JPH06135993A - Peptide having hemostatic action and biophylactic action - Google Patents

Abstract

PURPOSE:To provide the subject peptide having a specific amino acid sequence, inducing adhesive molecules from vascular endothelial cells to exhibit the hemostatic action, having a bacterium-removing effect, and effective for the therapy of infectious diseases or hemorrhagic diseases such as thrombin receptor paratrichosis, etc. CONSTITUTION:A protected peptide chain is synthesized on a solid carrier such as a resin in accordance with the amino acid sequence of the peptide by a solid synthetic method using a peptide synthesis device and using protected amino acids, cleaved out from the resin by a HE method, and simultaneously subjected to the removal of the protecting groups. The obtained peptide is separated and purified by chromatography comprising ion exchanging and/or gel filtration, by a high-performance liquid chromatography using a reverse phase column, etc., to provide the objective peptide of the formula inducing adhesive molecules from vascular endothelial cells, causing the activation of leukocytes without inhibiting plasmin, having a bacterium-removing effect, producing a tPA-inhibiting factor, and having a hemostatic action in a good yield and at a low cost.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide which is effective for treating hemorrhagic diseases associated with thrombin receptor abnormalities and for treating infectious diseases caused by leukocyte activation, and a drug containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art Physiological hemostasis occurs physiologically during bleeding during trauma or surgery, but there are cases in which it bleeds abnormally and does not stop. In such cases, there are vitamin K preparations (phytonadione, menaquinone, menadione) for promoting the production of vitamin K-dependent blood coagulation factors as agents that promote the hemostatic function. Thrombin is also a typical hemostatic agent, and fibrin (fibrin)
Inhibitors of the lytic enzyme plasmin are also included [Takudo Okuda, Akikazu Takada, Hiroshi Maeda: Physiology / Biochemistry for Understanding Diseases, 225-247, 1988, Kinhodo (Tokyo)],
For example, tranexamic acid is often used. Furthermore, an oligopeptide (SFLLRNPNPNDKY containing 14 amino acid residues at the N-terminus of the platelet thrombin receptor)
It has been reported that EPF) has a platelet aggregation action similar to thrombin, that is, a hemostatic action [T. -K. H.
Vu, D.D. T. Hung, V.D. I. Wheaton, a
ndS. R. Coughlin: Cell, 64, 10
57-1068, 1991].

The mechanism of the hemostatic action of thrombin is generally attributed to the platelet aggregation action and fibrin production action. In addition, the mechanism of hemostatic action of vitamin K preparations is said to cause blood coagulation by promoting production of blood coagulation factors such as prothrombin, factor III, factor IX, and factor X in the liver [Takudo Okuda] , Akazu Takada, Hiroshi Maeda: Physiology and biochemistry for understanding diseases 225
-247, 1988, Kinhodo (Tokyo)].

However, in thrombin receptor abnormalities in which thrombin receptors in platelets and vascular tissues lose their ability to bind thrombin, the platelet aggregation ability and the adhesion effect of platelets on blood vessels are reduced, resulting in continued bleeding. There is a risk of causing hemorrhagic diseases such as anemia and death. Vitamin K preparations are drugs that increase the production of thrombin in blood, and thus have little effect on hemorrhagic diseases caused by thrombin receptor abnormalities.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a hemorrhagic disease that cannot be sufficiently treated with existing hemostatic agents such as thrombin preparations, vitamin K preparations, and plasmin inhibitors, for example, thrombin receptor abnormality in platelets and vascular tissues. And a peptide for treating bleeding diseases due to a decrease in platelet aggregation ability and a decrease in platelet adhesion effect on blood vessels, and also for treatment of abnormal bleeding during surgery and bacterial infection due to leukocyte activation The purpose is to provide a drug containing

[0006]

The present inventors have proposed a peptide,
SFLLR (Ser-Phe-Leu-Leu-Arg), SFLLRNP (S
er-Phe-Leu-Leu-Arg-Asn-Pro), SFLLRNPND (S
er-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp) was synthesized and investigated, and it was found to have the following properties.

Actions similar to thrombin: 1) Leukocytes from vascular endothelial cells as well as thrombin,
Induces adhesion molecules such as ELAM-1 involved in monocyte adhesion.

2) Blocks the cause of bleeding by acting on vascular endothelial cells in the same manner as thrombin to promote the production of PAI [a protein that inhibits tissue plasminogen activator (tPA)]. .

3) However, it does not have the ability to directly inhibit plasmin, which is a fibrinolytic proteolytic enzyme (that is, it has no antiplasmin action).

Different action from thrombin: 4) It has no action of converting fibrinogen into fibrin to form a clot.

5) Since it does not activate protein C, it has no action of inhibiting the coagulation system.

It should be noted that the 14-amino acid residue peptide has so far acted on vascular endothelial cells to express adhesion molecules and PAI.
These are novel properties of the peptides of the present invention, as their production enhancing effects have not been found.

The above peptide is an N-terminal 14 amino acid residue oligopeptide (SFL) of the platelet thrombin receptor.
LRNPNPNDKYEPF) has 5 to 9 deleted C-terminal amino acid residues. It should be noted that, in this case, the peptide produced by deletion of 5 or less did not have a preferable biological activity, and it was unexpected that the above-mentioned property was obtained only when a short peptide as in the present invention was obtained.

Further, the above-mentioned peptides have a common skeleton SFLL.
It is considered that R contributes to each property. Therefore, in the longest peptide SFLLRNPND among the three, any amino acid from the C-terminal first D (Asp) to the fourth N (Asn) is deleted, or hydrophilic or hydrophobic,
Peptides substituted with other amino acids or modified amino acids that are acidic or basic or have similar properties in terms of molecular structure are also generally considered to have similar effects. Such peptides are also within the scope of the present invention as long as the above activity is retained. Examples of similar amino acid substitutions include the exchange of asparagine (Asn) for glutamine (Gln) or the exchange of aspartic acid (Asp) for glutamic acid (Glu). As an example of modification of an amino acid residue, an amino acid modified with a methyl group or a naphthyl group is considered.

The peptide of the present invention can be easily synthesized by a known commercially available method. For example, Applied Biosystems Model 43
Solid phase synthesis is performed using a protected amino acid using a 0A peptide synthesizer. Next, the HE is used to cut out the peptide from the resin and simultaneously remove the protecting group. Then, the target peptide can be separated and purified by chromatography such as ion exchange and / or gel filtration, high performance liquid chromatography using a reverse phase column, and the like.
The peptide of the present invention is composed of 9 or less amino acid residues,
Since it is smaller than the N-terminal 14 amino acid residues of the platelet thrombin receptor, it has the advantages of easy synthesis, good yield, and inexpensive production.

The present invention also relates to a therapeutic agent containing the above peptide alone or in combination with other agents as an active ingredient. The therapeutic agent of the present invention can be used for treating various hemorrhagic diseases.

In particular, the main subject of the present invention is a drug for promoting the production of tPA inhibitory protein (PAI) to treat various diseases. The PAI production promoter is used for the treatment of abnormal bleeding caused by the release of tPA from tissues during surgery, and for the treatment of bleeding disorders in abnormal thrombin receptors. In thrombin receptor abnormalities, stimulation is not transmitted even in the presence of thrombin, and PAI production in vascular endothelial cells due to thrombin stimulation does not occur, so the therapeutic agent of the present invention is effective.

Further, like the thrombin, the therapeutic agent of the present invention induces adhesion molecules such as ELAM-1 involved in adhesion of leukocytes and monocytes from vascular endothelial cells to induce interaction between vascular endothelial cells and leukocytes. It can enable and cause activation of white blood cells. Due to this effect of preventing or eliminating the infection of bacteria, the therapeutic agent of the present invention exerts a remarkable effect also in the treatment of hemorrhagic diseases and bacterial infections caused by bacterial infection.

Furthermore, the therapeutic agent of the present invention exerts a remarkable effect on the treatment of abnormal bleeding during surgery as well as thrombin and other hemostatic agents. Further, when used in combination with thrombin, an excellent hemostatic effect due to the combination of different hemostatic mechanisms can be expected.

The therapeutic agent of the present invention is, for example, a drip having a DDS (drug delivery system) or an injection, and the active ingredient peptide is 2-100 per day for an adult.
mg, either alone or in combination with pharmaceutically acceptable carriers or auxiliaries, for once or several times daily divided doses.

As used herein, a "pharmaceutically acceptable carrier" is a carrier that retains the desired biological activity of the peptide used and does not have untoward toxicity. Examples include water, ethanol, ethylene glycol, polyethylene glycol and the like.

[0022]

EXAMPLES Next, the present invention will be further described by way of examples, which are shown by way of example, and the scope of the present invention is not limited to these.

Example 1 of the peptide SFLLRNPND
This peptide was synthesized by Applied Biosystems (Applied Biosystems).
Biosystems) model 430A peptide synthesizer,
Synthesized and purified using the Boc method.

The analytical characteristics are as follows.

Properties: White powder Amino acid analysis: 6N hydrochloric acid, 110 ° C., 22 hours Asp (3) 2.92, Ser (1) 0.86, Pro (1) 1.
00, Leu (2) 2.01, Phe (1) 0.97, NH ₃ (2)
2.10, Arg (1) 0.98 Liquid Chromatography (HPLC) Analysis Column: YMC Pack A-302, 4.6 mm
I. D. × 150 mm Eluent: 0.1% TFA Gradient: CH ₃ CN 10% -60% (25
Min) Flow rate: 1 ml / min. Detection: 220 nm Purity: 98% The result of HPLC is shown in FIG.

Infrared absorption spectrum analysis The results of analysis using the KBr method are shown in FIG.

Example 2 Synthesis of Peptide SFLLRNP This peptide was synthesized, purified and analyzed by the same methods as in Example 1.

The analytical characteristics are as follows.

Properties: White powder Amino acid analysis: 6N hydrochloric acid, 110 ° C., 22 hours Asp (1) 0.98, Ser (1) 0.86, Pro (1) 1.
00, Leu (2) 2.01, Phe (1) 0.98, NH ₃ (1)
1.19, Arg (1) 0.98 Liquid Chromatography (HPLC) Analysis Column: YMC Pack A-302, 4.6 mm
I. D. * 150 mm Eluent: 0.1% TFA _{gradient: CH 3 CN 10% -60%} (25
Min) Flow rate: 1 ml / min. Detection: 220 nm Purity: 97% The result of HPLC is shown in FIG.

Infrared absorption spectrum analysis The results of analysis using the KBr method are shown in FIG.

Example 3 Synthesis of Peptide SFLLR This peptide was synthesized, purified and analyzed by the same methods as in Example 1.

The analytical characteristics are as follows.

Properties: White powder Amino acid analysis: 6N hydrochloric acid, 110 ° C., 22 hours Ser (1) 0.88, Leu (2) 2.06, Phe (1) 1.
00, Arg (1) 1.00 Liquid chromatography (HPL
C) Analytical column: YMC Pack A-302, 4.6 mm
I. D. * 150 mm Eluent: 0.1% TFA _{gradient: CH 3 CN 10% -60%} (25
Min) Flow rate: 1 ml / min. Detection: 220 nm Purity: 97% The result of HPLC is shown in FIG.

Infrared absorption spectrum analysis The results of analysis using the KBr method are shown in FIG.

Example 4 Effects on PAI production of vascular endothelial cells
Boss action (1) Cell culture and medium Human umbilical vein endothelial cells (HUVEC) and EGM-U
V medium was purchased from Kurashiki Spinning Co., Ltd. 3rd generation HUV
3.2 × 10 ³ cells were inoculated on a collagen-coated 24-well plate at a concentration of 3.2 × 10 ³ cells in the presence of 5% CO _2.
The cells were cultured at 7 ° C for about 1 week until they became confluent. During that time, the medium was replaced every 2 days or 3 days.

(2) Measurement of tPA and PAI The culture medium of HUVEC cultured at confluence was exchanged, 160 μM of the above synthetic peptide was added, and 5% CO 2 was added.
_The cells were cultured in the presence of _{2 at} 37 ° C. for 24 hours. Then, the amount of tPA and PAI in the culture supernatant was adjusted to Biopool.
It measured using the Imulyse (trademark) PAI ELISA KIT by the company. The results are shown in Table 1.

[0037]

[Table 1]

Thus, the synthetic peptide SFLLRNP
ND, like the 14-residue peptide SFLLRNPNDKYPEF, which has the same action as thrombin, is similar to HUVEC.
Significantly increased the PAI release amount in.

Example 5 Measurement of adhesion molecules on vascular endothelial cells
Expression of adhesion molecules on constant- vascular endothelial cells was determined by Cell ELI.
It was measured by the SA method. That is, the culture solution of human umbilical vein endothelial cells HUVEC (purchased from Kurashiki Spinning Co., Ltd.) cultured confluently was exchanged, and the above synthetic peptide 160 was exchanged.
μM was added, and the mixture was cultured at 37 ° C. for 6 hours in the presence of 5% CO ₂ . The culture supernatant is removed and phosphate buffered saline (PB
S, 2.7 mM KCl, 137 mM NaCl, 8.
1 mM Na ₂ HPO _4, washed 3 times with 1.5mM KH ₂ PO _4), fixed for 20 minutes with PBS containing 2% formaldehyde and blocked for 1 hour with PBS containing 2% BSA. Next, 0.5 μg / ml of anti-ELAM-1 mouse monoclonal antibody (primary antibody) was added and incubated for 1 hour. After washing with PBS, biotinylated anti-mouse IgG antibody (secondary antibody) was added for 30 minutes, and PB was further added.
After washing with S, avidin DH and biotinylated peroxidase were added and incubated for 1 hour. After washing with PBS, the peroxidase substrate ABTS [2, 2
-Azinobis (3-ethylbenztizoline sulfonic acid)] was added to cause color reaction, and the absorbance (OD) after 20 minutes
₄₀₅ ) was measured. Without adding the primary antibody at the same time, the absorbance when the reaction after the secondary antibody was made to be the same was measured as the absorbance due to non-specific binding, and the value subtracted from the absorbance of the sample was determined to be the true absorbance. . The results are shown in Table 2.

[0040]

[Table 2] Expression of adhesion molecules by synthetic peptides --------------------------------------- ----------------------- Peptide Absorbance (405nm) Control * 0.022 ± 0.012 SFLLR 0.050 ± 0.003 SFLLRNP 0.070 ± 0.006 SFLLRNPND 0.134 ± 0.012 SFLLRNPNDKYEPF 0.067 ± 0.029 ------------------------------- ------------------------------- * Control shows the case where no peptide was added to the reaction system.

Thus, the synthetic peptides SFLLR, S
FLLRNP and SFLLRNPND are similar to the 14-residue peptide SFLLRNPNDKYEPF in HUVE.
It was demonstrated to significantly increase the expression of adhesion molecule (ELAM-1) on C.

[0042]

INDUSTRIAL APPLICABILITY The peptide of the present invention induces adhesion molecules from vascular endothelial cells, causes leukocyte activation by the interaction between vascular endothelial cells and leukocytes without inhibiting plasmin, and has a bacterial elimination effect. , PAI (tPA inhibitor) is produced together with the action of preventing bacterial infection, and has an anti-bleeding action. In addition, the peptide of the present invention is composed of 9 or less amino acid residues, which is smaller than the N-terminal 14 amino acid residues of the platelet thrombin receptor, so that the peptide is easy to synthesize, the yield is good, and the manufacturing cost is low. There are advantages.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ser Phe Leu Leu Arg 5 1 5 SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid topology : Linear sequence type: peptide sequence Ser Phe Leu Leu Arg Asn Pro 7 15 SEQ ID NO: 3 sequence length: 9 sequence type: amino acid topology: linear sequence type: peptide sequence Ser Phe Leu Leu Arg Asn Pro Asn Asp 9 1 5

[Brief description of drawings]

1 is the peptide SFLLRNPN of Example 1. FIG.
It is a result of HPLC in the synthesis of D.

FIG. 2 shows the peptide SFLLRNPN of Example 1.
It is an analysis result using the KBr method of D.

FIG. 3 shows the results of HPLC in the synthesis of the peptide SFLLRNP of Example 2.

FIG. 4 shows the results of analysis of the peptide SFLLRNP of Example 2 using the KBr method.

FIG. 5 shows the results of HPLC in the synthesis of the peptide SFLLR of Example 3.

FIG. 6 shows the KB of the peptide SFLLR of Example 3.
It is an analysis result using the r method.

Claims (5)

[Claims] 1. A peptide represented by the formula: SFLLR (Ser-Phe-Leu-Leu-Arg). 2. The formula: SFLLRNP (Ser-Phe-Leu-Leu-Arg
-A peptide represented by (Asn-Pro). 3. The formula: SFLLRNPND (Ser-Phe-Leu-Leu
-Arg-Asn-Pro-Asn-Asp). 4. A formula: SFLLRNPND (Ser-Phe-Leu-Leu
-Arg-Asn-Pro-Asn-Asp) in the peptide represented by (Arg-Asn-Pro-Asn-Asp), in which any amino acid from the C-terminal to the first D (Asp) to the fourth N (Asn) is deleted or has similar properties. A peptide substituted with the amino acid of. 5. A pharmaceutical composition for treating a hemorrhagic disease, which comprises the peptide according to any one of claims 1 to 4 as an active ingredient.