JPH0859698A - New peptide having blood coagulation regulating ability - Google Patents

Abstract

PURPOSE: To obtain a new peptide having high stability in blood, derived from a tissue factor pathway inhibitor, having specific amino acid sequences, showing blood coagulation regulating ability, useful for treating diseases causing abnormality of blood coagulating fibrinolysis. CONSTITUTION: This new peptide is derived from TFPI (Tissue Factor Pathway Inhibitor), has amino acid sequences of formula I and formula II and blood coagulation fibrinolysis regulating ability, is useful for treating diseases causing abnormality of blood coagulating fibrinolysis such as arteriosclerosis, diabetic capillary vessel symptoms and DIC and has excellent stability in blood and low possibility of being deactivated and cleared in vlvo. The peptide is obtained by admitting that the 21 amino acids part of 27th to 47th of TFPI has a wide blood coagulation regulating ability and chemically synthesizing a new 21 amino acids peptide designed by using this part as a motif and by inverting its N end and C end.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide which affects the activity of various blood coagulation factors. More specifically, among Factor VIIa (hereinafter sometimes referred to as VIIa), Factor Xa (hereinafter sometimes referred to as Xa) and Activated Protein C (hereinafter sometimes referred to as APC), , Relating to peptides that act on multiple factors.

[0002]

2. Description of the Related Art Various factors having a role of regulating blood coagulation / fibrinolysis exist in a living body and are involved in cascade control of blood coagulation / fibrinolysis reaction. The origin of human blood extrinsic coagulation reaction is glycoprotein tissue factor (Tissue
Factor) expression. Factor VII becomes more activated by forming a complex with Tissue Factor and forms a Tissue Factor / VIIa complex. This complex has a functional serine protease-type activity of VIIa, acts specifically on Factor X and Factor IX, and activates each factor. As a result, coagulation factors are sequentially activated, leading to the formation of a fibrinoid thrombus. In addition, protein C is activated by the thrombin / thrombomodulin complex to become APC, and then factor V and VIII
By inhibiting the factor, it acts as a blood anticoagulant factor.

Conventionally, TFPI (Tissue Factor Pathway Inhibito) has been used as a factor for inhibiting the activity of VIIa and Xa.
r) is known (GJBroze et al: Nature, vol / 338,
518-520 (1989)). TFPI has three Kunitz-type inhibitory regions, and on the surface of blood endothelial cell membrane, Tissue Facto
It has been shown that it forms a tetramer with r, VIIa, Xa and inhibits the activity of VIIa and Xa, and that the binding site of TFPI with VIIa is the Kunitz 1 region (GJBroz
e et al; Biochemistry, vol / 29, 7539-7545 (1990)). Therefore, we focused on this Kunitz 1 region and examined the effect of the partial peptide on the blood coagulation reaction system.
^The 21 amino acid portion having a sequence of ²⁷ Ala to ⁴⁷ Phe is
We recognized that it suppresses the blood coagulation reaction induced by Factor, and filed an application for Japanese Patent Application No. 4-318496.

[0004] This time, as a result of extensive investigations, we investigated the effect of TFPI-derived peptides on blood coagulation and fibrinolysis. Surprisingly, the 21 amino acids derived from TFPI were found to be active in the Kunitz 1 region of TFPI from which they were derived. It was confirmed that not only the inferred VIIa inhibitory activity but also a wide range of blood coagulation regulation ability such as Xa inhibitory activity and APC inhibitory activity.

Therefore, based on this fact, we have made the above T
When a novel peptide was synthesized using 21 amino acids derived from FPI as a motif and the effect on the blood coagulation / fibrinolysis system was investigated, two new peptides (hereinafter referred to as peptide 1 and peptide 2) were identified as 21 amino acids derived from TFPI. Has been found to be a highly useful novel peptide having a different activity profile, leading to the present invention.

Use of the peptide 1 or peptide 2 according to the present invention makes it possible to treat all diseases which cause abnormalities in blood coagulation / fibrinolytic activity, such as arteriosclerosis, diabetic microangiopathy and DI.
In C, it can be expected to suppress the tendency of thrombus formation and deterioration of properties due to the Tissue Factor that appeared when tissue damage and blood vessel inflammation occurred.

Since it is a low molecular weight peptide,
Unlike the I protein, it has excellent stability in blood and is unlikely to be inactivated or cleared in the body. In addition, various dosage forms can be applied, and the range of administration route selection is extremely wide, and the utility is high.

[0008]

That is, the present invention has the following amino acid sequence:
It is a novel peptide having [I] and [II] and having a blood coagulation / fibrinolysis regulating ability.

[0009]

[Chemical Formula 3] NH ₂ -Ala-Phe-Lys-Ala-Asp-Asp-Gly-Pro-Cys-Arg-Ala-Ile-Met-Lys-Arg ... [I] 1 5 10 15 -Phe-Phe- Phe-Asn-Ile-Phe-COOH 20 NH ₂ -Phe-Ile-Asn-Phe-Phe-Phe-Arg-Lys-Met-Ile-Ala-Lys-Cys-Pro-Gly ... [II] 1 5 10 15 -Asp-Asp-Ala-Lys-Phe-Ala-COOH 20 The peptide according to the present invention is t-typed using a peptide synthesizer.
It can be synthesized by a conventionally known method such as Boc method or F-moc method. The synthesized peptide can be purified by reverse phase column chromatography or the like after cleaving the resin. The amino acid sequence of the purified peptide is
It can be confirmed using an amino acid sequencer.

When investigating the effect of the peptide of the present invention on the blood coagulation / fibrinolysis system, the lyophilized purified peptide should be weighed and then dissolved in ultrapure water to prepare an appropriate concentration for use. You can

The peptides [I] and [I] according to the present invention
[I] was synthesized, added to human plasma and reacted, and then human Tis
When the effect of the above peptide on extrinsic coagulation was confirmed by measuring the time until coagulation by adding sue factor and stirring, the coagulation prolongation effect was observed for both peptides [I] and [II]. The size of the peptide is Peptide [I]> Peptide [II].

VIIa is a factor that activates factor X and factor IX, and Xa is a factor that promotes blood coagulation by converting prothrombin into thrombin. VIIa is a synthetic substrate S-2288 and Xa is a synthetic substrate S-. 2222 can be used to measure its activity.

APC also deactivates PAI-1, which is an inhibitor of tissue plasminogen activator, as well as an anticoagulant activity that decomposes activated factor V and activated factor VIII which are procoagulant factors. Has a fibrinolytic activity due to A., and the activity of APC can be measured using the synthetic substrate S-2366.

The peptides [I] and [I according to the present invention
When [I] was synthesized and the inhibitory action on VIIa was examined using S-2288, the inhibitory action on VIIa was observed for both the peptides [I] and [II], and the magnitude of the action was peptide [I]> peptide [ II].

Further, when the inhibitory effect on Xa was examined using S-2222, the inhibitory effect on Xa was observed for both peptides [I] and [II], and the magnitude of the effect was as follows: peptide [I]> peptide [II] Is.

Further, when the APC inhibitory action is examined using S-2366, the peptide [I] has an APC inhibitory action.

That is, peptide [II] is a blood anticoagulant peptide that simultaneously inhibits VIIa and Xa, and peptide [I] is anticoagulant based on VIIa activity suppression, Xa activity suppression, and APC suppression activity. It is a multi-functional blood coagulation-regulating peptide that also has the ability to promote coagulation. At first glance, anticoagulant ability and anticoagulant ability are contradictory actions, but anticoagulant ability is exerted at a site with a thrombus-forming tendency, and it acts toward hypercoagulability at a site with a bleeding tendency, which results in complicated blood flow. It may be a peptide that skillfully balances the coagulation cascade reaction.

The peptides [I] according to the present invention, and [I]
[I] can be added not only by itself but also with appropriate modifying groups, protecting groups and the like to enhance the stability and activity of the peptide.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0020]

Example 1 Preparation of Synthetic Peptides The following No. 1 and No. 2 peptides were synthesized by the t-Boc method using a peptide synthesizer type 430A manufactured by Applied Biosystems.

[0021]

[Chemical 4] No. 1 NH ₂ -Ala-Phe-Lys-Ala-Asp-Asp-Gly-Pro-Cys-Arg-Ala-Ile-Met-Lys-Arg 1 5 10 15 -Phe-Phe-Phe- Asn-Ile-Phe-COOH No.2 NH ₂ -Phe-Ile-Asn-Phe-Phe-Phe-Arg-Lys-Met-Ile-Ala-Lys-Cys-Pro-Gly 1 5 10 15 -Asp-Asp -Ala-Lys-Phe-Ala-COOH No.1 dry resin containing peptide having amino acid sequence
1.5 g of anisole 2.25 ml, ethyl methyl sulfide
After adding 0.38 ml, the peptide was cut out from the resin by a type I HF processor manufactured by Peptide Institute. Then, use diethyl ether (anhydrous) on the glass filter (G3).
And washed with chloroform alternately 5 times. Finally, 50 ml of 2N acetic acid was added to obtain an extract containing the synthetic peptide.

300 μl of this peptide extract was diluted 10-fold with buffer A (1% acetonitrile, 0.1% TFA / water) and the reverse phase column (VYDAC, C18, 4.6 mm × 250 m) was used.
m) was used for purification. The elution conditions are

[0023]

[Table 1]

It was carried out at a flow rate of 0.5 ml / min by a linear concentration gradient in which buffer A and buffer B in the above table were mixed.

Under these conditions, the peptide having the No. 1 amino acid sequence was eluted in about 27 minutes, and the weight of the peptide-eluted fraction after freeze-drying was 5.3 mg.

After adding 2.25 ml of anisole and 0.38 ml of ethyl methyl sulfide to 1.5 g of a dry resin containing a peptide having the amino acid sequence of NO. 2, the peptide was cut out from the resin by a type I HF treatment device manufactured by Peptide Institute. Subsequently, the mixture was washed 5 times with diethyl ether (anhydrous) and chloroform alternately on a glass filter (G3).
Finally, 50 ml of 2N acetic acid was added to obtain an extract containing the synthetic peptide.

300 μl of this peptide extract was diluted 10-fold with buffer A, and a reverse phase column (VYDAC, C1
8, 4.6 mm x 250 mm). The elution conditions are

[0028]

[Table 2]

It was carried out at a flow rate of 0.5 ml / min by a linear concentration gradient obtained by mixing buffer A and buffer B in the above table.

Under this condition, the peptide having the No. 2 amino acid sequence was eluted in about 23 minutes, and the weight of the peptide-eluted fraction after freeze-drying was 5.3 mg.

[0031]

[Example 2] Measurement of peptide anticoagulant activity (prothrombin time) 100 µl of 100-fold diluted Thromborel S solution (manufactured by Beringberge ) containing 25 mM CaCl ₂ was added with 100 µl of a peptide solution of 125 µg / ml and reacted at 37 ° C for 10 minutes. Let 100 μl of human plasma was added to it, and the clotting time after stirring was measured by an automatic blood coagulation measuring device (Amelung-Coaguromete
r, KC10A). The results are shown in Table 3.

As a result, extension of blood coagulation time was observed for peptide No. 1.

[0033]

[Table 3]

[0034]

Example 3 Anticoagulant activity of peptide in the presence of APC
Measurement of Romboplastin Time) 100 μl of a solution containing various concentrations of peptides and 250 ng of APC was heated at 37 ° C. for 10 minutes, 100 μl of human plasma was added, and further heated at 37 ° C. for 1 minute. There Actin (made by Baxter Dade) 100μ
1 of the reaction mixture was added thereto and reacted at 37 ° C. for 2 minutes.
100 μl of CaCl ₂ was added, and the clotting time after stirring was measured using an automatic blood coagulation measuring device (Amelung-Coagurometer, KC10A). The results are shown in Fig. 1.

As a result, A was assigned to peptides No. 1 and No. 2.
The action of suppressing the anticoagulant activity of PC was recognized.

[0036]

Example 4 Measurement of Inhibitory Effect on VIIa Activity To 10 μl of 1 μg / ml VIIa solution, 30 μl of peptide solution having various concentrations was added, and after heating at 37 ° C. for 15 minutes, 2.5 mM of synthetic substrate S was added. -2288 (Daiichi Scientific Co., Ltd.)
10 μl) was added, and the change in absorbance (absorbance / minute) at 405 nm was measured. The value when the peptide concentration was 0 was defined as 100%, and the results of the relative values are shown in FIG.

As a result, VII was added to peptides No. 1 and No. 2.
It was observed that the activity of a was suppressed in a dose-dependent manner.

[0038]

Example 5 Measurement of Xa activity inhibitory effect 30 μl of peptide solutions of various concentrations were added to 10 μl of 1 μg / ml Xa solution and heated at 37 ° C. for 15 minutes, and then 2.5 mM of synthetic substrate S was added thereto. -2222 (Daiichi Pure Chemicals Co., Ltd.)
10 μl) was added, and the change in absorbance (absorbance / minute) at 405 nm was measured. The value when the peptide concentration was 0 was defined as 100%, and the results of the relative values are shown in FIG.

As a result, Xa was found in peptides No. 1 and No. 2.
It was confirmed that the activity of sucrose was suppressed in a dose-dependent manner.

[0040]

Example 6 Measurement of Inhibitory Effect on APC Activity 30 μl of peptide solutions of various concentrations were added to 10 μl of 1 μg / ml APC solution, and after heating at 37 ° C. for 15 minutes,
There, 2.5 mM synthetic substrate S-2366 (Daiichi Scientific Co., Ltd.)
(Manufactured by the company) was added, and the change in absorbance (absorbance / minute) at 405 nm was measured. The value when the peptide concentration was 0 was defined as 100%, and the results of the relative values are shown in FIG.

As a result, peptide No. 1 was found to have a dose-dependent inhibitory effect on APC activity.

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ile Met Lys Arg 1 5 10 15 Phe Phe Phe Asn Ile Phe 20

SEQ ID NO: 2 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Phe Ile Asn Phe Phe Phe Arg Lys Met Ile Ala Lys Cys Pro Gly 1 5 10 15 Asp Asp Ala Lys Phe Ala 20

[Brief description of drawings]

FIG. 1 shows the anticoagulant activity of the peptide of the present invention in the presence of APC.

FIG. 2 shows the inhibitory effect of activated seventh factor activity by the peptide of the present invention.

FIG. 3 shows the inhibitory effect of the activated factor 10 activity by the peptides of the present invention.

FIG. 4 shows the inhibitory effect of APC activity by the peptide of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Koji Suzuki 6-35, Kamehamacho, Tsu City, Mie Prefecture

Claims (2)

[Claims] 1. A peptide consisting of the following amino acid sequence that regulates human blood coagulation: embedded image NH ₂ -Ala-Phe-Lys-Ala-Asp-Asp-Gly-Pro-Cys-Arg-Ala-Ile-Met -Lys-Arg 1 5 10 15 -Phe-Phe-Phe-Asn-Ile-Phe-COOH 20 2. A peptide consisting of the following amino acid sequence that controls coagulation of human blood: embedded image NH ₂ -Phe-Ile-Asn-Phe-Phe-Phe-Arg-Lys-Met-Ile-Ala-Lys-Cys -Pro-Gly 1 5 10 15 -Asp-Asp-Ala-Lys-Phe-Ala-COOH 20