JPH07278185A - Peptide having calcium channel blocking action - Google Patents

Abstract

PURPOSE:To provide a new peptide having a specific amino acid sequence same as that of the partial fragment of a trypsin inhibitor separated and purified from human urine, effective for depressing blood pressure by the action to suppress the flow of calcium into cell and useful as a hypotensor, etc. CONSTITUTION:This new peptide has amino acid sequences of formula I and formula II same as those of the partial fragment of a trypsin inhibitor in human urine composed of a glycoprotein having a molecular weight of about 67,000 and separated and purified from human urine, exhibits calcium channel blocking action, depresses blood pressure by the action to suppress the flow of calcium into cells and is useful as a hypotensor. The peptide can be produced by successively bonding amino acids toward the N-terminal of a peptide by a solid-phase synthesis using a solid carrier such as styrene-divinylbenzene copolymer, treating the obtained protected peptide with trifluoromethanesulfonic acid, etc., to effect the deprotection and the removal of the peptide from the solid phase and purifying by a preparative high-performance liquid chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide for the prevention and / or treatment of diseases involving calcium channel block. The peptide has a blood pressure lowering action through this calcium channel blocking action.

[0002]

2. Description of the Related Art Molecular weight of about 6 isolated and purified from human urine
7,000 glycoproteins are known as human urinary trypsin inhibitor (UTI). This human urinary trypsin inhibitor has anti-pancreatic action and anti-shock action, and is widely used clinically as an indication for acute pancreatitis, acute exacerbation of chronic recurrent pancreatitis and acute circulatory failure. It is known that human urinary trypsin inhibitor has an inhibitory effect on blood coagulation factor Xa, in which the human urinary trypsin inhibitor itself has almost no activity in its partial fragment of the amino acid sequence (JP-A-5-84083). ).

[0003]

[Problems to be Solved by the Invention] In recent years, symptomatic hypertension and essential hypertension have been increasing more and more. Currently used anti-pressure agents include calcium channel blockers, ACE inhibitors,
There are alpha blockers, beta blockers, diuretics, vasodilators, and central sympathetic depressants. Among them, calcium blockers are most widely used, but they have some problems such as side effects and teratogenic effects. An object of the present invention is to provide a safer calcium blocker with less side effects, which is closer to the substance in the living body.

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted repeated studies for the purpose of developing safer calcium blockers, and found that a peptide having the same amino acid sequence as a partial fragment of human urinary trypsin inhibitor. The present invention was completed by discovering that Calcium has a calcium blocking action.

The present invention is a peptide represented by the formula (1): H-Asn-Leu-Pro-Val-Ile-Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile-Gln-Leu-OH (1) Consists of. Further, the present invention comprises a peptide represented by the formula (2) H-Arg-Ala-Phe-OH (2). Further, the present invention provides the above formula
It comprises a blood pressure lowering agent comprising the peptide represented by (1) or (2). The peptides (1) and (2) of the present invention are novel and can be obtained by chemical synthesis or by degrading a natural product with an enzyme.

The synthesis of these peptides is carried out by a method usually used in peptide synthesis, for example, a solid phase synthesis method using the t-Boc method or F-moc method or a stepwise extension method,
Although it is carried out by a liquid phase synthesis method such as a fragment condensation method, it is easy to operate by solid phase synthesis using a peptide synthesizer.

The peptide of the present invention can be produced by the solid phase synthesis method, for example, a polymer which is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer, an amino acid corresponding to the C-terminal of the desired peptide or an amide thereof. Are bonded via an α-COO- group or an α-CONH- group obtained by removing a hydrogen atom from an α-COOH or the like or an α-CONH ₂ group, respectively, which are then bound to the amino acid or its amide. Operation for binding the corresponding amino acid or peptide fragment toward the N-terminal direction of the corresponding peptide after protecting functional groups such as α-amino group other than α-COOH group possessed by the amino acid or peptide fragment, and condensing And an amino group forming a peptide bond such as an α-amino group in the bound amino acid or peptide fragment By repeating the operation of removing the protecting group sequentially, the peptide chain is extended to form a peptide chain corresponding to the target peptide, and then the peptide chain is released from the polymer, and the protected functional group is released. It is carried out by removing the protecting group from the group to obtain the peptide of interest and then purifying it.

The peptide of the present invention can be obtained by subjecting a trypsin inhibitor obtained by purification from urine or the like and an inter-α-trypsin inhibitor obtained by purification from plasma to a limited decomposition with an enzyme or the like. You can also Furthermore, the antihypertensive agent of the present invention is a substance obtained by subjecting the above-mentioned novel peptide to chemical decoration such as sugar addition, alkylation, oxidation, reduction, and water splitting, or a salt with a pharmacologically acceptable acid or base. The substance and the like obtained by forming
The above-mentioned peptide is usually mixed with a pharmaceutically acceptable auxiliary component to give a pharmaceutical composition.

The dose of the pharmaceutical composition of the present invention is appropriately selected depending on the content of the active ingredient, the condition of the patient to be treated, the age, the body weight and the like. The pharmaceutical composition of the present invention is preferably administered in an effective amount of 0.1 to 1000 mg / kg, more preferably 0.2 to 100 mg / kg. In addition, the pharmaceutical composition of the present invention may be administered orally, intramuscularly, intraperitoneally, intradermally, subcutaneously, intravenously, intraarterially, rectally, or even intranasally depending on the patient's condition. Although it can be used for mucosal absorption and the like, it is preferably used by a method of intravenous administration.

[0010]

EXAMPLES Next, the present invention will be described more specifically by showing examples. Example 1 A polypeptide of the formula (1) was synthesized using a peptide synthesizer. Using Biosystems 430A PAM leucine, t-Boc-L-Leu 0.5 mmol, amino acids were sequentially bound toward the N-terminal of the peptide. In the binding reaction, t-Boc asparagine, t-Boc leucine and t-B manufactured by Applied Biosystems, Inc. of the United States are used.
oc proline, t-Boc isoleucine, t-Boc valine, t-Boc arginine, t-Boc glycine, t
-Boc cysteine, t-Boc alanine, t-Boc
Phenylalanine and t-Boc glutamine were used, respectively.

The obtained protected peptide was used as a TFM manufactured by ABI.
Deprotection and removal from the solid phase were performed using SA and trifluoromethanesulfonic acid. The Beckman preparative high performance liquid chromatograph for the crude product (column: Tosoh Corp.,
It was purified by TSK gel ODS 22FX, 260 mm). The obtained purified peptide was applied to a Beckman high performance liquid chromatograph [column: Tosoh Corp., TSK gel ODS 80TM CTR, mobile phase: acetonitrile / water mixed solvent containing 0.1% by volume of trifluoroacetic acid (acetonitrile concentration: (Changed from 5% by volume to 50% by volume in 30 minutes)]],
A single peak was shown at 210 nm at 27.1 minutes (Figure 1). This peptide is an amino acid analyzer: Hitachi 835-
Consistent with the composition analyzed by type 50. Furthermore, it was also confirmed that the amino acid sequences were matched by a protein sequencer (ADI 476A).

Example 2 The peptide of formula (1) and the intracellular calcium influx inhibitory action were measured. In human neutrophil cells, it was examined whether or not the above peptides suppress the increase of intracellular calcium concentration in response to stimulation with endotoxin (LPS). Human venous blood collected with a syringe whose inner wall is wet with heparin 1
Physiological saline was added to 0 ml, mixed and left at room temperature for 30 minutes. The upper layer of plasma and white blood cells was taken and gently layered on a centrifuge tube containing a Ficoll density gradient solution in advance. This was centrifuged for 5 minutes, and the platelet layer separated by the density gradient was aspirated. Further, the white blood cell layer was taken, the polynuclear cell layer separated by the Ficoll density gradient solution was taken, washed with physiological saline, and then suspended and suspended in Hanks solution.

This suspension suspension is placed on a Flexipem Disc, which is a slide glass surrounded so that the liquid does not get wet.
It was adjusted to be 0 ⁶ . The medium of this cell liquid was replaced with a serum-free medium, and a fluorescent dye Fura 2-AM that specifically binds to calcium ions was added to them, and the cells were cultured in the CO ₂ incubator for 60 minutes in the dark. Then, the above peptide was added to the cell culture medium to a concentration of 100 μM. Then, LPS was added to the cell liquid medium to a concentration of 100 μg / ml. Using a Nicon TMD fluorescence microscope (with epifluorescence device) (manufactured by Nicon), changes in calcium concentration of arbitrarily selected single cells were measured with fluorescence intensities of 340 nm and 380 nm (F340 / F380). On the other hand, without adding the peptide to the above-mentioned cell fluid,
The calcium concentration was measured in the same manner as in the case of adding only the stimulation by, and this was used as a control.

As a result, in the cells to which the peptide of formula (1) was added, the increase of intracellular calcium concentration was almost completely suppressed by LPS stimulation. Next, the peptide of formula (2), which is a further partial fragment of the peptide of formula (1), was synthesized in the same manner as in Example 1, and the intracellular calcium influx inhibitory effect was also measured. The peptide of (2) has the formula (1)
The same inhibitory effect as that of the peptide was obtained. Combined formula (2)
When the peptide of Example 1 was analyzed by high performance chromatography (the analysis conditions were the same as those of the peptide of the above formula (1)), a single peak was shown at 19.1 minutes (FIG. 2).

FIG. 3 shows the intracellular calcium influx suppressing effect of each peptide on LPS stimulation. (A) is a single cell stimulated with LPS but no peptide was added (control), and (B) is LPS in 4 single cells when the peptide of formula (1) was added. Intracellular calcium concentration by stimulation, (C) is the same formula
It is a result in three single cells when the peptide of (2) is added. In addition, in the same manner as in the above human neutrophil cells, leukemia cell lines U-937, H
The effect of suppressing calcium influx on LPS stimulation was measured in each of L60 and normal human fibroblasts, and similar results were obtained.

Example 3 Confirmation of antihypertensive effect on animals The peptide of formula (1) was administered to 5 male SHR rats weighing 7 to 10 weeks and weighing 140 to 200 g to examine the antihypertensive effect. 2 mg of the peptide of formula (1) was administered from the rat tail vein under non-anesthesia, and blood pressure and heart rate were observed over time by a non-invasive method, and compared with two control groups. As shown in FIG. 4, in the group to which the peptide was administered, the blood pressure decreased immediately after the administration, reached the lowest level in about 20 minutes, and recovered to an average of 80% after 60 minutes. Under anesthesia (Nembutal anesthesia, intraperitoneal administration 0.05m
In g), the same examination as above was performed with one control group and two administration groups. The same hypotensive effect was observed under anesthesia as under non-anesthesia (Fig. 5). Heart rate did not change significantly under anesthesia and non-anesthesia. In addition, a dose of 0.5
The hypotensive effect was examined 10 minutes after administration of 1, 2 and 5 mg. A hypotensive effect of about 30 mmHg was shown at a dose of 1 mg or more (Fig. 6).

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, a novel peptide having an action of suppressing intracellular calcium influx is provided. INDUSTRIAL APPLICABILITY The novel peptide of the present invention lowers blood pressure through its intracellular calcium influx suppressing action and is therefore useful as a hypotensive agent.

[0018]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: None Sequence: Asn-Leu-Pro-Val-Ile-Arg-Gly-Pro-Cys-Arg- Ala-Phe-Ile-Gln-Leu 1 5 10 15

[Brief description of drawings]

1 shows a high performance liquid chromatogram of the peptide of formula (1).

FIG. 2 shows a high performance liquid chromatogram of the peptide of formula (2).

FIG. 3 is a graph showing the intracellular calcium influx suppressing effect of peptides on LPS stimulation.

FIG. 4 is a graph showing the blood pressure lowering action of the peptide of formula (1).

FIG. 5 is a graph showing the hypotensive action of the peptide of formula (1) under anesthesia.

FIG. 6 is a graph showing the degree of hypotension 10 minutes after the administration of the peptide of formula (1).

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area C07K 5/09 8318-4H

Claims (3)

[Claims] 1. A peptide represented by the formula (1): H-Asn-Leu-Pro-Val-Ile-Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile-Gln-Leu-OH (1). 2. A peptide represented by the formula (2): H-Arg-Ala-Phe-OH (2). 3. An antihypertensive agent comprising the peptide according to claim 1 or 2.