. A known amount of the test material was dissolved in isoperated saline and the material was administered as a single intravenous injection when the dog secreted gastric juices at a uniform rate. Suppression of gastric juice secretion was shown for certain doses. Biological potency can be determined from the results obtained at several doses.

Amino acid analysis was performed using a Locane amino acid analyzer [Locane manufactured by Lgensha, 24mpero Gate
, Mountain SW. It was carried out using ``Mannen''.

All column chromatography was performed at 4°C and all buffers were saturated with toluene. Example 1 A polypeptide (216 Sanyu) consisting of a single polypeptide chain with three internal dizasulfide bonds, having 47 amino acid residues in the following proportions, and exhibiting the following properties was prepared using 0.1 mole of ammonium bicarbonate. Dissolve carboxybeptidase A (carboxybef) in 0.1 molar ammonium bicarbonate solution (4 meth) (116 f.).

A solution of Tidase A-DFP 4) was added. The mixture was incubated at 370°C for 3 hours and the sample was analyzed for amino acids. Furthermore, an enzyme (12 pm) was added, the temperature was maintained for an additional 3 hours, and the sample was analyzed for amino acids. Each assay indicated the presence of free leucine in the reaction mixture, with the second assay indicating an amount equal to 1/4 of the leucine content of the starting polypeptide. That is, the enzyme cleaves one leucine residue from the C-terminus of the starting polypeptide, thus making it similar to the starting polypeptide in its amino acid ratio only in that it has three leucine residues instead of four in the starting polypeptide. It is shown that polypeptides with 46 different amino acid residues are generated. A sample of the polypeptide solution thus obtained produced a biological activity with an inhibition rate of 42% in a single measurement at a dose equal to 0.6 xylene/kg of starting material. The polyJ peptide used as starting material was obtained as follows: 0.1 molar ammonium bicarbonate buffer (200 molar) at pH 8.2 and 0.01 molar magnesium chloride solution (50 molar). A solution of 8-urogastrone (9 of 1.0) in a mixture with AM in water (200 l)
The mixture was incubated for 1 hour at 37q0 with protease (60r).

The resulting solution was applied to a column (35xl) of porous cross-linked dextran gel (Sephadex G-25).
The column was developed with 0.05 molar ammonium acetate at a flow rate of 8 k/hr. Collect the 1' fractions and add the peptide material to fractions 12-20 and 38.
Detected at ~52.

The material in fractions 38-52 was biologically inert, and the samples were hydrolyzed with acid, base or oylamine/peptidase.

Analysis of the resulting solution revealed that the original peptides were in the following proportions: GIe1, Leu 1, Lys 1, Trp 2
, Arg 1 was shown to contain amino acids. Dansylation showed the presence of lysine at the N-terminus.
The material from fractions 12-20 is biologically active, and when these fractions are combined and hydrophilized,
A white, water-soluble acidic polypeptide was produced with the following properties: 1 Biological activity in a single measurement: 30% inhibition at a dose equal to 0.7 buds/k9 of starting material; 2
Amino acid ratio when analyzing hydrolyzate: Asp 7, Se
r3, Glu4, Pro1, GIy4, AIa
2, Va1 3, Cys 6, Met l, lie2,
Leu 4, T 5, His 2, Lys 1, Ar
g23 n-butanol:acetic acid:pyridiso:water (30:
A single spot with a mobility of 0.90 for phenylalanine on paper chromatography at 6:24:20).

4 Acetic acid/formic acid at pH 2.1 (20% formic acid, 80% acetic acid
by diluted with water) by paper electrophoresis.
Comets with a tip mobility of 1.58 for P-lysine.

5 Promophenol flu-
A single spot with a mobility of 0.81.

Dansylation of the product showed the presence of aspartic acid at the N-terminus.

Example 2 A polypeptide (1.5 x 9) consisting of a single polypeptide chain with 3 internal disulfide bonds, having 47 amino acid residues in the following proportions and exhibiting the properties described in Example 1: moles of ammonium bicarbonate (200 French)
0.1 molar ammonium bicarbonate solution (60
A solution of carboxybeptidase A (carboxybeptidase A-DFP60 muta) in 2000 was added.

The mixture was kept at temperature for 2 hours at 3 o, then further enzyme (60 minutes) was added, and kept at temperature for 2 more hours. The resulting solution was mixed with a porous polyacrylamide gel [Biogel P6 (
Bio bag 1-P6): 200-400 mesh] column (100 x 0.9 skin). The column was developed with the same solvent and fractions 1 and 1 were collected. Biologically active material was detected in fractions 27-34, which, when combined and hydrophilized, yielded a white, water-soluble acidic polypeptide with the following properties: 1 Amino acids upon analysis of the hydrolyzate Ratio: Asp 7, Ser 3, Glu 4
, Pro l, GIy4, AIa 2, Va1 3, C
ys 6, Met l, lie2, Leu 3, TM
5, His 2, Lys l, Arg2.

2 n-butanol:acetic acid:pyridine:water (30:6:
24:20) single spot RFO. 62.

3 Acetic acid/formic acid at pH 2.1 (formic acid 20%, acetic acid 80%
Please use the paper electrophoresis method (soak the tabletop in water overnight).
A comet with a tip mobility of 1.67 for P-lysine.

4 A single spot with a mobility of 0.77 for promophenol blue in acrylamide gel electrophoresis in 0.1 molar Tris-HCl buffer at pH 8.9.

Example 3 The polypeptide obtained in Example 2 (300 ml) was dissolved in water and urea (25 ml), ethylenediaminetetra-acetic acid (9 of 0.5) and 1.5 molar Tris-HCl buffer (pH 8.6). 25 Buddhas) were added.

The solution was diluted 60 flasks with water, washed with oxygen-free nitrogen, and mercaptoethanol (1 flask) was added. The resulting solution was kept in the refrigerator for 3 hours and then divided into two equal parts. The first aliquot was divided into a column (1
00x0.9) and the column was developed with 0.05M ammonium acetate, the cystine residues were reduced to cysteine residues.

A solution of tide was formed. The second aliquot was treated with a solution of 14C iodoacetamide (3.6 μg) in water (10 μg) for 30 minutes at room temperature.

The resulting solution was applied to a column (35 x 1 c) of porous crosslinked dextran gel (Sephadex G-25) and the column was developed with 0.05M ammonium acetate. A single radioactive product was detected, which yielded carpoxymethylcysteine rather than cystine in the amino acid analysis of the acidic hydrolyzate, and therefore reaction with mercaptoethanol suggested that cystine residues were reduced to cysteine residues. The results show that the polypeptides were produced using polypeptides. Example 4 A polypeptide as described in Example 1 or 2, or its reduction product in which the original cystine residue has been reduced to cysteine, is dissolved in a pyrogen-free 5% w/v dextrose solution to give the final The concentration was set to 40 Mta/gu'.

Pour this solution into a vial using a sterile membrane filtration system, e.g.
Each sample was dispersed into 2.5' aliquots through a French Mmipore filter. The contents of each vial were then made hydrophilic and the vials were capped and sealed under aseptic conditions. Vials containing the sterile mixture of polypeptide and dextrose were stored at 4°C. Example 5 To a vial prepared as described in Example 4, immediately before use, add 2.5 g/' of sterile water to a sterile injection solution of 40 m/' of polypeptide in 5% w/v dextrose solution. did.

Example 6 A polypeptide as described in Example 1 or 2, or its reduction product in which the original cystine residue has been reduced to cysteine (10 females), is dissolved in pyrogen-free water (5 females); The solution was filtered into ampoules through a sterile membrane filtration system, such as a 0.22 h French Millipol filter.

Each ampoule received 0.5 desk. The contents of each ampoule were then made hydrophilic and the ampoules were sealed under aseptic conditions. Ampules each containing 100 μm of sterile polypeptide were kept at -2000. Example 7 The ampoule contents prepared as described in Example 6 were
The polypeptides were dissolved in a sterile, pyrogen-free 5% w/v dextrose solution to give a solution containing 1 to 5 polypeptides in a 5% w/v dextrose solution.

This solution is suitable for administration by injection. If a solution suitable for injection was required, the contents of the ampoule were dissolved in a sterile, pyrogen-free 5% w/v dextrose solution to give a solution containing polypeptide 5-50.

Additionally, the 5% w/v dextrose solution can be replaced by isocular saline.

Claims (1)

[Scope of Claims] 1. A white water-soluble acidic polypeptide consisting of a single polypeptide chain having three internal disulfide bonds, having aspartic acid at the N-terminus in the dancillation, and having a molecular weight of approximately 5500. and the following physical properties: n-
Butanol: Acetic acid: Pyridine: Water (30:6:24:2
Paper chromatography at 0): single spot R_F
0.62, paper electrophoresis in acetic acid/formic acid at pH 2.1: "Comets" with a tip mobility of 1.67 for ε-DNP-lysine, 0.1 molar Tris/formic acid at pH 8.9. Acrylamide gel electrophoresis in hydrochloric acid buffer: has a single spot migrating towards the anode with a mobility of 0.77 for bromophenol blue, with the following amino acid ratio: aspartic acid 7 for analysis of hydrolysates. , serine 3, glutamic acid 4, proline 1, glycine 4, alanine 2, valine 3, cysteine 6, methionine 1, isoleucine 2, leucine 3, tyrosine 5, histidine 2, lysine 1 and arginine 2 are obtained. Value is 0
．． A polypeptide represented by the formula ▲ (numerical formula, chemical formula, table, etc. available), or a reduction product in which its cystine residue is reduced to cysteine residue, within the range of 2 to 1 μg/kg. 2 A white water-soluble acidic polypeptide consisting of a single polypeptide chain with three internal disulfide bonds, an aspartic acid at the N-terminus in the dancillation, a molecular weight of approximately 5500, and the following physical property: n-
Butanol: Acetic acid: Pyridine: Water (30:6:24:2
Paper chromatography at 0): single spot R_F
0.62, paper electrophoresis in acetic acid/formic acid at pH 2.1: "Comets" with a tip mobility of 1.67 for ε-DNP-lysine, 0.1 molar Tris/formic acid at pH 8.9. Acrylamide gel electrophoresis in hydrochloric acid buffer: has a single spot migrating towards the anode with a mobility of 0.77 for promophenol blue, and in the analysis of the hydrolyzate the following amino acids: aspartic acid 7 , serine 3, glutamic acid 4, proline 1, glycine 4, alanine 2, valine 3, cysteine 6, methionine 1, isoleucine 2, leucine 3, tyrosine 5, histidine 2, lysine 1 and arginine 2 are obtained. Value is 0
．． A polypeptide represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or its reduction product in which cysteine residues are reduced to cysteine residues within the range of 2 to 1 μg/kg, and pharmaceutical A pharmaceutical composition for inhibiting the secretion of acidic gastric juices, comprising a diluent or excipient acceptable to . 3. The pharmaceutical composition according to claim 2, which is in a form suitable for parenteral administration. 4. The pharmaceutical composition according to claim 3, which is a sterile injectable solution or suspension. 5 polypeptide or its reduction product from 0.5 to 500
A pharmaceutical composition according to claim 4 containing μg/ml. 6. A pharmaceutical composition according to claim 3, which is in a sterile injectable depot or slow release formulation. 7. A pharmaceutical composition according to claim 6, comprising up to 2 mg of the polypeptide or its reduction product.