JP2768564B2 - Anticoagulant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anticoagulant containing the polypeptide represented by SEQ ID NO: 1 as an active ingredient.

[0002]

2. Description of the Related Art It is well known that when a blood vessel is damaged, two mechanisms work to stop the blood. One is a thrombus due to platelets (primary hemostasis), and the other is a blood clot (secondary hemostasis) formed by fibrin taking in red blood cells. 1
In the next hemostasis, the platelets adhere to the collagen and the like exposed by the blood vessel damage, and then deform to form a strong thrombus and stop the hemostasis.
At this time, serotonin is released from platelets to contract blood vessels. It is also known that thrombi in blood vessels through which blood flows quickly consist mainly of small amounts of fibrin and platelets.

[0003]

Cathepsin G is known as one of the substances that induces a series of platelet activations (Laboratory Investigation, 62 , 409, 1990).
That is, cathepsin G released by activated neutrophils causes platelet aggregation and serotonin release. Therefore, a drug that inhibits cathepsin G is considered to suppress platelet aggregation and serotonin release and prevent thrombosis, and is therefore expected as an antiplatelet agent or antithrombotic agent.

On the other hand, human polymorphonuclear leukocyte elastase inhibitor (hereinafter referred to as SLPI) is present in human saliva and semen, and its structure has been determined (RC Thompson et al., Nu.
cl. Acid Res. 14 , 7883-7896, 1986;
No. 2).

[0005] This SLPI has an inhibitory activity on chymotrypsin-like enzymes such as leukocyte elastase and cathepsin G, but also has an inhibitory activity on physiologically important trypsin-like enzymes such as trypsin. For this reason, SLPI is therefore difficult to use as a therapeutic agent for human diseases, despite having excellent inhibitory activities leading to elastase inhibitors and cathepsin G inhibitors.

[0006] For this reason, S by genetic engineering techniques
The first half domain (Ala at position 1-Pro at position 54) was removed from the LPI (Ala at position Ser-107), and the second half domain (55
Position of Asn - 107 of the Ala; hereinafter referred to as (Asn ⁵⁵ -Ala ¹⁰⁷⁾ SLPI) is prepared, this (Asn ⁵⁵ -Ala ¹⁰⁷⁾ SLPI is, SL
It has already been confirmed that SLPI does not have the trypsin-like enzyme inhibitory activity while maintaining the elastase inhibitory activity of PI (WO89 / 06239). However, whether or not such an SLPI second half domain maintains the cathepsin G inhibitory activity of SLPI has not been clarified so far.

[0007] Therefore, the present inventors have determined that the SLPI late domain has cathepsin G inhibitory activity,
The PI late domain has been found to potently inhibit cathepsin G-mediated platelet aggregation and serotonin release, and have reached the present invention.

[0008]

That is, according to the present invention,
Substantially different from the amino acid sequence represented by SEQ ID NO: 1.
That the polypeptide or one of the amino acid sequence, or
An anticoagulant is provided which comprises, as an active ingredient, a polypeptide having a biologically equivalent amino acid sequence in which a plurality of amino acids have been deleted or added .

In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 refers to SEQ ID NO: 1.
A polypeptide consisting of 53 amino acids represented by
Say.

[0010] The polypeptides having these biologically equivalent amino acid sequence, SEQ ID NO: amino acid sequence or found one or more amino acids represented by 1 is deleted, to the amino acid sequence a polypeptide consisting of one or more amino acids added to it are the amino acid sequence,
It has biological properties as the anticoagulant of the present invention.

[0011] Such polypeptides include, for example, 1 from Asn at position 55 of SLPI represented by SEQ ID NO: 1.
Has an amino acid sequence up to Ala at position 07 (Asn ⁵⁵ -Al
a ¹⁰⁷ ) SLPI. Such a polypeptide can be obtained, for example, by isolating and purifying natural SLPI present in airway secretions, saliva, nasal secretion, sexual fluid, uterine mucus, etc., and treating it with proteolytic enzymes, or the amino acid sequence of the polypeptide. Can be expressed alone or as a fusion protein and, if necessary, treated with hydroxylamine or thrombin to obtain the desired polypeptide. Among them, for example, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI is preferably obtained by a genetic engineering technique as described in WO 89/06239.

The anticoagulant of the present invention can be administered orally or parenterally such as intravenously, subcutaneously, intramuscularly, transdermally, and rectally. Examples of the dosage form for oral administration include tablets, pills, granules, powders, suspensions, capsules and the like.

For tableting, excipients such as lactose, starch and crystalline cellulose; binders such as carboxymethylcellulose, methylcellulose and polyvinylpyrrolidone; disintegrants such as sodium alginate, sodium hydrogencarbonate and sodium lauryl sulfate And the like, and can be molded by an ordinary method. Pills, powders,
Granules can also be formed by a usual method using the above-mentioned excipients and the like.

Solutions and suspensions include, for example, tricaprylin,
It can be formed by a usual method using glycerin esters such as triacetin and alcohols such as ethanol. Capsules can be formed by filling granules, powders, solutions or the like into capsules such as gelatin.

As a dosage form for subcutaneous, intramuscular, or intravenous administration, an injection in the form of an aqueous or non-aqueous solution is known. Physiological saline or the like is used as the aqueous solution. As the water-insoluble solution, for example, propylene glycol, polyethylene glycol, olive oil, ethyl oleate and the like are used, and if necessary, a preservative, a stabilizer and the like are added. The injection is sterilized by appropriate treatment such as filtration through a bacteria-retaining filter and blending of a bactericide.

As the dosage form for transdermal administration, for example, ointments,
Creams and the like can be mentioned, and ointments are formed by a usual method using a fatty oil such as castor oil or olive oil; vaseline or the like, and creams are formed from a fatty oil; be able to. For rectal administration, usual suppositories such as gelatin soft capsules are used.

The dosage of the polypeptide of the present invention varies depending on the administration route, age of the patient, sex, and the degree of the disease.
The dose is usually about 1 to 1000 mg / dose,
It is preferable to adjust the preparation so as to satisfy such conditions. The anticoagulant of the present invention can be used in combination with an existing drug.

[0018]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

[0019] In accordance with an embodiment of Reference Example 1 (Asn ⁵⁵ -Ala ¹⁰⁷⁾ SLPI manufacturing WO89 / 06239 Pat (Asn ⁵⁵ -Al
a ¹⁰⁷ ) SLPI was manufactured. Represented by SEQ Table 1 (Asn ⁵⁵ -
Ala ¹⁰⁷ ) A DNA fragment encoding SLPI was chemically synthesized in E. coli using appropriate codons. Leu-Val-Pro-Ar that can cleave the human growth hormone gene with thrombin
through the DNA sequence coding for the g fused to (Asn ⁵⁵ -Ala ^107). The expression vector (plasmid pGH-TE) thus obtained was introduced into E. coli HB101. Culturing the transformed cells (E. coli HB 101 strain (pGH-TE), to give a capsule combined, and purified by thrombin treatment and S-S reconfiguration further chromatography, the objective (Asn ⁵⁵ -Ala ¹⁰⁷⁾
Got SLPI. The E. coli HB101 strain (pGH-TE)
It has been internationally deposited on December 1, 1988, with the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology, as Microfabrication Research Institute No. 2168 (FERM BP-2168).

[0020] for Example 1 Human neutrophil elastase and cathepsin G and (Asn ⁵⁵ -Ala ¹⁰⁷⁾ SLPI inhibitory activity was measured by a synthetic substrate method. The enzyme concentrations of human neutrophil elastase and cathepsin G in the measurement solution were 0.2 μM, and the synthetic substrates were N-succinyl-ala-ala-ala-p-nitroanilide and N-suc, respectively.
Using cinyl-ala-ala-pro-phe-p-nitroanilide, the measurement results are shown in FIG. 1 (% human neutrophil elastase activity) and FIG. 2 (% cathepsin G activity). In the figure, * indicates a significant difference (p <0.05). As is clear from FIGS. 1 and 2, (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI inhibited both enzymes in a concentration-dependent manner. Furthermore, the concentration (IC ₅₀ value) of the polypeptide exhibiting a 50% inhibitory concentration was determined, and the results are shown in Table 1.

[0021]

[Table 1]

Example 2 Activation of human platelets induced by cathepsin G, ie, (As) on human platelet aggregation and serotonin release
n ⁵⁵ -Ala ¹⁰⁷ )
The measurement was performed by changing the concentration of (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI at 0.2 μM, and the measurement results are shown in FIG. 3 (platelet aggregation rate%) and FIG. 4 (serotonin release rate%). In the figure, * indicates a significant difference (p <0.05).

As apparent from FIGS. 3 and 4, (Asn
⁵⁵ -Ala ¹⁰⁷ ) SLPI inhibited platelet aggregation and serotonin release in a concentration-dependent manner. Furthermore, the concentration (IC ₅₀ value) of the polypeptide exhibiting a 50% inhibitory concentration was determined, and the results are shown in Table 2.

[0024]

[Table 2]

Example 3 The inhibitory activity of (Asn ⁵⁵ -Ala ¹⁰⁷ ) SLPI on human platelet aggregation by cathepsin G released by neutrophils was measured in a system in which neutrophils and human platelets coexist. In a test tube in which neutrophils and human platelets are simultaneously present, the concentration was changed in the range of 0 to 2.5 μM as shown in FIGS. 5 and 6 (Asn ⁵⁵ -Al
a ¹⁰⁷ ) Add SLPI, and 1 minute later, add neutrophil stimulant formy
After adding 0.5 μM of l-Met-Leu-Phe (FMLP), platelet aggregation and serotonin release were measured. The results are shown in FIG. 5 (platelet aggregation rate%) and FIG. 6 (serotonin release rate%). In the figure, * indicates a significant difference (p <0.05).

As apparent from FIGS. 5 and 6, (Asn
^55- Ala ¹⁰⁷ ) SLPI inhibited platelet aggregation and serotonin release in a concentration-dependent manner, with significant (p <0.05) minimum inhibitory concentrations of 1.0 μM and 0.75 μM, respectively. In addition, 50%
Determine the concentration of the polypeptide indicating the inhibitory concentration (IC ₅₀ value), and the results are shown in Table 3.

[0027]

[Table 3]

On the other hand, β from neutrophils stimulated by FMLP
-glucuronidase release did not change by (Asn ⁵⁵ -Ala ^{1 07)} SLPI added. Thus, rather than direct action on (Asn ⁵⁵ -Ala ¹⁰⁷⁾ SLPI platelet aggregation inhibitory activity neutrophils, F
It was understood to be due to the inhibition of cathepsin G released by neutrophils upon MLP stimulation.

[Sequence list]

SEQ ID NO: 1 Sequence length: 53 Sequence type: amino acid Topology: linear Sequence type: protein Fragment type: C-terminal fragment Sequence: Asn Pro Thr Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln 1 5 10 15 Cys Leu Met Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys 20 25 30 Lys Arg Asp Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val 35 40 45 Ser Pro Val Lys Ala 50

[Brief description of the drawings]

FIG. 1 is a graph showing the esterase inhibitory activity of a polypeptide in Example 1.

FIG. 2 shows the polypeptide cathepsin G in Example 1.
It is a graph which shows an inhibitory activity.

FIG. 3 is a graph showing the platelet aggregation inhibitory activity of a polypeptide in Example 2.

FIG. 4 is a graph showing the serotonin release inhibitory activity of a polypeptide in Example 2.

FIG. 5 is a graph showing the platelet aggregation inhibitory activity of a polypeptide in Example 3.

FIG. 6 is a graph showing the serotonin release inhibitory activity of a polypeptide in Example 3.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Michel Sinore, France, 75724 Paris CedEx 15 Lou de Dr. Lou, 25, Institut Pasteur (72) Inventor Patricia Lenest, France, 75724 Paris Sedex 15 Lou de Dr. Lou, 25, Institue Pasteur (56) References JP-A-62-501291 (JP, A) European Patent Application Publication 373335 (EP, A) Laboratory Investigation, Vol. 62, No. 4 (1990) p. 409-416 (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/43-38/58 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] An amino acid sequence represented by SEQ ID NO: 1.
Or an amino acid sequence consisting essentially of
May have one or more amino acids deleted
Or a blood coagulation inhibitor comprising, as an active ingredient, a polypeptide comprising a biologically equivalent amino acid sequence . 2. The blood coagulation inhibitor according to claim 1, which is an antiplatelet aggregation disease agent. 3. The anticoagulant according to claim 1, which is an antithrombotic disease agent. 4. The anticoagulant according to claim 1, wherein the polypeptide is a polypeptide obtained by a genetic engineering technique.