JPH0940699A - Polypeptide and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polypeptide, having a specific amino acid sequence, capable of manifesting glucagonlike activities and useful as a hyperglycemic agent, a suppressant for enterokinesis, a testing agent, etc., for endocrinic functions for treating hypoglycemia, the pretreatment for roentgenoscopy of an alimentary canal, testing, etc., of the endocrinic functions. SOLUTION: This new polypeptide has an amino acid sequence of the formula (X is an amino acid residue other than Met; Hse is homoserine or homoserine- lactone residue) and glucagonlike activities. The polypeptide is useful as an active ingredient of a hyperglycemic agent, a suppressant for enterokinesis, a testing agent and a diagnostic agent for endocrinic functions and used for treating hypoglycemia, the pretreatment for roentgenoscopy or endoscopy of an alimentary canal, testing, etc., of the endocrinic functions. The polypeptide is obtained as a glucagon-analog by replacing methionine residue at the 27th position in an amino acid sequence of glucagon with leucine residue and synthesizing a peptide chain having homoserine or homoserine-lactone at the C-terminal by using a solid-phase synthetic method, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide and its use. This polypeptide has a physiological activity similar to that of glucagon, and therefore, as a pharmaceutical ingredient, in particular, as a pretreatment for treatment of hypoglycemia, pretreatment of gastrointestinal X-ray examination or gastrointestinal endoscopy, and further examination of endocrine function. It can be used as an active ingredient of agents and diagnostic agents.

[0002]

2. Description of the Related Art Glucagon is a peptide hormone that has been crystallized by Staub et al. In 1953 from which amorphous porcine insulin was marketed.
In 1957, Bromer et al. Revealed that the primary amino acid structure of porcine glucagon was as follows (SEQ ID NO: 2), and it was subsequently discovered that the amino acid structure is the same in human and bovine. His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 5 10 15 Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 The physiological actions of glucagon are adrenaline-like glycogenolysis and gluconeogenesis. Promotion, suppression of glycogen synthesis, decomposition of neutral fat into free fatty acids and glycerol, promotion of electrolyte excretion, suppression of gastrointestinal motility and gastric acid secretion, suppression of gastrin secretion, promotion of secretion of growth hormone, promotion of secretion of catecholamines, etc. Are known.

Since glucagon has various physiological effects as described above, glucagon is used for the treatment of hypoglycemia by utilizing the action of promoting glycogenolysis and gluconeogenesis, and is digested by utilizing the action of suppressing gastrointestinal motility and gastric acid secretion. It is used as an active ingredient of endocrine function test agents and diagnostic agents by utilizing various actions exerted on the endocrine system as a tube motility inhibitor.

Position 27 in the amino acid sequence of glucagon is a methionine (Met) residue.
It is also known that a polypeptide having an amino acid structure converted to an eu) or norleucine (Nle) residue exhibits the same physiological activity as glucagon.

[0005]

SUMMARY OF THE INVENTION According to the prior art, glucagon must rely on synthetic methods or extraction from biological samples to obtain glucagon, and thus to prepare the substance inexpensively and in large quantities. It was extremely difficult.

The present invention has been made in view of the above problems in the prior art, and the main object of the present invention is to:
It is an object of the present invention to provide a polypeptide that can be mass-produced easily and can be provided at a low price, and that has a physiological activity equivalent to or higher than that of natural glucagon. An additional but important object of the present invention is to provide a drug containing the above-mentioned polypeptide having glucagon-like activity as an active ingredient, particularly a hypoglycemic agent, a gastrointestinal motility suppressor, an endocrine function test agent and a diagnostic agent. To provide.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that they have an amino acid sequence similar to glucagon, except that the 27th position is an amino acid residue other than methionine (Met) and glucagon is present. Is composed of 29 amino acids as described above, but homoserine or homoserine lactone residue at position 30 (hereinafter, when both homoserine and homoserine lactone residue are simply referred to as “Hse” residue) It has been found that the polypeptide further having the above) has physiological activity equivalent to or higher than that of natural glucagon, and thus the present invention has been basically completed.

Therefore, the peptide according to the present invention has an amino acid sequence (corresponding to SEQ ID NO: 1) of His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 5 10 15 Ser Arg Arg Ala Gln Asp Phe Val Gln Trp. Leu X Asn Thr Hse 20 25 30 (X means amino acid residue other than Met in the above sequence, Hse means homoserine or homoserine lactone residue) and has glucagon-like activity This peptide achieves the above-mentioned main purpose.

In the above-mentioned polypeptide according to the present invention, the reason that the 27th position of the amino acid sequence is defined as an amino acid residue other than Met is that the 27th position of Met is an obstacle to microproduction using fermentation or chemical synthesis. However, it is not suitable for large-scale production by the following method (when the produced fusion protein or inclusion body is treated with bromocyanine, cleavage also occurs at the Met site at position 27, which may lead to the desired physiological activity. I can't get the glucagon analog). The above-mentioned polypeptide according to the present invention, although not specifically described herein, was proposed by researchers of the applicant company with regard to another peptide hormone, motilin analog, and
7-22517 (JP-A-3-80096) In the same manner as the technique disclosed in Japanese Patent Laid-Open No. 3-80096, a DNA fragment corresponding to the leader peptide (both the amino acid residues at the N- and C-terminals thereof are Met residues) is added to the above-mentioned polypeptide. A polypeptide having the same amino acid sequence as the above (however, the C-terminal amino acid residue is Met, not Hse) is placed at the N-terminal side, while the C-terminal side is provided with a DNA fragment encoding a spacer peptide. Via a DNA corresponding to a plurality of polypeptides having the same amino acid sequence as the above-mentioned polypeptide (however, the amino acid residue at the C-terminal thereof is Met, not Hse, as in the above). The fragment is ligated in tandem, and the thus-formed and double-stranded DNA fragment is integrated into a vector such as a plasmid, and a microorganism such as a large-scale DNA is transformed with this gene recombinant vector. Bacteria or animal cells such as CHO cells are transformed, and this transformant is cultured to produce a fusion protein or an inclusion body. Cleavage at the site to separate individual monomer fragments (where the C-terminal Met residue is H
(changed to se residue) can be produced easily and extremely efficiently, and thus can be provided at low cost. The above-mentioned additional object according to the present invention is achieved by using the above-mentioned polypeptide as an active ingredient and formulating it by a method known per se.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail and specifically with reference to Production Examples and Pharmacological Activity Test Examples.
In the following, in the amino acid sequence of glucagon
A polypeptide in which the methionine (Met) residue at position 27 has been replaced with a leucine (Leu) residue ([Leu ²⁷ ] -glucagon-Hse)
However, using the fermentation method, 27th position is Met.
It should be noted that other glucagon-like polypeptides that are amino acid residues other than Leu or can be similarly prepared.

Synthesis of Production Example ([[Leu ²⁷ ] -Glucagon-Hse) Peptide Synthesizer manufactured by Perkin Elmer
(Model 431A) was used to synthesize a polypeptide ([[Leu ²⁷ ] -glucagon-Hse) having homoserine or homoserine lactone at the C-terminus by t-Boc solid phase synthesis method and Fmoc solid phase synthesis method as follows. I went to. (a) t-Boc solid-phase synthesis method Amino acid has α-amino group with t-butyloxycarbonyl (t-Boc)
What was protected with a group and the side chain active group was protected with a suitable protecting group was used. A commercially available resin, aminomethylated polystyl
Chemically synthesized N-Boc-0-benzyl-L-homoseryl-4- (oxymethyl) p that gives homoserine to the C-terminus in 0.5 mmol of ene ・ HCl
2.0 mmol of henylacetic asid was added to dicyclohexylcarbodiimide (DCC) / hydroxybenzotriazole (HOBt)
A peptide bond was formed by carrying out a condensation reaction with. After washing with methylene chloride, t-Boc was measured with TFA.
By removing the protecting group and repeating the above condensation reaction operation, amino acids were sequentially introduced from the carboxy terminal to synthesize. Regarding the polypeptide synthesized in this manner, trifluoromethanesulfonic acid low-high
Deprotection and resin removal were carried out by the method. That is, 500 mg of the synthesized resin-coated polypeptide was added to 100 μl of ethanedithiol, 400 μl of m-cresol, and 1.5 m of dimethyl sulfide.
l, TFA 2.5 ml and TFMSA 500 μl, and add 3 at 0 ° C.
The reaction was allowed to stir for a period of time. After precipitating with ethyl ether and filtering with a filter, thioanisole 1.0 ml, ethanedithiol 500 μl, TFA 5.0 ml and TFMSA 1.0 ml were added to the residue.
Was added, and the mixture was stirred at 0 ° C. for 1 hour and reacted to obtain a polypeptide (glucagon analog, [[Leu ²⁷ ] -glucagon-Hse) having a desired glucagon-like physiological activity.

(B) Fmoc solid-phase synthesis method Amino acid has 9-fluorenylmethyloxycarbony
The l (Fmoc) group was used for protection, and the side chain active group was protected with an appropriate protecting group. 4-hydroxymeth which is a commercially available resin
Chemically synthesized N- (9-f) that gives homoserine to the C-terminus of ylphenoxymethyl-copolystyrene-1% divinylbenzene resin 0.25 mmol using dicyclohexylcarbodiimide (DCC) / dimethylaminopyridine (DMAP) as an activator
luorenylmethoxycarbonyl) -O-trityl-L-homoserine 1.0
mmol was introduced. Then, the Fmoc protecting group was removed with piperidine, and 1.0 mmol of the amino acid was further added to [2- (1H-benzotri
azol-1-yl) -1,1,3,3-tetramethyluronium hexafluoroph
The peptide bond was formed by performing a condensation reaction with osphate] (HBTU). After washing with N-methylpyrrolidone (NMP), the above operation was repeated to successively introduce amino acids from the carboxy terminus to synthesize.
The polypeptide synthesized in this manner was deprotected and deresined by the trifluoroacetic acid (TFA) method.
That is, 0.75 g of phenol crystals, 250 μl of ethanedithiol, 500 μl of thioanisole, 500 μl of deionized water and 5.0 ml of TFA were added to 500 mg of the synthesized resin-coated polypeptide and reacted at room temperature for 3 hours with stirring. Next, ethyl ether was added, the mixture was filtered through a 3 μm filter, the residue was dissolved in TFA, precipitated in ethyl ether, and the precipitate was recovered by centrifugation. The resulting crude polypeptide was purified by reverse phase HPLC under the conditions shown below. Column: Waters μ-bondasphere 5 μm C1
8 100A (19 x 150 mm) Eluent: linear gradient of 75% to 40% acetonitrile in 0.012N hydrochloric acid, 35 minutes Flow rate: 7.0 ml / min The main peak by HPLC was collected and freeze-dried. When a part thereof was examined with a protein sequencer (model 476A) manufactured by Perkin-Elmer, it was confirmed that it was chemically synthesized correctly. In addition, FAB with a mass spectrometer (JMS-SX102A / DA7000) manufactured by JEOL Ltd.
Mass spectrometry confirmed that the theoretical molecular weight was structurally correct. The C-terminal of the polypeptide obtained in the above process was homoserine or homoserine lactone.

Biological Activity Test Example 1 (Measurement of Blood Glucose Elevating Activity In Vitro ) The polypeptide (glucagon analog) obtained in the above production example and synthetic human glucagon purchased from Peninsula Laboratories, Inc. were tested. As a substance, the amount of glucose released from cultured rat hepatocytes was measured and measured in vitro.
The blood glucose increasing activity at ro was evaluated. That is, rat free hepatocytes were first prepared by the collagenase perfusion method, and cultured in a plastic plate at 37 ° C. for 24 hours to give primary cultured hepatocytes. The culture medium of this primary culture hepatocytes was exchanged with a buffer solution containing the test substance and further cultured at 37 ° C, and the reaction was stopped 10 minutes, 20 minutes, and 30 minutes after the medium was exchanged, respectively, in the buffer solution. The amount of glucose released to the was measured by the glucose oxidase method. Figure 1 plots the relationship between various concentrations of glucagon analog and synthetic glucagon, and the amount of glucose released to the exchange medium (buffer) after 20 minutes of culture from medium exchange. As is clear from this figure, the glucagon analog according to the present invention has 10
^-10 - 10 have substantially the same glucose release effect and synthetic glucagon in the range of ^-7 M was found.

Biological activity test example 2 (Measurement of blood glucose elevation activity in vivo ) Polypeptide (glucagon analog) obtained in the above-mentioned production example and synthetic human glucagon purchased from Peptide Institute Co., Ltd. Was used as a test substance and was intramuscularly administered to anesthetized rats, and then blood was collected over time, and the glucose concentration in plasma was measured to evaluate the effect of increasing blood glucose in vivo . That is, rats anesthetized with sodium pentobarbital were first allowed to stand for 30 minutes to stabilize the blood glucose level, and then the test substance was dissolved in physiological saline at a concentration of 20 μg / ml.
It was administered intramuscularly at a rate of 5 μg / kg. After administration, 100 μl of heparin-added blood was collected from the jugular vein over time. Blood sampling is performed 10 minutes and 1 minute before the administration of the test substance and from the administration.
It was carried out after 5, 10, 15, 20, 30, 40, 50 and 60 minutes. Glucose level in plasma as blood glucose level Wako Pure Chemical Industries, Ltd.
The measurement was performed using a diagnostic glucose measurement kit purchased from the company. The result indicated by the amount of increase in blood glucose level after administration to the average value of the two measurements performed before administration of the test substance is
As shown in FIG. 2, it was revealed that the glucagon analog according to the present invention has a higher blood glucose-elevating activity in vivo than the native glucagon.

Biological activity test example 3 (Measurement of gastrointestinal motility inhibitory activity in vivo) Polypeptide (glucagon analog) obtained in the above-mentioned production example and "for injection" purchased from Yamanouchi Pharmaceutical Co., Ltd. Using glucagon derived from animal pancreas extracted from "Glucagon Novo" as a test substance, the contractile movement of the antrum of the stomach of the rat under anesthesia was measured using a strain gauge force transducer, and its inhibitory effect on contraction was evaluated. did. That is, a rat anesthetized by inhaling ethyl ether was subjected to a laparotomy along the midline to first expose the stomach. Strain gauge force transducer (Model F-061S made by Star Medical Co., Ltd.)
The serosal surface was sewn with surgical silk so that the contractile movement of the antrum of the antrum could be measured. After the abdomen was closed, the rat was fixed in a ball-man cage in a state of being laid down, and an indwelling needle for administering the test substance was placed in the tail vein. The transducer was connected to an organ movement analysis system (manufactured by Star Medical Co., Ltd.) to measure rat stomach movements. At the time when gastric motility of rats became noticeable after laparotomy, physiological saline 5
ml / kg was intravenously administered as a control without administration of the test substance.
After that, the test substance dissolved in physiological saline was added every 2 hours.
Rats were intravenously administered at a rate of 0, 100 or 500 μg / kg. Since gastric motility is recorded as a contraction waveform, the value obtained by integrating this waveform is taken as the amount of exercise, and the amount of exercise for 30 minutes before administration is calculated.
When the value converted per 5 minutes was set to 100%, the amount of exercise every 5 minutes after administration was calculated up to 30 minutes after administration. The results are shown in FIG. 3, and as is clear from this figure, the glucagon analogue according to the present invention is superior to the natural glucagon in the strength and duration of the gastrointestinal motility suppressing effect. There was found.

Formulation Example Aseptically distributed into vials so as to contain 1 mg of the glucagon analog according to the present invention, and the water is removed by freeze-drying, followed by sealing. This lyophilized powder is dissolved in distilled water for injection before use. In addition, human serum albumin and the like can be used as a stabilizer for the glucagon analog according to the present invention, as in general peptide preparations.

[0017]

Polypeptide according to the present invention, according to the present invention is, in vitro
Tested showed comparable bioactive and glucagon, in vivo
Tests show superior bioactivity. Probably, this polypeptide is a method developed by researchers of the applicant company.
[JP-A-3-80096 (Japanese Patent Publication No. 7-22517)] makes it possible to prepare easily and extremely efficiently. Therefore, the present invention provides glucagon which is useful as a main component of a drug.
It provides analog at a low price.

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Topology: Linear sequence type: Peptide Other information: Xaa ¹ : Amino acid residue other than Met (methionine), Xaa ² : Hse (homoserine or homoserine・ Lactone residue) Sequence His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 5 10 15 Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa ¹ Asn Thr Xaa ² 20 25 30 SEQ ID NO: 2 of the sequence Length: 29 Sequence type: Amino acid topology: Linear sequence type: Peptide sequence His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 5 10 15 Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25

[Brief description of drawings]

FIG. 1 Glucagon, a polypeptide according to the present invention.
3 is a graph showing the results of measuring the glucose release promoting action from rat primary cultured hepatocytes, which are exhibited by these substances, using analog and synthetic glucagon as test substances.

FIG. 2: Glucagon, a polypeptide according to the present invention.
FIG. 3 is a graph showing the results of measuring the blood glucose elevation effect by the glucose oxidase method when these substances were administered intramuscularly to anesthetized rats using analog and synthetic glucagon as test substances.

FIG. 3: Glucagon, a polypeptide according to the present invention.
FIG. 6 is a graph showing the results of examining the inhibitory effect of gastric antrum contraction movement when these substances were intravenously administered to anesthetized rats using analog and synthetic glucagon as test substances.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihiko Tagi 35, Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Stock Company, Sanwa Chemical Research Institute (72) Inventor, Koji Mineda, 35, Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Inside the Sanwa Chemical Research Institute (72) Inventor Tatsushi Maeda 35 Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Shares Inside the Sanwa Chemical Research Institute (72) Inventor Shoji Hoshino 35 Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Shares 3 Inside the Waseda Institute

Claims (2)

[Claims] 1. Amino acid sequence (corresponding to SEQ ID NO: 1)
Is His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 5 10 15 Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu X Asn Thr Hse 20 25 30 (where X is an amino acid residue other than Met in the above sequence) Group, and Hse is homoserine or homoserine lactone residue) and has glucagon-like activity. 2. A blood glucose elevation agent, a gastrointestinal motility suppressor, an endocrine function test agent, or an endocrine function diagnostic agent, which comprises the polypeptide according to claim 1 as an active ingredient.