JP2001278894A - Medicinal composition for preventing and/or treating blood circulation disorder and peptide as active ingredient for the composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicinal composition which contains as an active ingredient a peptide having a structure common to HSP 20 peptide known as one of human stress proteins and is effective for blood circulation disorders, cerebrovascular disorders, peripheral vascular disorders, and the like. SOLUTION: It was found that an amino acid sequence placed within the amino acid range of -5 to +6 from serine placed at No. 16 amino acid position from the NH2-terminal of human HSP 20, or a certain amino acid sequence among amino acid sequences starting from tryptophan at No. 9 amino acid position from the NH2-terminal of the amino acid sequence of αB crystallin highly homologous to the human HSP 20 had a high blood platelet coagulation inhibiting action and/or a blood coagulation time-extending action due to the stimulation of prothrombinds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition effective for circulatory disorders, cerebrovascular disorders, peripheral vascular disorders, etc., in particular, a part of human HSP20 which is known as one of human stress proteins. The present invention relates to a pharmaceutical composition comprising, as an active ingredient, a peptide having the same structure as the above peptide.

[0002]

Conventionally, it has been considered that a stress protein (heat shock protein) acts as a molecular chaperone in a cell. On the other hand, HSP20, a human-derived protein having a molecular weight of 20 kilodaltons, has been purified as a new low-molecular-weight HSP, its characteristics have been identified, and its amino acid sequence has been reported (Kato, K., et al., J. Am. Biol. Che
m. 269: 15302-15309, 1994: Reference 1). Furthermore, it has been known that the phosphorylation of a protein having a molecular weight of 20 kilodaltons is involved in the cyclic nucleotide-dependent relaxing action in vascular smooth muscle cells. (Beall, AC, etal., J. Biol. Chem. 272).
: 11283-11287, 1997: Reference 2). The present inventors have reported that human HSP20 can be used as extracellular botrocetin (Botrocetin).
has been reported to inhibit platelet aggregation induced by tin) and thrombin (thrombin) (Ma
tsuno, H. et al., FEBS Letters, 429: 327-329, 1998:
Reference 3). However, one of the low molecular weight HSPs
The species HSP27 showed no activity.

[0003] According to subsequent research, among the peptides that constitute the HSP20, NH ₂ - from terminal methionine 1
It was revealed that serine located at the sixth position was phosphorylated, and the amino acid sequence containing the serine, Trp Leu Arg Arg Ala Ser Al
a Serine residue of a peptide consisting of Pro Leu Pro Gly Leu Lys (an amino acid sequence from the methionine at the NH ₂ -terminal to the 11th to 22nd amino acids and a lysine bound to the COOH-terminal of the 22nd leucine) Has been reported to relax vascular smooth muscle cells upon phosphorylation (Beall, A., et al., J. Biol. Chem. 274: 11344-).
11351, 1999: Reference 4). However, none of the above reports discloses that a specific amino acid sequence among the amino acid sequences of peptides constituting human HSP20 exhibits an activity of inhibiting platelet aggregation. Of course, there is no description suggesting that a specific amino acid sequence has such an effect.

[0004]

PROBLEM TO BE SOLVED BY THE INVENTION Pharmaceutical compositions effective for circulatory disorders, cerebrovascular disorders, peripheral vascular disorders, etc., particularly human HS which is known as one of human stress proteins
An object of the present invention is to provide a pharmaceutical composition effective for circulatory disorders, cerebrovascular disorders, peripheral vascular disorders, and the like, which contains a peptide having an amino acid sequence common to the amino acid sequence of the peptide of P20 as an active ingredient.

[0005]

Means for Solving the Problems Accordingly, the present inventors have developed a peptide having a structure common to a part of human HSP20, and which is a medicament effective for cardiovascular disorders, cerebrovascular disorders, peripheral vascular disorders and the like. As a result of various investigations to find a composition,
NH human HSP20 ₂ - NH of the to -5 serine amino acid sequence located +6, or HSP20 peptides and amino acid sequence of αB crystallin showing high homology around the serine located 16 penultimate ₂
A certain amino acid sequence among the amino acid sequences starting from tryptophan located at the ninth position from the terminal has a strong inhibitory effect on thrombin-stimulated platelet aggregation and / or a prolonging effect of prothrombin-stimulated blood clotting time; It has been found that the present invention has been completed.

Specifically, peptides useful as active ingredients for a pharmaceutical composition effective for circulatory disorders, cerebrovascular disorders, peripheral vascular disorders, and the like have been found, and a pharmaceutical composition containing these peptides has been found to be effective in treating circulatory disorders. , Cerebrovascular disorders, prevention and treatment of peripheral vascular disorders, especially in the field of cardiovascular disorders,
Prevention of unstable angina, myocardial infarction, prevention of coronary artery occlusion after PTCA (percutaneous coronary angioplasty), prevention of graft occlusion after coronary artery bypass surgery, prevention of cerebral embolism by atrial fibrillation,
And the prevention of thrombus formation after artificial valve replacement, and in the field of cerebrovascular disease, transient ischemic attack (TI
A) prevention of recurrence, and in the field of peripheral vascular disorders, treatment and prevention of lower deep vein thrombosis and pulmonary obstruction,
The present invention has been found to be promising for prevention of obstruction after revascularization surgery, prevention and treatment of obstructive thromboangitis, obstructive arteriosclerosis, and the like, and completed the present invention.

Thus, according to the present invention, human HSP
Of 20 NH ₂ - among the amino acids comprising an amino acid sequence of the serine located 16th from terminal methionine from -5 counted as a reference to +6 (SEQ ID NO: 1), the N
H ₂ - distally, at least -5 3 3 contiguous amino acid sequence Trp Leu located Arg (SEQ ID NO: 10), or located -4 +2 7 contiguous amino acid sequence Leu Ar
g Arg AlaSer Ala Pro (SEQ ID NO: 13) Peptide containing the amino acid shown in SEQ ID NO: 13, or the amino acid sequence of αB crystallin showing high homology to HSP20 peptide,
NH ₂ - begins tryptophan located ninth counted from terminal methionine, comprising an amino acid comprising an amino acid sequence of up to phenylalanine located 17th least 6 contiguous, and, the NH ₂ - distally, At least one from the 9th tryptophan
A medicament for preventing and / or treating a blood circulation disorder, comprising as an active ingredient a peptide containing an amino acid represented by the six consecutive amino acid sequence Trp Ile Arg Arg Pro Phe (SEQ ID NO: 26) up to the fourth phenylalanine. A composition for use.

Further, the amino acid sequence Trp Leu Arg
A peptide comprising an amino acid sequence represented by SEQ ID NOS: 1 to 10 and a pharmaceutical composition for preventing and / or treating blood circulation disorders, comprising the peptide as an active ingredient; A peptide comprising Leu Arg Arg Ala Ser Ala Pro comprising an amino acid represented by the amino acid sequence shown in SEQ ID NOs: 11 to 13, and a blood circulation disorder prevention and / or treatment characterized by containing the peptide as an active ingredient. Pharmaceutical composition, the amino acid sequence Trp Ile Ar
g A peptide comprising an amino acid sequence represented by the amino acid sequence represented by SEQ ID NOS: 23 to 26, and a peptide comprising the amino acid sequence represented by SEQ ID NOS: 23 to 26, and a medicament for preventing and / or treating a blood circulation disorder, comprising the peptide as an active ingredient. A composition will be provided.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present specification, human HSP20 refers to a human-derived protein having a molecular weight of 20 kilodalton, which is a member of the low molecular weight stress protein family. The symbol-or + represents human HSP known as a phosphorylation site involved in cyclic nucleotide-dependent vasorelaxation in the amino acid sequence of human HSP20 reported in Reference 4.
Of 20 NH ₂ - serine located 16th counted from the terminus of methionine as a reference (0), NH ₂ - to position when the counted end side, - a, when the counted COOH- terminus The position is indicated by adding +.

Accordingly, in a human HSP20, for example, -5 occurring amino acids of, NH ₂ serine located 16th - when counted distally, amino acids located in the fifth, i.e., NH ₂ - terminal methionine 11 counted from
It means tryptophan located at the second position.
In addition, the amino acid sequence from -5 to +6 is NH _2-
Refers to the amino acid sequence from tryptophan located at position 11 from the terminal methionine to serine at position 16 and leucine at position 22, unless otherwise specified. It should be understood that it means an amino acid sequence containing the above contiguous sequences.

By the way, the present inventors counted from methionine at the NH ₂ -terminal of human HSP20, which is known as a phosphorylation site involved in the cyclic nucleotide-dependent vasorelaxant action of human HSP20 reported in Reference 4. Attention was paid to serine located at the 16th position, and its physiological activity was tested mainly on a peptide consisting of a series of amino acid sequences included in the amino acid sequence from -5 to +6 counted from the serine. Furthermore, among the amino acid sequences of αB crystallin having high homology to the amino acid sequence of human HSP20, a peptide having an amino acid sequence starting from tryptophan, which is located at the ninth position from the NH ₂ -terminal methionine, was also used for the assay. These peptides and amino acids are shown in Tables 1 and 2.

That is, these peptides and amino acids were chemically synthesized, and the platelet aggregation inhibitory effect of each test substance was assayed under the following test conditions. In addition, among the peptides shown in Tables 1 and 2, the peptides having four or more amino acids bonded exhibited all the effects irrespective of the effect, and the peptides consisting of three amino acids showed an effective action. The amino acid sequences are shown in the sequence listing as SEQ ID NOS: 1 to 27. In addition, peptides are Atherton,
Solid phase peptide synthesis by E. et al .: a pract
Synthesized according to the F-moc solid phase method described in IRS PRESS (1989) PP. 25-161. Purification was performed by reverse phase HPLC using a column manufactured by Rainin Instrument Co., Inc. The confirmation was performed using Kraros Kompact MALDI 2 manufactured by Shimadzu Corporation.

[0013]

[Table 1]

[0014]

[Table 2]

[0015] The inhibitory effects of these peptides on the platelet aggregation test and blood coagulation reaction (blood coagulation test) were evaluated by the following methods. 1. Blood used Blood was collected from healthy people (18, ages 21 to 34) (citrate concentration in blood collection tube: 3.15%, volume ratio 1/10) and used for thrombin-stimulated platelet aggregation test and prothrombin-stimulated blood coagulation test did.

2. Separation of platelets The collected whole blood was dispensed into centrifuge tubes in an amount of 5 to 6 ml each, and
Centrifuge at xg for 12 minutes. After centrifugation, the supernatant is collected and used as platelet-rich plasma (PRP). The precipitate is centrifuged again at 1500 × g for 10 minutes and the supernatant is collected. This is referred to as platelet poor plasma (PRP).

3. Preparation of Washed Platelets An equal amount of a phosphate buffer (wash solution) containing an analog of PGI ₂ (final concentration 10 μM) is added to PRP, and mixed well. Then, centrifuge at 500 × g for 7 minutes and discard the supernatant. Add 5 ml of the above washing solution to the precipitated platelets, mix well, and stir. This operation is repeated twice. After the third centrifugation, the supernatant is discarded, 2 ml of phosphate buffer is added, and the number of platelets is counted. Then, a phosphate buffer is added to adjust the number of platelets to be constant (4.0 × 10 ⁸ / ml).

4. Platelet aggregation test Using a platelet aggregation measuring instrument (Hema Tracer 801; NBS), thrombin-stimulated platelet aggregation is assayed using light transmittance as an index. 200 μl of the PRP prepared in the above item 3 is weighed into a micro cuvette, and the cuvette is placed in a measurement site of a measuring instrument to measure the transmittance. At this time, the light transmittance is set to 100%. Then, PRP (PRP160
μl + 40 μl of phosphate buffer) is placed in a separate cuvette and measured, which reduces light transmission due to platelets floating in solution. The transmittance at this time is set to 0%. During this time, the PRP was stirred in the cuvette for 8
Stirred at 00 rpm. When thrombin is added as a substance that stimulates platelets, the stimulated platelets form aggregates and precipitate below. As a result, the number of floating platelets decreases, and the light transmittance increases.

From this change, the platelet aggregation ability is calculated. The amount of thrombin to be added is 10% by volume (20 μm).
l), the concentration is 0.06 units / ml as the final concentration in the solution.
0.2 units / ml. This concentration sets the concentration of thrombin that results in an increase in transmission of 80-90% for each blood sample. The concentration of the synthetic peptide as each test material is 10 to 300 μM (the concentration in the final cuvette), and the volume ratio to be added is 10% (20 μl). PRP (160 μl of PRP + 20 μl of peptide) to which a synthetic peptide solution of each concentration is added is placed in a cuvette and incubated at 37 ° C. for 3 to 4 minutes. Then
To examine the inhibitory effect of platelet aggregation on the change in light transmittance by adding thrombin.

[5] Blood coagulation test The PRP prepared in 3 above is used as a sample for testing. Blood coagulation time is measured by Blood Coagulation Anal
Measure using yzer CA-100 (Sysmex). Human prothrombin (thromboplastin C plus,
Sysmex). 100 μl of PRP prepared in 3 above
In a dedicated cuvette and incubate at 37 ° C. for 3 minutes. Next, 200 μl of the reagent is added, and the mixture is quickly and gently stirred with a dedicated vortex, and then the cuvette is set in the measurement section. The measurement measures the blood clotting time based on the change in light transmittance. The average measurement time of a healthy person is 11 seconds to 12
The meter is pre-adjusted to be in the range of seconds.

6. Results Using the peptides shown in Tables 1 and 2, the platelet aggregation inhibitory effect of thrombin stimulation and the coagulation time elongation effect of prothrombin stimulation were examined. FIG. 1 is a graph showing the results obtained when 300 μM of the peptide samples of each of the test substance numbers 1 to 27 were used. However, the result is
Test substance numbers 1 to 27 are shown using the average value of the measured values of each blood sample. Note that a buffer was used as a control. The lower the platelet aggregation rate compared to the control, the stronger the platelet aggregation inhibitory effect of the peptide is suggested.

Test substance number 1 shown in Tables 1 and 2
In the peptides of ９０5, 16, 17 (SEQ ID NOS: 1 to 5, 11, 12), platelet aggregation was suppressed by 90 to 98%. Then, test substance numbers 6 to 10 and 18 (SEQ ID NOs: 6 to 1)
In 0, 13), 20 to 70% was suppressed. No suppression was observed with the other peptides. On the other hand, the prolongation of the clotting time (prothrombin time) was 19 to 2 times longer than that of the control.
The test substance Nos. 1 to 10 (SEQ ID NOs.
0) and nothing else was observed. In order to investigate the effects of serine phosphorylation, phosphorylation of serine in these peptide sequences was also used.However, the presence or absence of phosphorylation had no effect on the platelet aggregation inhibitory effect or the coagulation time extending effect. Did not.

Starting from tryptophan, which is located at the ninth position from the NH ₂ -terminal methionine of αB crystallin, which is highly homologous to the amino acid sequence of human HSP20,
Test substance No. 28-32 which is a peptide having an amino acid sequence up to phenylalanine located at the 17th position
(SEQ ID NOs: 23 to 27) were also chemically synthesized, and the thrombin-stimulated platelet aggregation inhibitory effect and the prothrombin-stimulated coagulation time-extending effect were examined. Each peptide 3
The results when 0 μM was used are shown in the graph. Buffer was used as a control. Test substance numbers 28, 29, 30, and 31
The platelet aggregation inhibitory effect was observed for the peptides of (SEQ ID NOS: 23 to 26). The coagulation time was prolonged for the peptides of test substance numbers 28 to 32 (SEQ ID NOs: 23 to 27). The inhibitory effects of test substance numbers 28, 29, 30, and 31 (SEQ ID NOS: 23 to 26) on platelet aggregation are as follows:
Among the human HSP20 peptides, the peptide was 10-fold more potent than the peptides of the aforementioned test substances Nos. 1 to 10 (SEQ ID Nos. 1 to 10) having the specific amino acid sequence described in detail below.

From the results shown in FIGS. 1 and 2, methionine at the NH ₂ -terminal of human HSP20 known as a phosphorylation site involved in the cyclic nucleotide-dependent vasorelaxant action of human HSP20 reported in Reference 4 is shown. among the peptides represented by -5 nucleotides to + 6 up to the amino acid sequence of counted from serine located 16th and (SEQ ID NO: 1) counted from, NH ₂ human HSP20 - located 16th counted from the terminal methionine From -5, counting from serine
Up to three consecutive amino acid sequences TrpLeu Arg (SEQ ID NO: 1)
0), and in some cases, a peptide that may further comprise an amino acid sequence from -2 to +6, or a 7-consecutive amino acid sequence from -4 to +2
ΑB crystallin containing at least Leu Arg Arg Ala Ser Ala Pro (SEQ ID NO: 13) and optionally containing a sequence up to the longest + 6th leucine or HSP20 peptide of the amino acid sequence, NH ₂ - begins tryptophan located ninth counted from terminal methionine, 6 contiguous amino acids sequence from phenylalanine located at least 14 th Trp Ile Arg Arg Pro Phe (SEQ ID NO: 26), if In some cases, a peptide having an amino acid sequence from phenylalanine at the 15th position to phenylalanine at the 17th position has an inhibitory effect on platelet aggregation and / or an effect of prolonging the blood clotting time stimulated by prothrombin as compared to a control. It became clear to have.

That is, from the serine located at the 16th position from the methionine at the NH ₂ -terminal of human HSP20,
_A peptide comprising at least a 3-contiguous amino acid sequence Trp Leu Arg located at -5 to -3 as counted on the ₂ -terminal side, and optionally further comprising an amino acid sequence from -2 to +6, or A peptide comprising at least the 7-consecutive amino acid sequence Leu Arg Arg Ala Ser Ala Pro (SEQ ID NO: 13) from -4 to +2, and in some cases, the sequence up to the longest + 6th leucine, or Among the amino acid sequences of αB crystallin showing high homology to the HSP20 peptide, NH ₂
A six-consecutive amino acid sequence Trp Ile Arg starting from tryptophan at the ninth position counted from the terminal methionine and extending to at least the 14th position phenylalanine
Arg Pro Phe (SEQ ID NO: 26), and in some cases, a peptide which may include an amino acid represented by the amino acid sequence from phenylalanine located at position 15 to phenylalanine located at the longest position at position 17 can be used to prevent blood circulation disorders and / or Or it turned out to be effective as a therapeutic drug.

More specifically, the peptide consisting of the amino acid sequence Trp Leu Arg from -5 to -3 counted from the 16th serine as a minimum unit includes Trp Leu Arg Arg Ala of SEQ ID NO: 1. Ser Ala Pro
Leu Pro Gly Leu, Trp Leu Arg Arg Ala S of SEQ ID NO: 2
er Ala Pro Leu Pro Gly, Trp Leu Arg Ar of SEQ ID NO: 3
g Ala Ser Ala Pro Leu Pro, Trp Leu Arg of SEQ ID NO: 4
Arg AlaSer Ala Pro Leu, Trp Leu Arg A of SEQ ID NO: 5
rg Ala Ser Ala Pro, Trp Leu Arg Arg Al of SEQ ID NO: 6
a Ser Ala, Trp Leu Arg Arg Ala Ser of SEQ ID NO: 7, Trp Leu Arg Arg Ala of SEQ ID NO: 8, Trp Leu of SEQ ID NO: 9
Arg Arg, and peptides composed of the amino acids of Trp Leu Arg of SEQ ID NO: 10.

7 consecutive amino acid sequences Leu Ar from -4 to +2 counting from the 16th serine from the NH ₂ -terminal
g Peptide containing Arg Ala Ser Ala Pro as a minimum unit includes Leu Arg Arg Ala Ser Ala Pr of SEQ ID NO: 11.
o Leu Pro Gly Leu, Leu Arg Arg Ala S of SEQ ID NO: 12
er Ala Pro Leu Pro, LeuArg Arg Ala S of SEQ ID NO: 13
Peptides composed of the amino acids of er Ala Pro.

Further, in the amino acid sequence of αB crystallin showing high homology to the HSP20 peptide, NH ₂ −
Six consecutive amino acid sequences Trp Ile Arg Ar starting from tryptophan located at the ninth position counted from the terminal methionine and extending to at least the 14th position of phenylalanine
The peptide containing g Pro Phe (SEQ ID NO: 26) as a minimum unit includes Trp Ile Arg Arg Pro Ph of SEQ ID NO: 23.
e Phe ProPhe, Trp Ile Arg Arg Pro Ph of SEQ ID NO: 24
e Phe Pro, Trp Ile Arg Arg Pro Phe P of SEQ ID NO: 25
he, and a peptide consisting of Trp Ile Arg Arg Pro Phe of SEQ ID NO: 26.

The pharmaceutical composition containing one or more of the peptides according to the present invention as an active ingredient is prepared using carriers, excipients, and other additives that are usually used in pharmaceutical formulation. Is done. The carrier or excipient for the preparation may be either solid or liquid, such as lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, acacia, olive oil, sesame oil, cocoa butter, ethylene glycol and the like. Other commonly used ones are also included.

Administration is usually performed by injection, such as intravenous injection, suppository,
Any form of parenteral administration such as transdermal is employed. The dose is appropriately determined depending on the individual case in consideration of the symptoms, age of the administration subject, sex, etc., but is usually in the range of 1 to 1000 mg per adult per day, once to several times a day. Intravenous administration may be carried out, or continuous intravenous administration may be performed for 1 hour to 24 hours per day. Of course, as described above, the dosage varies under various conditions, so that an amount smaller than the above dosage range may be sufficient.

[0031] Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline. Water-insoluble solutions,
Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80. Such compositions may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, stabilizing (eg, lactose) and solubilizing agents (eg, glutamic acid, aspartic acid). These are sterilized by, for example, filtration through a bacteria retaining filter, blending of a bactericide or irradiation. In addition, these can be used by preparing a sterile solid composition and dissolving it in sterile water or a sterile injection solvent before use.

[0032]

According to the present invention, there is provided a peptide having a partial amino acid sequence of human HSP20 or αB crystallin exhibiting high homology thereto, comprising 3 to 12 amino acids having a specific amino acid sequence as described above. These peptides were found to exhibit an extremely high platelet aggregation inhibitory effect and / or an effect of prolonging prothrombin-stimulated blood coagulation time as compared to the control, even when compared to HSP27 as a control. Thus, it has been strongly suggested that pharmaceutical compositions containing these peptides as active ingredients are promising for the prevention and treatment of cardiovascular disorders, cerebrovascular disorders, peripheral vascular disorders, and the like.

[0033]

[Sequence List] SEQUENCE LISTING <110> Kozawa, Osamu; Matsuno, Hiroyuki; Niwa, Masayuki; Kato, Kanefusa Uematsu, Toshihiko <120> A medical composition for therapeutical and / or preventive treatmen t of impairment in blood circluation and related organs and a peptide usable as an active ingredient therefore <130> WP-3187 <140> <141> <160> 27 <170> PatentIn Ver. 2.0 <210> 1 <211> 12 <212> PRT <213> Homo sapiens < 400> 1 Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu 1 5 10 <210> 2 <211> 11 <212> PRT <213> Homo sapiens <400> 2 Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly 1 5 10 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro 1 5 10 <210> 4 <211> 9 <212> PRT < 213> Homo sapiens <400> 4 Trp Leu Arg Arg Ala Ser Ala Pro Leu 1 5 <210> 5 <211> 8 <212> PRT <213> Homo sapiens <400> 5 Trp Leu Arg Arg Ala Ser Ala Pro 1 5 <210> 6 <211> 7 <212> PRT <213> Homo sapiens <400> 6 Trp Leu Arg Arg Ala Ser Ala 1 5 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Trp Leu Arg Arg Ala Ser 1 5 <210> 8 <211> 5 <212> PRT <213> Homo sapiens <400> 8 Trp Leu Arg Arg Ala 1 5 <210> 9 <211> 4 <212> PRT < 213> Homo sapiens <400> 9 Trp Leu Arg Arg 1 <210> 10 <211> 3 <212> PRT <213> Homo sapiens <400> 10 Trp Leu Arg 1 <210> 11 <211> 11 <212> PRT <213> Homo sapiens <400> 11 Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu 1 5 10 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Leu Arg Arg Ala Ser Ala Pro Leu Pro 1 5 <210> 13 <211> 7 <212> PRT <213> Homo sapiens <400> 13 Leu Arg Arg Ala Ser Ala Pro 1 5 <210> 14 <211> 6 <212> PRT <213> Homo sapiens <400> 14 Leu Arg Arg Ala Ser Ala 1 5 <210> 15 <211> 5 <212> PRT <213> Homo sapiens <400> 15 Leu Arg Arg Ala Ser 1 5 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <400> 16 Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu 1 5 10 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Arg Ala Ser Ala Pro Leu Pro Gly Leu 1 5 <210> 18 <211> 8 <212> PRT <213> Homo sapiens <400> 18 Ala Ser Ala Pro Leu Pro Gly Leu 1 5 <210> 19 < 211> 7 <212> PRT <213> Homo sapiens <400> 19 Ser Ala Pro Leu Pro Gly Leu 1 5 <210> 20 <211> 6 <212> PRT <213> Homo sapiens <400> 20 Ala Pro Leu Pro Gly Leu 1 5 <210> 21 <211> 5 <212> PRT <213> Homo sapiens <400> 21 Pro Leu Pro Gly Leu 1 5 <210> 22 <211> 4 <212> PRT <213> Homo sapiens < 400> 22 Leu Pro Gly Leu 1 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Trp Ile Arg Arg Pro Phe Phe Pro Phe 1 5 <210> 24 <211> 8 <212 > PRT <213> Homo sapiens <400> 24 Trp Ile Arg Arg Pro Phe Phe Pro 1 5 <210> 25 <211> 7 <212> PRT <213> Homo sapiens <400> 25 Trp Ile Arg Arg Pro Phe Phe 1 5 <210> 26 <211> 6 <212> PRT <213> Homo sapiens <400> 26 Trp Ile Arg Arg Pro Phe 1 5 <210> 27 <211> 5 <212> PRT <213> Homo sapiens <400> 27 Trp Ile Arg Arg Pro 1 5

[Brief description of the drawings]

FIG. 1 is a graph showing the platelet aggregation rate and prothrombin time extension rate of test substance numbers 1 to 27 in comparison with a control.

FIG. 2 is a graph showing platelet aggregation rates and prothrombin time elongation rates of test substance numbers 28 to 32 in comparison with a control.

Continuation of the front page (51) Int.Cl. ⁷ Identification code FI Theme coat II (Reference) C07K 7/06 A61K 37/02 (71) Applicant 593232446 Kato Kanebo 20-1 Tomioka, Meito-ku, Nagoya-shi, Aichi Prefecture (71) Application 500500301 Toshihiko Uematsu 3680-15 Honjo, Gifu City, Gifu Prefecture (72) Inventor Osamu 3-47, Shinoki-cho, Kasugai-shi, Aichi Prefecture (72) Inventor Hiroyuki Matsuno 2-60, Takaya Shiroki, Kitakata-cho, Motosu-gun, Gifu Prefecture (72) Inventor Masayuki Niwa 1-628, Iwata Nishi, Gifu City, Gifu Prefecture (72) Inventor Kanebo Kato, 20-1 Fuigaoka, Meito-ku, Nagoya City, Aichi Prefecture (72) Inventor Toshihiko Uematsu 3680-15 Honjo, Gifu City, Gifu Prefecture F-term (reference) 4C084 AA02 AA03 AA07 BA01 BA02 BA10 BA15 BA16 BA17 BA23 NA14 ZA362 ZC022 4H045 AA10 AA30 BA12 BA13 BA14 BA15 BA16 CA40 EA23

Claims (8)

[Claims] 1. A NH human HSP20 ₂ - among the amino acids represented by the amino acid sequence from -5 counting serine located 16 penultimate methionine as a reference to +6 (SEQ ID NO: 1), the NH ₂ - At the terminal side, at least three consecutive amino acid sequences Trp Leu Arg located at -5 to -3
(SEQ ID NO: 10) or a peptide containing an amino acid represented by the seven-consecutive amino acid sequence Leu Arg Arg Ala Ser Ala Pro (SEQ ID NO: 13) located from -4 to +2, or αB showing high homology to the HSP20 peptide The amino acid sequence of the crystallin contains at least six consecutive amino acids represented by the amino acid sequence starting from tryptophan located at the ninth position from the methionine at the NH ₂ -terminus and extending to phenylalanine at the 17th position. At the NH ₂ -terminal side, a six-consecutive amino acid sequence Trp Ile ArgArg P from at least the ninth tryptophan to the fourteenth phenylalanine
A pharmaceutical composition for preventing and / or treating a blood circulation disorder, comprising a peptide containing an amino acid represented by ro Phe (SEQ ID NO: 26) as an active ingredient. 2. The method according to claim 1, wherein the peptide containing the amino acid sequence Trp Leu Arg comprises the amino acids represented by the amino acid sequences shown in SEQ ID NOs: 1 to 10. Or a therapeutic pharmaceutical composition. 3. The amino acid sequence Leu Arg
Peptides containing Arg Ala Ser Ala Pro are SEQ ID NOS: 11 to
The pharmaceutical composition for preventing and / or treating a blood circulation disorder according to claim 1, wherein the composition comprises an amino acid represented by the amino acid sequence shown in 13. 4. The amino acid sequence Trp Ile Arg Arg Pro
The pharmaceutical composition for preventing and / or treating a blood circulation disorder according to claim 1, wherein the peptide containing Phe consists of the amino acid represented by the amino acid sequence shown in SEQ ID NOS: 23 to 26. 5. An amino acid sequence represented by the amino acid sequence (SEQ ID NO: 1) located from -5 to +6 with reference to serine located at the 16th position from the methionine at the NH ₂ -terminus of human HSP20. , Its NH
_{At the 2} -terminal side, at least three consecutive amino acid sequences Trp Leu Arg (SEQ ID NO: 10) located at -5 to -3 or seven consecutive amino acid sequences Leu Ar located at -4 to +2
g Among the amino acid sequence of the peptide containing the amino acid represented by Arg Ala SerAla Pro (SEQ ID NO: 13) or αB crystallin showing high homology to the HSP20 peptide,
NH ₂ - begins tryptophan located ninth counted from terminal methionine, comprising an amino acid comprising an amino acid sequence of up to phenylalanine located 17th least 6 contiguous, and, the NH ₂ - distally, At least one from the 9th tryptophan
A peptide containing an amino acid represented by the six consecutive amino acid sequence Trp Ile Arg Arg Pro Phe (SEQ ID NO: 26) up to phenylalanine located at the fourth position. 6. A peptide comprising an amino acid represented by the amino acid sequence shown in SEQ ID NOs: 1 to 10. 7. A peptide comprising an amino acid represented by the amino acid sequence shown in SEQ ID NOs: 11 to 13. 8. A peptide comprising the amino acids represented by the amino acid sequences shown in SEQ ID NOs: 23 to 26.