HK1130209B - Therapeutic and/or ameliorating agent for disseminated intravascular coagulation - Google Patents

Description

Therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome

Technical Field

The present invention relates to a therapeutic agent and/or an ameliorating agent for disseminated intravascular coagulation syndrome, which has a higher therapeutic effect and contains thrombomodulin as an active ingredient. Further, the present invention relates to a method for selecting patients with disseminated intravascular coagulation syndrome to whom thrombomodulin should be administered.

Background

Disseminated intravascular coagulation syndrome (hereinafter sometimes abbreviated as DIC) is one of coagulation diseases, and is a disease in which: a large amount of blood vessel coagulation-promoting substances flow out due to tissue disorders in various diseases, the function of the coagulation system is extremely enhanced, small thrombi are generated in blood vessels of the whole body, and the small blood vessels are blocked; at the same time, platelets or coagulation factors necessary for controlling bleeding are consumed and become insufficient, resulting in coagulation abnormality. DIC causes circulatory disorders and shock, due to ischemia caused by accumulation of blood coagulation factors in the capillaries of the kidney and lung. Thus, tachypnea, dyspnea, tachycardia, bradycardia, cold limbs, oliguria, spasm and the like may occur, and sudden death due to shock may occur in some cases. At the same time, the consumption of blood coagulation factors and platelets produces bleeding tendency, which causes purpura, punctate bleeding, hemoptysis, hematemesis, hematuria, bloody stool, epistaxis, and the like of the skin. As therapeutic agents for DIC, heparin, antithrombin (also referred to as antithrombin III, hereinafter, also referred to as AT or ATIII) and the like are used.

On the other hand, thrombomodulin is known to be a substance that specifically binds to thrombin, has a function of inhibiting the blood coagulation activity of thrombin and remarkably promoting the protein C activation ability of thrombin, and is known to have a potent blood coagulation inhibitory action. Thrombomodulin is known to prolong the time of coagulation by thrombin and to inhibit platelet aggregation by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation and fibrinolysis system, and is activated by the action of thrombin to become activated protein C. It is known that the activated protein C inactivates factor V and factor VIII, which are active forms of coagulation factors, in vivo, and that the activated protein C is involved in the production of plasminogen activator having thrombolytic activity (non-patent document 1). Therefore, thrombomodulin promotes activation of protein C by this thrombin, and is useful as an anticoagulant or thrombolytic agent, and there has been a report on animal experiments which explain that thrombomodulin is effective for treatment and prevention of diseases accompanied by hypercoagulability (non-patent document 2).

Conventionally, thrombomodulin has been discovered and obtained as a glycoprotein expressed on vascular endothelial cells of various animal species including humans, and has been successfully cloned. That is, a gene of a human thrombomodulin precursor containing a signal peptide is cloned from a human lung cDNA library by a genetic engineering method, and then the entire gene sequence of thrombomodulin is analyzed to find an amino acid sequence of 575 residues containing a signal peptide (usually, 18 amino acid residues are mentioned) (patent document 1). It is known that a mature thrombomodulin in which a signal peptide is cleaved is composed of 5 regions of an N-terminal region (positions 1 to 226; the same is hereinafter assumed for the position where the signal peptide is 18 amino acid residues), a region having 6 EGF-like structures (positions 227 to 462), an O-type sugar chain-attached region (positions 463 to 498), a transmembrane region (positions 499 to 521), and an intracytoplasmic region (positions 522 to 557) from the N-terminal side of the mature peptide, and that the portion having the same activity as that of the full-length thrombomodulin (i.e., the minimum active unit) is a portion mainly composed of the 4 th, 5 th, and 6 EGF-like structures from the N-terminal side among the regions having 6 EGF-like structures (non-patent document 3).

In contrast to soluble thrombomodulin, which is difficult to dissolve unless a surfactant is present, and requires the addition of a surfactant as a preparation, soluble thrombomodulin is present which can be dissolved smoothly even in the absence of a surfactant. It is only necessary to prepare soluble thrombomodulin not containing at least a part or all of the transmembrane region, and it has been confirmed that, for example, soluble thrombomodulin composed of only 3 regions of the N-terminal region, the region having 6 EGF-like structures and the O-type sugar chain addition region (i.e., the region composed of the amino acid sequences at positions 19 to 516 of SEQ ID NO. 9) can be obtained by applying a recombinant technique, and that the recombinant soluble thrombomodulin has the activity of natural thrombomodulin (patent document 1). Some other examples of soluble thrombomodulin have been reported (patent documents 2 to 9). In addition, examples of natural forms include soluble thrombomodulin derived from human urine and the like (patent documents 10 and 11).

Incidentally, in the gene, as has been confirmed in many cases, a variety of variations are found in humans by natural variations or acquired variations, and a sequence in which the amino acid at the 473 rd position of the human thrombomodulin precursor composed of the above-mentioned 575-residue amino acid sequence is Val and a sequence in which the amino acid is Ala have been confirmed. In the nucleotide sequence encoding this amino acid, the nucleotide sequence has a mutation at position 1418 into T and C, respectively (non-patent document 4). However, there was no difference in activity and physical properties at all, and it was judged that both were substantially the same.

Thrombomodulin has been reported to be effective in the treatment of DIC (non-patent document 5). In addition to the above, the use of thrombomodulin is expected to be used for the treatment and prevention of diseases such as Acute Coronary Syndrome (ACS), thrombosis, peripheral vascular occlusion, arteriosclerosis obliterans, vasculitis, functional disorders secondary to cardiac surgery, complications of organ transplantation, angina pectoris, transient ischemic attack, gestational toxicosis, diabetes, Liver VOD (Liver Veno-ischemic disease; hepatic venous occlusion after fulminant hepatitis or bone marrow transplantation), deep vein thrombosis (DVT; deep venous thrombosis), and sepsis or Adult Respiratory Distress Syndrome (ARDS).

Patent document 1: japanese laid-open patent publication No. Sho 64-6219

Patent document 2: japanese laid-open patent publication No. 2-255699

Patent document 3: japanese laid-open patent publication No. 3-133380

Patent document 4: japanese laid-open patent publication No. 3-259084

Patent document 5: japanese laid-open patent publication No. 4-210700

Patent document 6: japanese patent laid-open publication No. 5-213998

Patent document 7: WO92/00325

Patent document 8: WO92/03149 publication

Patent document 9: WO93/15755 publication

Patent document 10: japanese laid-open patent publication No. 3-86900

Patent document 11: japanese laid-open patent publication No. 3-218399

Non-patent document 1: suzuki hong zhi, medicine Zi あゆみ (progress of medicine), Vol 125, p 901 (1983)

Non-patent document 2: good mi et al, Blood 75.1396-1399(1990)

Non-patent document 3: M.Zushi et al, J.biol.chem., 246, 10351-10353(1989)

Non-patent document 4: D.Z.Wen et al, Biochemistry, 26, 4350-4357(1987)

Non-patent document 5: S.M.Bates et al, Br.J.of Pharmacol, 144, 1017-

Disclosure of Invention

The present invention addresses the problem of providing a therapeutic agent and/or an ameliorating agent for DIC having a higher therapeutic effect, or a method for treating and/or ameliorating DIC. Another object of the present invention is to provide a method for selecting patients with disseminated intravascular coagulation syndrome to whom thrombomodulin should be administered.

As a treatment and/or improvement of DIC, an anticoagulant therapy using an anticoagulant, or a supplement of concentrated platelets and fresh frozen plasma is generally performed. Since the actual state of DIC is excessive activation of the coagulation system, anticoagulant therapy to inhibit it is widely performed in parallel with treatment for underlying diseases. Heparin and/or AT preparations are often used in the treatment and/or amelioration of DIC. Heparin is considered to be expressed by significantly promoting the blood coagulation factor inhibition rate of AT (Majerus PW, Tollefsen DM. "anticoagulant, thrombolytic およ" anti-platelet drug ", yokukan, fotemain, akashizhu shoji, goodman gilman pharmacology, 10 th edition, tokyo, guanchuan bookshop, 2003, p1937-63), so that heparin (yokukan jeidaho" disseminated intravascular coagulation syndrome と multiple organ failure "(disseminated intravascular coagulation syndrome and multiple organ failure), pharmaceutical Journal corporation) is used for patients having a high AT activity value In plasma, for example, about 80%. In addition, in patients whose AT activity value in plasma is, for example, about 70% or less, AT preparations are exemplified as typical drugs (Anthrobin P and Neuart are accompanied by instructions: administration and dose), and a method for treating and/or improving DIC by promoting an anticoagulant effect by increasing the AT activity value in plasma is mainly used. In this case, heparin may be used in combination as needed.

As described above, in order to treat and/or improve DIC, it is preferable to supplement to maintain AT activity value in plasma of patients with DIC AT least 70% or more, and in addition, it is considered that increasing AT activity value in plasma is common knowledge in treating and/or improving DIC, and in such a case, the present inventors have unexpectedly confirmed that thrombomodulin has higher effectiveness in patients having low AT activity value in plasma, such as an AT activity value in plasma of less than 50%, on the contrary, and thus completed the present invention. As a result, even though it was estimated in the past, it was unexpected that high effects were found in DIC patients having low plasma AT activity values of less than 50%.

If an AT preparation is administered, the AT preparation to be administered is a preparation made from precious human blood as a raw material, and further, the risk of transmission of infectious diseases due to the use of human blood as a raw material cannot be completely eliminated (Anthrobin P and Neuart's accompanying instructions: use notes), and there is a fear that AT is further expensive as a blood preparation, but the present invention can provide a preferable drug or DIC therapeutic agent and/or improving agent for solving these problems.

That is, the present invention includes the following embodiments.

A therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome, which is a drug containing thrombomodulin as an active ingredient, for administration to patients with disseminated intravascular coagulation syndrome having an antithrombin activity value in plasma of less than 50%;

the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to [1], wherein the thrombomodulin is soluble thrombomodulin;

the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to [1] or [2], wherein the thrombomodulin is a peptide obtained from transformed cells prepared by transfecting host cells with DNA encoding the amino acid sequence represented by SEQ ID NO. 1, 3, 5, 7, 9 or 11;

[3-2] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to [1] or [2], wherein the thrombomodulin is a peptide obtained from transformed cells prepared by transfecting host cells with DNA encoding the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 3;

[3-3] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to [1] or [2], wherein the thrombomodulin is a peptide obtained from transformed cells prepared by transfecting host cells with DNA encoding the amino acid sequence represented by SEQ ID NO. 5 or SEQ ID NO. 7;

[3-4] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to [1] or [2], wherein the thrombomodulin is a peptide obtained from transformed cells prepared by transfecting host cells with a DNA encoding the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 11;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is any one of a peptide having the 19 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3, a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity, or a mixture thereof;

[4-2] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having the sequence at positions 19 to 132 in each of SEQ ID Nos. 1 and 3, or a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity;

[4-3] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 in each of SEQ ID Nos. 1 and 3;

[4-4] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is any one of a peptide having the sequence at positions 19 to 132 or 17 to 132 in each of SEQ ID NO. 1 or SEQ ID NO. 3, a peptide having the same variant sequence of the above sequence and having thrombomodulin activity, or a mixture thereof;

[4-5] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 or 17 to 132 in each of SEQ ID NO. 1 or SEQ ID NO. 3, or a mixture thereof;

[4-6] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 132 th positions or 17 th to 132 th positions of SEQ ID NO. 1, or a mixture thereof;

[4-7] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence of 19 th to 132 th positions or 17 th to 132 th positions in SEQ ID NO. 3, or a mixture thereof;

[4-8] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 132 th positions or from the 17 th to 132 th positions of SEQ ID NO. 1, or a mixture thereof;

[4-9] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-2], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 132 th positions or from the 17 th to 132 th positions of SEQ ID NO. 3, or a mixture thereof;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is any one of a peptide having the 19 th to 480 th positions of SEQ ID NO. 5 or SEQ ID NO. 7, a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity, or a mixture thereof;

[5-2] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having the sequence at positions 19 to 480 in each of SEQ ID Nos. 5 and 7, or a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity;

[5-3] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence at position 19 to 480 in each of SEQ ID Nos. 5 and 7;

[5-4] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is any one of a peptide having the sequence at positions 19 to 480 or 17 to 480 in each of SEQ ID Nos. 5 and 7, a peptide having the same variant sequence of the above sequence and having thrombomodulin activity, or a mixture thereof;

[5-5] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 480 or 17 to 480 in each of SEQ ID Nos. 5 and 7, or a mixture thereof;

[5-6] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence of 19-480 th position or 17-480 th position in SEQ ID NO. 5, or a mixture thereof;

[5-7] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence of 19-480 th position or 17-480 th position in SEQ ID NO. 7, or a mixture thereof;

[5-8] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 480 th positions or from the 17 th to 480 th positions of SEQ ID NO. 5, or a mixture thereof;

[5-9] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-3], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 480 th positions or from the 17 th to 480 th positions of SEQ ID NO. 7, or a mixture thereof;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516 in SEQ ID NO. 9 or SEQ ID NO. 11, respectively; a peptide having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the above-mentioned peptide, and having thrombomodulin activity; or any one of their mixtures;

[6-2] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516 in SEQ ID NO. 9 or SEQ ID NO. 11; or a peptide having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the above-mentioned peptide, and having thrombomodulin activity;

[6-3] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516 in SEQ ID NO. 9 or SEQ ID NO. 11;

[6-4] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at position 19 to 516, position 19 to 515, position 17 to 516 or position 17 to 515 of SEQ ID NO. 9 or SEQ ID NO. 11, respectively; a peptide having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the above-mentioned peptide, and having thrombomodulin activity; or mixtures thereof;

[6-5] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at position 19 to 516, position 19 to 515, position 17 to 516 or position 17 to 515 of SEQ ID NO. 9 or SEQ ID NO. 11, respectively; or mixtures thereof;

[6-6] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515 of SEQ ID NO. 9; or mixtures thereof;

[6-7] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515 of SEQ ID NO. 11; or mixtures thereof;

[6-8] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide comprising a sequence from the 19 th to 516 th position, the 19 th to 515 th position, the 17 th to 516 th position, or the 17 th to 515 th position of SEQ ID NO. 9; or mixtures thereof;

[6-9] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide comprising a sequence from the 19 th to 516 th position, the 19 th to 515 th position, the 17 th to 516 th position or the 17 th to 515 th position of SEQ ID NO. 9;

[6-10] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide comprising a sequence from the 19 th to 516 th position, the 19 th to 515 th position, the 17 th to 516 th position or the 17 th to 515 th position of SEQ ID NO. 11; or mixtures thereof;

[6-11] the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1], [2] and [3-4], wherein the thrombomodulin is a peptide comprising a sequence from the 19 th to 516 th position, the 19 th to 515 th position, the 17 th to 516 th position or the 17 th to 515 th position of SEQ ID NO. 11;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [6-11], wherein the patients with disseminated intravascular coagulation syndrome have an antithrombin activity value in plasma of 40% or less;

note that the term numbers cited as in the above-mentioned [1] to [6-11] are indicated by ranges, and when an item having a branch number such as [6-2] is disposed in the ranges, it means that an item having a branch number such as [6-2] is also cited. The same applies to the following.

The therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [7], wherein the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome is a non-orally administered agent;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [8], wherein the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome is an intravenous administration agent;

the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [9], which is characterized by being continuously intravenously instilled at an administration amount of 0.02 to 0.08mg/kg per day for 4 hours or less;

the therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [10], which is not administered in combination with antithrombin;

the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [11], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome derived from hematopoietic malignant tumor or from infectious disease;

[12-1] the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of the above [1] to [11], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome derived from a hematopoietic malignancy;

[12-2] the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome according to any one of [1] to [11], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome caused by infection;

[12-3] A medicament for reducing the possibility of death of a human patient suffering from disseminated intravascular coagulation syndrome, wherein the medicament comprises thrombomodulin as an active ingredient for administration to disseminated intravascular coagulation syndrome patients having an antithrombin activity value in plasma of less than 50%;

[12-4] the pharmaceutical agent according to [12-3], wherein the thrombomodulin is the peptide according to any one of [1] to [6-11 ];

[12-5] the pharmaceutical agent according to [12-3], which has any one of the characteristics of [1] to [12-2 ];

a method of selecting a patient having disseminated intravascular coagulation syndrome to whom thrombomodulin should be administered, the method comprising the steps of: measuring an antithrombin activity value in plasma of a patient with disseminated intravascular coagulation syndrome, selecting a patient with disseminated intravascular coagulation syndrome having an antithrombin activity value of less than 50%, and determining that the patient is a patient with disseminated intravascular coagulation syndrome to whom thrombomodulin should be administered;

[13-2] the method of selecting a patient with disseminated intravascular coagulation syndrome to whom thrombomodulin should be administered, wherein the thrombomodulin is a peptide according to any one of the above [1] to [6-11 ];

[13-3] A therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome, which is a drug containing thrombomodulin as an active ingredient, for administration to patients with disseminated intravascular coagulation syndrome after determination of the antithrombin activity value in plasma;

[13-4] an agent for treating and/or ameliorating disseminated intravascular coagulation syndrome, which is a drug comprising thrombomodulin as an active ingredient, said drug being for administration to a patient suffering disseminated intravascular coagulation syndrome when the antithrombin activity value in plasma is measured and said antithrombin activity value is less than 50%;

a method for treating and/or improving disseminated intravascular coagulation syndrome, which comprises the step of administering thrombomodulin to a patient with disseminated intravascular coagulation syndrome whose antithrombin activity value in plasma is less than 50%;

the method according to [14] above, wherein the thrombomodulin is soluble thrombomodulin;

the method according to [14] or [15], wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO. 1, 3, 5, 7, 9 or SEQ ID NO. 11;

[16-2] the method according to [14] or [15], wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 3;

[16-3] the method according to [14] or [15], wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence represented by SEQ ID NO. 5 or SEQ ID NO. 7;

[16-4] the method according to [14] or [15], wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 11;

the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is any one of a peptide having the sequence at positions 19 to 132 of SEQ ID NO. 1 or SEQ ID NO. 3, a peptide having the same variant sequence of the above-mentioned sequence and having thrombomodulin activity, or a mixture thereof;

[17-2] the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is a peptide having the sequence at positions 19 to 132 of SEQ ID NO. 1 or SEQ ID NO. 3, or a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity;

[17-3] the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 of SEQ ID NO. 1 or SEQ ID NO. 3;

[17-4] the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is any one of a peptide having the sequence at the 19 th to 132 th positions or the 17 th to 132 th positions in SEQ ID NO. 1 or SEQ ID NO. 3, a peptide having the same variant sequence of the above sequence and having thrombomodulin activity, or a mixture thereof;

[17-5] the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO. 1 or SEQ ID NO. 3, respectively, or a mixture thereof;

[17-6] the method according to any one of [14], [15] or [16-2], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 132 th positions or 17 th to 132 th positions in SEQ ID NO. 1, or a mixture thereof;

[17-7] the method according to any one of the above [14], [15] or [16-2], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 132 th positions or 17 th to 132 th positions of SEQ ID NO. 3, or a mixture thereof;

[17-8] the method according to any one of [14], [15] and [16-2], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 132 th positions or from the 17 th to 132 th positions of SEQ ID NO. 1, or a mixture thereof;

[17-9] the method according to any one of the above [14], [15] or [16-2], wherein the thrombomodulin is a peptide having a sequence from the 19 th to 132 th positions or the 17 th to 132 th positions of SEQ ID NO. 3, or a mixture thereof;

the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is any one of a peptide having a sequence from position 19 to 480 in sequence No. 5 or sequence No. 7, a peptide having the same variant sequence of the above sequence and having thrombomodulin activity, or a mixture thereof;

[18-2] the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is a peptide having the sequence at position 19 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7, or a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity;

[18-3] the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7;

[18-4] the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is any one of a peptide having a sequence from the 19 th to the 480 th or from the 17 th to the 480 th positions of SEQ ID NO. 5 or SEQ ID NO. 7, a peptide having the same variant sequence of the above sequence and having thrombomodulin activity, or a mixture thereof;

[18-5] the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is a peptide having a sequence of 19 th to 480 th or 17 th to 480 th positions in SEQ ID NO. 5 or SEQ ID NO. 7, respectively, or a mixture thereof;

[18-6] the method according to any one of the above [14], [15] or [16-3], wherein the thrombomodulin is a peptide having a sequence of 19 th to 480 th positions or 17 th to 480 th positions in SEQ ID NO. 5, or a mixture thereof;

[18-7] the method according to any one of the above [14], [15] or [16-3], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 480 th positions or 17 th to 480 th positions of SEQ ID NO. 7, or a mixture thereof;

[18-8] the method according to any one of [14], [15] or [16-3], wherein the thrombomodulin is a peptide consisting of the 19 th to 480 th or 17 th to 480 th position sequence of SEQ ID NO. 5, or a mixture thereof;

[18-9] the method according to any one of the above [14], [15] or [16-3], wherein the thrombomodulin is a peptide consisting of the 19 th to 480 th or 17 th to 480 th position sequence of SEQ ID NO. 5, or a mixture thereof;

the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is any one of a peptide having the sequence at positions 19 to 516 of SEQ ID NO. 9 or SEQ ID NO. 11, a peptide having the same variant sequence of the above-mentioned sequence and having thrombomodulin activity, or a mixture thereof;

[19-2] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide having the sequence at positions 19 to 516 of SEQ ID NO. 9 or SEQ ID NO. 11, or a peptide having the same variant sequence of the aforementioned sequence and having thrombomodulin activity;

[19-3] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516 of SEQ ID NO. 9 or SEQ ID NO. 11, respectively;

[19-4] the method according to any one of the above [14], [15] or [16-4], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions in SEQ ID NO. 9 or SEQ ID NO. 11, respectively; a peptide having the same variant sequence as the above sequence and having thrombomodulin activity; or any one of their mixtures;

[19-5] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions in SEQ ID NO. 9 or SEQ ID NO. 11, respectively; or mixtures thereof;

[19-6] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide having a sequence of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions in SEQ ID NO. 9; or mixtures thereof;

[19-7] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515 of SEQ ID NO. 11; or mixtures thereof;

[19-8] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide consisting of a sequence from the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions of SEQ ID NO. 9; or mixtures thereof;

[19-9] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide consisting of a sequence from the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions of SEQ ID NO. 9;

[19-10] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide consisting of a sequence from the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions of SEQ ID NO. 11; or mixtures thereof;

[19-11] the method according to any one of [14], [15] or [16-4], wherein the thrombomodulin is a peptide consisting of a sequence from position 19 to 516, from position 19 to 515, from position 17 to 516 or from position 17 to 515 of SEQ ID NO. 11;

the method according to any one of the above [14] to [19-11], wherein the thrombomodulin is administered to a patient with disseminated intravascular coagulation syndrome whose antithrombin activity value in plasma is 40% or less;

the method according to any one of the above [14] to [20], wherein the thrombomodulin is administered non-orally;

the method according to any one of the above [14] to [21], wherein the thrombomodulin is administered intravenously;

the method according to any one of the above [14] to [22], wherein the intravenous drip of thrombomodulin is continued for 4 hours or less at an administration amount of 0.02 to 0.08mg/kg per day;

the method according to any one of the above [14] to [23], wherein the thrombomodulin is not administered in combination with antithrombin;

the method according to any one of the above [14] to [24], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome derived from a hematopoietic malignancy or from an infectious disease;

[25-1] the method according to any one of the above [14] to [24], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome derived from a hematopoietic malignancy;

[25-2] the method according to any one of the above [14] to [24], wherein the disseminated intravascular coagulation syndrome is disseminated intravascular coagulation syndrome caused by an infectious disease;

a method for reducing the likelihood of mortality in a human patient suffering from disseminated intravascular coagulation syndrome, in which method thrombomodulin is administered to disseminated intravascular coagulation syndrome patients having an antithrombin activity value in plasma of less than 50%;

[26-2] the method according to [26], wherein the thrombomodulin is the peptide according to any one of [1] to [6-11 ];

[26-3] A method for administration according to the method of [26] above, which comprises any one of the features of [1] to [25-2 ];

a method for treating and/or improving disseminated intravascular coagulation syndrome, which comprises the step of administering thrombomodulin to a patient with disseminated intravascular coagulation syndrome after determining an antithrombin activity value in plasma;

[27-2] A method for treating and/or ameliorating disseminated intravascular coagulation syndrome, which comprises the steps of: determining the antithrombin activity value in the plasma, and administering thrombomodulin to patients with disseminated intravascular coagulation syndrome when the antithrombin activity value is less than 50%.

By using the thrombomodulin-containing preparation of the present invention, it is possible to effectively treat and/or improve the symptoms of DIC patients having an AT activity value of less than 50% in plasma, preferably without using it in combination with AT.

Detailed Description

The present invention will be specifically described below.

According to the present invention, there is provided a therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome (DIC), which is a drug containing thrombomodulin as an active ingredient and which is administered to patients with disseminated intravascular coagulation syndrome having an antithrombin activity value in plasma of less than 50%.

That is, the therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome of the present invention is a therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome of patients with disseminated intravascular coagulation syndrome who have an antithrombin activity value in plasma of less than 50%.

Furthermore, the present invention also provides a medicament for reducing the likelihood of death in a human patient suffering from disseminated intravascular coagulation syndrome.

As one of preferable effects of treating and/or improving DIC, for example, "prevention of death of a patient due to DIC" is mentioned, and "prevention of deterioration of the general condition of a patient due to DIC" is also mentioned as a preferable effect.

DIC is a disease or syndrome as follows: a large amount of blood vessel coagulation-promoting substances are discharged due to tissue disorders in various diseases, the function of the coagulation system is extremely enhanced, and small thrombi (microthrombosis) are generated in blood vessels of the whole body to block the small blood vessels; at the same time, platelets or coagulation factors necessary for controlling bleeding are thereby consumed and become insufficient, resulting in abnormal hemostasis. Specifically, it has been found that, due to the formation of fibrin in blood vessels, hemorrhage symptoms are caused by consumption coagulation fibrinolysis or organ failure symptoms are caused by the formation of microthrombus. DIC is sometimes referred to as disseminated intravascular coagulation syndrome or generalized intravascular coagulation syndrome.

In diseases based on malignant tumors such as hematopoietic malignant tumors and solid cancers, DIC is caused by excessive activation of the coagulation system due to contact of blood with tissue factor expressed on tumor cells.

In addition, DIC is sometimes caused in the extension of systemic inflammatory response syndrome (hereinafter sometimes abbreviated as SIRS) caused by serious clinical invasion regardless of infectivity and noninfectivity.

SIRS is a pathological condition caused by an increase in the production of inflammatory cytokines associated with severe tissue damage or severe infection, and when this reaction proceeds excessively, DIC is caused by activation of neutrophils or activation of vascular endothelial cells. That is, SIRS is a preparatory stage for DIC (hereinafter, sometimes simply referred to as "preDIC"), and the therapeutic and/or ameliorating agent for DIC of the present invention can be effectively used for preDIC, and the therapeutic and/or ameliorating agent for DIC of the present invention also includes the treatment and/or amelioration of preDIC.

Clinical symptoms of DIC vary depending on the type of underlying disease, and it is preferable to diagnose DIC by scoring DIC with several test values as described below and scoring at a certain or higher score, in addition to observing bleeding symptoms and organ symptoms. Examples of the test values include the number of platelets in blood, the concentration of plasmin-decomposed fibrin and fibrinogen decomposition products (hereinafter sometimes abbreviated as "FDP"), the concentration of D-dimer, the fibrinogen concentration, and the prothrombin time. Furthermore, a diagnosis of PREDIC (a Chinese Yak doctor, China sick intravascular coagulation (DIC)) by a platelet lowering, D-dimer or FDP concentration increase or the like without scoring DIC may be performed by using に Seki する to investigate (an investigation report on a diagnostic standard using Disseminated Intravascular Coagulation (DIC)) an investigation class of blood coagulation disorders of specific disorders in major life, expressed as an 11-year study report, 1999: 65-72, export of an investigation class of blood coagulation disorders of specific disorders in early stages of DIC (an agent on a standard for starting early treatment of DIC), expressed as an 11-year study report, 1999: 73-77, a study report on blood of Chinese Yak and Japanese doctors, expressed as a state of DIC outage- アンケ - ト (a research report on DIC diagnosis of clinical results), 1999, 40: 362-364). Furthermore, it is also possible to diagnose preDIC by measuring the concentration of soluble fibrin or the concentration of thrombin-antithrombin complex in blood. Specific examples of diagnostic criteria for scoring DIC include the diagnostic criteria for Overt DIC (Taylor FB et al, Thromb Haemost2001, 86: 1327-.

The diagnostic method for DIC is not particularly limited as long as it is a diagnostic method for scoring DIC. In addition, there are other methods in which diagnosis of DIC is preferable when DIC needs to be treated from an early stage in terms of therapeutic effects, medical costs, and QOL (quality of life) of patients.

As a diagnostic method for scoring DIC, a similar diagnostic method for scoring DIC may be used in addition to the above diagnostic method.

The therapeutic and/or ameliorating agent for DIC of the present invention is not limited to the kind of DIC, but is preferably used for DIC derived from malignant hematopoietic tumors or DIC derived from infectious diseases, and more preferably used for DIC derived from infectious diseases. As a specific example of DIC derived from infectious diseases, DIC derived from sepsis is cited as a preferable example. In addition, another preferable embodiment is also an application to DIC derived from hematopoietic malignant tumor.

The therapeutic and/or ameliorating agent of the present invention may be used for sepsis. Sepsis is also localized to SIRS caused by serious clinical invasion of infectivity, and has a close relationship with DIC which is a disease causing infection. DIC is frequently complicated in sepsis, and the drug of the present invention may be used for patients with such DIC complicated sepsis. That is, in the present invention, it may be used for patients having or suspected of having either or both of DIC and sepsis.

Sepsis is known to be a severe systemic infection as follows: diseases such as infection, malignant tumor, liver cirrhosis, renal failure, diabetes, and abnormal childbirth; or treatment for wounds or diseases such as indwelling catheters, transfusion instruments, dialysis, tracheotomy, etc., microorganisms continuously or intermittently invade the blood from the focus of infection. When the symptoms worsen, septic shock is induced, that is, systemic shock is induced by rapid blood pressure reduction and peripheral circulation failure, and death is caused by the failure of important organs such as the lung, kidney, liver, heart, digestive tract, and central nervous system. In addition, as a complication accompanying sepsis, DIC is induced, or Adult Respiratory Distress Syndrome (ARDS) characterized by edema, hemorrhage or acute respiratory failure of the pulmonary interstitium is induced due to pulmonary capillary disorder accompanied by activation of neutrophils and accumulation of migration to the lung parenchyma, and the prognosis becomes very poor.

There are several methods as a diagnostic method of sepsis. This is described in Levy m.et al, crit.care.med., 31: 1250 and 1256. Including, for example, the method of making the diagnosis by a physician or the use of a test valueAnd the like. As an example of the latter, the following method is included: at 1) body temperature > 38 ℃ or < 36 ℃; 2) heart rate > 90/min; 3) the respiratory rate is more than 20/min or artificial respiration is necessary; 4) white blood cell number > 12000/mm³Or < 4000/mm³Or > 10% of blasts; within these 4, SIRS was diagnosed when 2 was satisfied, and SIRS proved to be or suspected to be causative of microorganisms was diagnosed as sepsis [ Larosa S., The homepage of Cleveland Clinic]. Methods similar to this are described in Members of the American College of Chest Physicians/Society of clinical Care Medicine Consenses Conference: crit Care Med, 20, 864-874 (1992).

Examples of the condition of a sepsis patient include bacteremia, sepsis (septicema), Systemic Inflammatory Response Syndrome (SIRS), sepsis (SIRS which is proved to be or suspected to be caused by microorganisms), severe sepsis, septic shock, refractory septic shock, and multiple organ dysfunction (hereinafter sometimes referred to as MODS) (harrison's original science 15 th edition 124P 828-833 Medical Sciences International). The above conditions may be indicated as symptoms effective as the therapeutic agent and/or the ameliorating agent of the present invention.

As the bacteremia, a state in which the presence of bacteria in blood was confirmed can be cited, and the presence of the bacteria was confirmed by positive blood culture.

As sepsis (septicema), a state in which the presence of microorganisms or other toxins in blood is confirmed can be cited.

The Systemic Inflammatory Response Syndrome (SIRS) is in the state of being ready for DIC as described above.

Examples of severe sepsis include sepsis accompanied by one or more of metabolic acidosis, acute encephalopathy, oliguria, hypoxemia, organ failure such as disseminated intravascular coagulation, and hypotension.

Examples of septic shock include septic shock accompanied by organ failure and unresponsiveness to resuscitation by fluid replacement at low blood pressure (blood pressure of 90mmHg or less or 40mmHg lower than normal blood pressure).

Examples of the refractory septic shock include a refractory septic shock which lasts for 1 hour or more and is not responsive to a fluid replacement or pressure raising agent.

Examples of the multi-organ dysfunction (MODS) include multi-organ dysfunction in which 1 or more organs are present and medical intervention is required to maintain homeostasis.

The therapeutic and/or ameliorating agent for DIC of the present invention can be effectively used in patients with AT-lowering. An AT-reduced patient refers to a patient whose plasma AT activity value is 70% or less and whose AT activity value is reduced as compared with that of a normal person. Examples of patients with reduced AT include patients with congenital AT deficiency and patients with acquired AT deficiency. Congenital AT deficiency patients refer to patients with a lower natural AT activity value than normal people, about 35-75%, and acquired AT deficiency patients refer to patients with an AT activity value which is reduced to below 70% in the day after the day. Specific examples of the cause of the decrease in the AT activity value in the latter day include liver diseases such as chronic liver failure, kidney diseases such as postoperative disease, shock, severe infection and nephrotic syndrome, digestive tract diseases such as inflammatory bowel disease, decrease in drug activity of heparin, decrease in age, premature neonates, diabetes, behcet's disease and malnutrition. In the present invention, whether an AT-reduced patient is congenital or acquired is not particularly limited, and may be any patient whose AT is reduced. The species of the patient is not particularly limited, but is preferably Japanese.

Among them, the therapeutic and/or ameliorating agent for DIC containing thrombomodulin as an active ingredient of the present invention is effective for treating and/or ameliorating DIC symptoms in patients having an AT activity value of less than 50% in plasma.

The AT activity is defined as an activity of neutralizing the blood coagulation activity of thrombin or an active type factor X, and as a general example, an AT activity value is represented by a relative value to an AT activity obtained from plasma obtained from a healthy adult (hereinafter referred to as normal plasma). The normal plasma to be used is not particularly limited, and any normal plasma that is generally used by those skilled in the art may be used, and international Standards of normal plasma (NIBSC) that are established by the Biological Standardization expert committee (expert committee on Biological Standardization (hereinafter, sometimes abbreviated as ECBS)) of the World Health Organization (WHO), managed and distributed by the National Institute for Biological Standards and control (hereinafter, sometimes abbreviated as NIBSC)) of the uk may be used. Further, normal plasma added to a normal AT activity value measurement kit or normal plasma specified by an AT activity value measurement kit may be used as the normal plasma added to a normal AT activity value measurement kit or normal plasma specified by an AT activity value measurement kit, and a normal plasma "first" (first chemical) may be used as a preferable example. The AT activity value was determined from the AT activity value based on the first "(first chemical).

The normal plasma is preferably an international standard for NIBSC. In addition, there are other ways to preferably use commercially available standard plasma with corrected AT activity using international standards for NIBSC. A more preferable example of the normal plasma includes a normal plasma agent "first" (first chemical). For example, the case where the value of AT activity in plasma is 50% means that the proportion of AT activity relative to the above normal plasma is 50%.

In order to effectively treat and/or ameliorate symptoms of DIC, the AT activity value in plasma of patients with DIC is not particularly limited, but is preferably less than 50%, the upper limit is preferably 48% or less, more preferably 46% or less, further preferably 44% or less, particularly preferably 42% or less, most preferably 40% or less, the lower limit is not particularly limited, and is preferably not less than the detection limit of the above-described measurement method, and is 0.1% or more, preferably 10% or more, more preferably 15% or more, further preferably 20% or more, particularly preferably 25% or more, and is most preferably 30% or more in view of the AT activity value of the patient group of test example 1 in the examples.

Further, according to the present invention, there is provided a therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome, which is a drug containing thrombomodulin as an active ingredient, for administration to patients with disseminated intravascular coagulation syndrome after measurement of the antithrombin activity value in plasma. The therapeutic agent and/or ameliorating agent of the present invention is not particularly limited as long as it is a drug to be administered to patients with disseminated intravascular coagulation syndrome after measurement of the antithrombin activity value in plasma, but is preferably a therapeutic agent and/or ameliorating agent for disseminated intravascular coagulation syndrome to patients with disseminated intravascular coagulation syndrome when the measured antithrombin activity value is less than 50%. In this way, it is preferable to administer thrombomodulin to patients with disseminated intravascular coagulation syndrome after measuring the antithrombin activity value in plasma, from the viewpoint of sufficiently exerting the drug effect.

The therapeutic and/or ameliorating agent for DIC of the present invention may be used for patients suffering from AT-lowering associated with liver diseases such as chronic liver failure, postoperative diseases, shock, severe infectious diseases, and the like.

The thrombomodulin of the present invention is known to have (1) an effect of selectively binding to thrombin and (2) an effect of promoting the activation of thrombin-based protein C. Preferably, (3) an effect of prolonging the thrombin-based coagulation time and/or (4) an effect of inhibiting the thrombin-based platelet aggregation are also usually confirmed. The action that these thrombomodulins have is sometimes referred to as thrombomodulin activity.

The thrombomodulin activity preferably has the effects of (1) and (2) above, and more preferably all the effects of (1) to (4) above.

The activity of promoting the activation of protein C by thrombin can be easily confirmed by, for example, a test method which is described in various publicly known documents as typified by Japanese patent application laid-open No. Sho 64-6219, and the amount of activity or the presence or absence of the activity promoting the activation of protein C. In addition, the effect of prolonging the thrombin-based coagulation time and/or the effect of inhibiting the thrombin-based platelet aggregation can also be easily confirmed.

The thrombomodulin in the present invention is not particularly limited as long as it has a thrombomodulin activity, and is preferably soluble thrombomodulin. Preferable examples of the solubility of the soluble thrombomodulin include solubility in water, for example, distilled water for injection (usually in the vicinity of neutrality in the absence of a surfactant such as triton X-100 or lauromacrogol), of 1mg/mL or more, or 10mg/mL or more, preferably 15mg/mL or more, or 17mg/mL or more, more preferably 20mg/mL or more, 25mg/mL or more, or 30mg/mL or more, particularly preferably 60mg/mL or more, and in some cases, 80mg/mL or more, or 100mg/mL or more, respectively. When judging whether or not the soluble thrombomodulin is dissolved, the index is clear and contains no clearly identifiable insoluble substance when visually observed at a position of about 1000 lux brightness immediately below a white light source after the dissolution. Further, the presence or absence of the residue may be checked by filtration.

The thrombomodulin in the present invention is preferably one that contains the amino acid sequences at the 19 th to 132 th positions of SEQ ID NO. 1, which are known as the central part of thrombomodulin activity in human thrombomodulin, and is not particularly limited as long as it contains the amino acid sequences at the 19 th to 132 th positions of SEQ ID NO. 1. The amino acid sequences 19 to 132 of SEQ ID NO. 1 may be naturally or artificially mutated, and one or more amino acids may be substituted, deleted, or added to the amino acid sequences 19 to 132 of SEQ ID NO. 1, as long as they have an action of promoting the activation of thrombin-based protein C, i.e., thrombomodulin activity. The allowable degree of variation is not particularly limited as long as it has thrombomodulin activity, and examples of the amino acid sequence include 50% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. Such amino acid sequences are referred to as identical variant sequences. Such a mutation can be easily obtained by using a general gene manipulation technique as described below.

The sequence of SEQ ID NO. 3 is a sequence in which the 125 th amino acid Val of the sequence of SEQ ID NO. 1 is mutated to Ala, and preferably contains the 19 th to 132 th amino acid sequences of SEQ ID NO. 3 as a thrombomodulin in the present invention.

As described above, the thrombomodulin in the present invention is not particularly limited as long as it contains the 19 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3, or a peptide sequence having at least the same variant sequence of the above sequences and having at least thrombomodulin activity, and preferable examples thereof include peptides composed of the 19 th to 132 th sequences or 17 th to 132 th sequences in SEQ ID NO. 1 or SEQ ID NO. 3, or peptides composed of the same variant sequence of the above sequences and having at least thrombomodulin activity, and more preferable peptides composed of the 19 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3. There are also other embodiments of a peptide having at least thrombomodulin activity, which is more preferably composed of the same variant sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO. 1 or SEQ ID NO. 3, respectively.

In addition, as the thrombomodulin in another embodiment of the present invention, it is preferable to include the amino acid sequences at the 19 th to 480 th positions of SEQ ID NO. 5, and the amino acid sequences are not particularly limited as long as they include the amino acid sequences at the 19 th to 480 th positions of SEQ ID NO. 5. The amino acid sequence of 19 th to 480 th positions of SEQ ID NO. 5 may be the same variant sequence as long as it has an action of promoting activation of thrombin-based protein C, i.e., thrombomodulin activity.

The sequence of SEQ ID NO. 7 is a sequence in which the amino acid Val at the 473 rd position of the sequence of SEQ ID NO. 5 is mutated to Ala, and preferably contains the amino acid sequences at the 19 th to 480 th positions of SEQ ID NO. 7 as the thrombomodulin in the present invention.

As described above, the thrombomodulin in the present invention is not particularly limited as long as it contains the 19 th to 480 th sequences of SEQ ID NO. 5 or SEQ ID NO. 7, or a peptide sequence having at least the same variant sequence of the above sequences and having at least thrombomodulin activity, and preferable examples thereof include a peptide comprising the 19 th to 480 th or 17 th to 480 th sequences of SEQ ID NO. 5 or SEQ ID NO. 7, or a peptide comprising the same variant sequence of the above sequences and having at least thrombomodulin activity, and more preferable a peptide comprising the 19 th to 480 th sequences of SEQ ID NO. 5 or SEQ ID NO. 7. There are also other embodiments of a peptide having at least thrombomodulin activity, which is more preferably composed of the same variant sequence at positions 19 to 480 or 17 to 480 in SEQ ID NO. 5 or SEQ ID NO. 7, respectively.

In addition, as the thrombomodulin in another embodiment of the present invention, it is preferable to include the amino acid sequences at the 19 th to 515 th positions of SEQ ID NO. 9, and there is no particular limitation as long as it includes the amino acid sequences at the 19 th to 515 th positions of SEQ ID NO. 9. The amino acid sequence of 19 th to 515 th positions of SEQ ID NO. 9 may be the same variant sequence as long as it has an action of promoting activation of thrombin-based protein C, i.e., thrombomodulin activity.

The sequence of SEQ ID NO. 11 is a sequence in which the amino acid Val at position 473 of the sequence of SEQ ID NO. 9 is mutated to Ala, and preferably contains the amino acid sequences at positions 19 to 515 of SEQ ID NO. 11 as a thrombomodulin in the present invention.

As described above, the thrombomodulin in the present invention is not particularly limited as long as it contains the 19 th to 515 th sequences of SEQ ID NO. 9 or SEQ ID NO. 11, or a peptide sequence having at least the same variant sequence of the above sequences and having at least thrombomodulin activity, and preferable examples thereof include a peptide comprising the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th sequences in SEQ ID NO. 9 or SEQ ID NO. 11, or a peptide comprising the same variant sequence of the above sequences and having at least thrombomodulin activity, and particularly preferable examples thereof include a peptide comprising the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th sequences in SEQ ID NO. 9. Mixtures thereof may be mentioned as preferred examples. In addition, there are other embodiments in which a peptide having a sequence from the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions in SEQ ID NO. 11 is particularly preferable. Mixtures thereof may be mentioned as preferred examples. Further, a peptide having at least thrombomodulin activity, which is formed of the same variant sequence as the above-mentioned sequence, is also exemplified as a preferable example.

As described above, the peptides having the same variant sequence are peptides in which one or more (i.e., one or two or more, and more preferably a plurality of (e.g., 1 to 20, preferably 1 to 10, more preferably 1 to 5, and particularly preferably 1 to 3)) amino acids can be substituted, deleted, or added to the amino acid sequence of the target peptide. The allowable degree of variation is not particularly limited as long as it has thrombomodulin activity, and examples of the amino acid sequence include 50% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

Furthermore, as the thrombomodulin of the present invention, a peptide composed of the sequence of SEQ ID NO. 14 (462 amino acid residues), a peptide composed of the sequence of SEQ ID NO. 8 (272 amino acid residues), or a peptide composed of the sequence of SEQ ID NO. 6 (236 amino acid residues) in Japanese patent laid-open No. 64-6219 can be cited as preferable examples.

The thrombomodulin in the present invention is not particularly limited as long as it has at least the amino acid sequence of the 19 th to 132 th positions of SEQ ID NO. 1 or SEQ ID NO. 3, and among them, a peptide having at least the amino acid sequence of the 19 th to 480 th positions of SEQ ID NO. 5 or SEQ ID NO. 7 is preferable, and a peptide having at least the amino acid sequence of the 19 th to 515 th positions of SEQ ID NO. 9 or SEQ ID NO. 11 is more preferable. As the peptide having at least the 19 th to 515 th amino acid sequences of SEQ ID NO. 9 or 11, there may be mentioned, as a more preferred example, peptides having the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th amino acid sequences of SEQ ID NO. 9 or 11, respectively. Further, a mixture of peptides of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th, or 17 th to 515 th sequences of SEQ ID NO. 9 or 11, respectively, with respect to SEQ ID NO. 9 or 11, is more preferable.

In the case of the above mixture, the mixing ratio of the peptide from the 17 th position to the peptide from the 19 th position in each of SEQ ID NO. 9 and SEQ ID NO. 11 is (30: 70) to (50: 50), preferably (35: 65) to (45: 55).

The mixing ratio of the peptide terminating at position 515 and the peptide terminating at position 516 in SEQ ID NO. 9 or 11 is (70: 30) to (90: 10), preferably (75: 25) to (85: 15).

The mixing ratio of these peptides can be determined by a usual method.

The 19 th to 132 th sequences of the sequence number 1 correspond to the 367 th to 480 th sequences of the sequence number 9, and the 19 th to 480 th sequences of the sequence number 5 correspond to the 19 th to 480 th sequences of the sequence number 9.

The 19 th to 132 th bit sequences of sequence No. 3 correspond to the 367 th to 480 th bit sequences of sequence No. 11, and the 19 th to 480 th bit sequences of sequence No. 7 correspond to the 19 th to 480 th bit sequences of sequence No. 11.

Further, the sequences at positions 1 to 18 in each of the sequence numbers 1, 3, 5, 7, 9 and 11 are all the same sequence.

As described later, these thrombomodulin of the present invention can be obtained from transformed cells prepared by transfecting DNA encoding the peptide of SEQ ID NO. 1, 3, 5, 7, 9 or 11 (specifically, the base sequence of SEQ ID NO. 2, 4, 6, 8, 10 or 12, respectively) into host cells with a vector.

Further, these peptides may have the above amino acid sequence, and may have a sugar chain added thereto or not, and this point is not particularly limited. In addition, in gene manipulation, the kind, position or degree of addition of the sugar chain varies depending on the kind of host cell used, and it can be used arbitrarily. The binding position and type of the sugar chain are known from Japanese patent application laid-open No. 11-341990, and the same sugar chain may be added to the same position to the thrombomodulin of the present invention. As described later, it is not limited to the case of obtaining by gene manipulation, but when obtaining by gene manipulation, the signal sequence that can be used for expression may be a nucleotide sequence encoding the amino acid sequence at positions 1 to 18 of the aforementioned SEQ ID NO. 9, a nucleotide sequence encoding the amino acid sequence at positions 1 to 16 of the SEQ ID NO. 9, or other known signal sequences (for example, a signal sequence of human histotypic plasminogen activator (International publication No. 88/9811, Japanese patent application laid-open No. 11-341990)).

When the DNA sequence encoding thrombomodulin is introduced into a host cell, a method of introducing the DNA sequence encoding thrombomodulin into a vector (particularly preferably an expression vector which can be expressed in animal cells) is preferable. The expression vector refers to a DNA molecule comprising a promoter sequence, a sequence which imparts a ribosome binding site to mRNA, a DNA sequence encoding a protein to be expressed, a splicing signal, a terminator sequence for termination of transcription, a replication initiation sequence, etc., and examples of preferred animal cell expression vectors include pSV2-X reported by R.C. Mullgan et al [ Proc.Natl.Acad.Sci.U.S.A.78.2072(1981) ], and pBP69T (69-6) reported by P.M.Howleuss et al [ Methods in enzymology, 101, 387, Academic Press (1983) ], etc.

Examples of host cells that can be used for producing these peptides include Chinese Hamster Ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, dog kidney-derived MDCK cells, hamster AV-12-664 cells, and human-derived cells include HeLa cells, W138 cells, and human 293 cells. CHO cells are generally highly preferred, and among CHO cells, DHFR-CHO cells are more preferred.

In addition, in the genetic engineering process or the peptide preparation process, microorganisms such as Escherichia coli are also used in many cases, and it is preferable to use a suitable host-vector system for each, and an appropriate vector system can be selected from among the above host cells. The gene of thrombomodulin used in gene recombination technology has been cloned, and examples of production of thrombomodulin by gene recombination technology have been disclosed, and purification methods for obtaining purified products are also known [ Japanese patent application laid-open No. Sho 64-6219, Japanese patent application laid-open No. Hei 2-255699, Japanese patent application laid-open No. Hei 5-213998, Japanese patent application laid-open No. Hei 5-310787, Japanese patent application laid-open No. Hei 7-155176, J.biol.chem., 264: 10351-10353(1989)]. Therefore, the thrombomodulin used in the present invention can be produced by using the method described in the above report or according to the method described therein. For example, Japanese patent application laid-open No. Sho 64-6219 discloses Escherichia coli K-12 strain DH5(ATCC deposit No. 67283) containing plasmid pSV2TMJ2 containing DNA encoding full-length thrombomodulin. Furthermore, a strain (Escherichia coli DH5/pSV2TMJ2) (FERM BP-5570) which was restored from the life research (Collection of International patent organism, national institute of advanced Industrial science and technology). The thrombomodulin of the present invention can be produced by a known gene manipulation technique using the DNA encoding the full-length thrombomodulin as a raw material.

The thrombomodulin used in the present invention may be produced by a conventionally known method or according to a conventionally known method, and for example, the method described in the above-mentioned Shanben et al (Japanese patent application laid-open No. Sho 64-6219) or Japanese patent application laid-open No. Hei 5-213998 can be referred to. That is, the human thrombomodulin gene may be further modified as necessary after it is prepared, for example, as a DNA encoding the amino acid sequence of SEQ ID NO. 9 by gene manipulation techniques. For example, in order to prepare a DNA encoding the amino acid sequence of SEQ ID NO. 11 (specifically, the base sequence of SEQ ID NO. 12), the nucleotide sequence of the codon encoding the 473 rd amino acid (specifically, the base of 1418) of the amino acid sequence of SEQ ID NO. 9 was modified by using Method in Enzymology, 100: 468 (1983), Academic Press, the method described therein, carries out site-specific mutagenesis. For example, a DNA obtained by converting the base T at position 1418 of SEQ ID NO. 10 into the base C can be formed using a synthetic DNA for mutation having the base sequence represented by SEQ ID NO. 13.

The DNA thus prepared may be introduced into, for example, Chinese Hamster Ovary (CHO) cells to prepare transformed cells, which may be appropriately selected, and purified thrombomodulin may be prepared from a culture solution obtained by culturing the cells by a known method. As described above, it is preferable to transfect the DNA encoding the amino acid sequence of SEQ ID NO. 9 (SEQ ID NO. 10) into the above host cell. The method for producing thrombomodulin to be used in the present invention is not limited to the above-mentioned methods, and for example, it may be extracted and purified from urine, blood, other body fluids, etc., or extracted and purified from a tissue producing thrombomodulin, a tissue culture solution thereof, etc., or may be subjected to cleavage treatment with a protease, if necessary.

When the thrombomodulin of the present invention is produced by the above cell culture method, the N-terminal amino acid may be found to have diversity by post-translational modification of the protein. For example, the amino acid at position 17, 18, 19 or 22 of SEQ ID NO. 9 may form the N-terminus. In addition, for example, the N-terminal amino acid may be modified as in the case of converting glutamic acid at position 22 into pyroglutamic acid. Preferably, the amino acid at position 17 or 19 forms the N-terminus, and more preferably the amino acid at position 19 forms the N-terminus. In addition, there are other ways in which the amino acid at position 17 is preferably N-terminal. The same examples as those of SEQ ID NO. 11 can be given for the above modifications, diversifications, and the like.

Further, when thrombomodulin is produced using a DNA having the base sequence of SEQ ID NO. 10, a diversity of C-terminal amino acids may be found, and a peptide having 1 amino acid residue in less may be produced. That is, the C-terminal amino acid may be modified as in the case where the amino acid at position 515 forms the C-terminus and the amino acid at position 515 is amidated. Therefore, it is possible to prepare a peptide or a mixture thereof rich in diversity of the N-terminal amino acid and the C-terminal amino acid. Preferably, the amino acid at position 515 forms the C-terminus. In addition, there are other ways in which it is preferable that the amino acid at position 516 form the C-terminus. The above modifications, diversifications, and the like are also the same for the DNA having the base sequence of SEQ ID NO. 12.

The thrombomodulin obtained by the above method may be a mixture of peptides having diversity at the N-terminus and the C-terminus. Specifically, there can be mentioned a mixture of peptides having the sequence of SEQ ID NO. 9 at positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515.

Next, the method for isolating and purifying thrombomodulin from the culture supernatant or the culture obtained as described above can be carried out according to a known method [ Horikoshu et al, basic experiment method for protein-enzyme ]. For example, a chromatographic carrier having a functional group having a charge opposite to that of thrombomodulin immobilized thereon, and ion exchange chromatography or adsorption chromatography utilizing an interaction between thrombomodulins are preferably used. In addition, affinity chromatography utilizing specific affinity with thrombomodulin is also a preferred example. As a preferred example of the adsorbent, thrombin, which is a ligand of thrombomodulin, or an antibody against thrombomodulin, is used. As such an antibody, an antibody of thrombomodulin having an appropriate property or recognizing an appropriate epitope can be used, and examples thereof include those described in, for example, Japanese patent publication No. 5-42920, Japanese patent application laid-open No. 64-45398, Japanese patent application laid-open No. 6-205692, and the like. Further, gel filtration chromatography or ultrafiltration using the molecular weight of thrombomodulin can be mentioned. Further, there are a chromatographic carrier having a hydrophobic group immobilized thereon and a hydrophobic chromatogram utilizing hydrophobic bonding with a hydrophobic site possessed by thrombomodulin. Furthermore, hydroxyapatite can be used as a carrier for adsorption chromatography, and examples thereof include those described in Japanese patent application laid-open No. 9-110900. These methods may be appropriately combined. The degree of purification may be selected depending on the intended use, and is preferably such that a single band is obtained as a result of electrophoresis (preferably SDS-PAGE), or a single peak is formed as a result of gel filtration HPLC or reverse phase HPLC of the separated purified product. Of course, when two or more thrombomodulin are used, it is preferable that substantially only a band of thrombomodulin is formed, and it is not required that a single band is formed.

Specifically, the purification method of the present invention includes a method of purifying thrombomodulin using its activity as an index, and examples thereof include the following methods: the culture supernatant or the culture was roughly purified by Q-sepharose Fast Flow on an ion exchange column to recover a fraction having thrombomodulin activity, followed by main purification on a DIP-thrombin-agarose (dissopropylthrombin agar) column of an affinity column to recover a fraction having strong thrombomodulin activity, concentration of the recovered fraction, and gel filtration to obtain a thrombomodulin active fraction in a pure form [ good k.et al, Blood, 75: 1396-1399(1990)]. Examples of the thrombomodulin activity as an index include a promoting activity of thrombin-based activation of protein C. Further, preferred purification methods are as follows.

Selecting proper ion exchange resin with good adsorption condition with thrombomodulin, and carrying out ion exchange chromatography purification. A particularly preferred example is a method using Q-Sepharose Fast Flow equilibrated with 0.02M Tris-hydrochloric acid buffer (pH7.4) containing 0.18M NaCl. After the appropriate washing, the thrombomodulin can be obtained as a crude purified product by, for example, elution with 0.02M Tris-hydrochloric acid buffer solution (pH7.4) containing 0.3M NaCl.

Then, for example, it is possible to specifically bind thrombomodulinThe affinity substance is immobilized on a resin for affinity chromatography purification. As preferable examples, there may be mentioned a DIP-thrombin-agarose column and an anti-thrombomodulin monoclonal antibody column. The DIP-thrombin-agarose column is equilibrated in advance with, for example, 20mM Tris hydrochloric acid buffer solution (pH7.4) containing 100mM NaCl and 0.5mM calcium chloride, and the crude purified product is packed, washed as appropriate, and eluted with, for example, 20mM Tris hydrochloric acid buffer solution (pH7.4) containing 1.0M NaCl and 0.5mM calcium chloride, whereby thrombomodulin can be obtained as a purified product. In addition, in the antithrombotic monoclonal antibody chromatographic column, the following methods can be mentioned: 0.1M NaHCO containing 0.5M NaCl dissolved with an anti-thrombomodulin monoclonal antibody₃The buffer (pH8.3) was contacted with Sepharose 4FF (GE Healthcare Bio-Sciences) previously activated by CNBr, and the column packed with the resin in which Sepharose 4FF was coupled with the anti-thrombomodulin monoclonal antibody was equilibrated in advance with, for example, a 20M phosphate buffer solution (pH 7.3) containing 0.3M NaCl, washed appropriately, and then eluted with, for example, 100mM glycine hydrochloride buffer solution (pH 3.0) containing 0.3M NaCl. The eluate is neutralized with an appropriate buffer solution to obtain a purified product.

The purified product thus obtained was adjusted to pH 3.5, and then packed in a cation exchanger (preferably a strong cation exchanger SP-Sepharose FF (GE Healthcare Bio-Sciences)) equilibrated with 100mM glycine hydrochloride buffer (pH 3.5) containing 0.3M NaCl, and washed with the same buffer to obtain a non-adsorbed fraction. The obtained fraction is neutralized with an appropriate buffer solution to obtain a purified product of high purity. They are preferably concentrated by ultrafiltration.

Further, it is preferable to perform buffer exchange by gel filtration. For example, a high-purity purified product concentrated by ultrafiltration is packed in a Sephacryl S-300 column or S-200 column equilibrated with a 20mM phosphate buffer solution (pH 7.3) containing 50mM NaCl, separation is performed with a 20mM phosphate buffer solution (pH 7.3) containing 50mM NaCl, the activity of promoting the activation of thrombin-based protein C is confirmed, and the active fraction is recovered to obtain a buffer-exchanged high-purity purified product. The thus obtained high-purity purified product is preferably filtered with an appropriate virus-removing membrane, for example, Planova 15N (asahi chemical industry co., ltd.) for safety, and then concentrated to a desired concentration by ultrafiltration. Preferably, filtration is finally carried out through sterile filtration membranes.

The thrombomodulin thus obtained can be prepared into a freeze-dried preparation by a usual method. Namely, the following methods can be mentioned: the solution containing thrombomodulin and, if necessary, additives is frozen and dried by sublimating water under reduced pressure.

In the present invention, the AT activity value in plasma may be an AT activity value in artery-derived plasma or an AT activity value in vein-derived plasma, preferably an AT activity value in vein-derived plasma. In addition, AT activity values in plasma derived from arteries are also preferable in some cases. Generally refers to the value of AT activity in plasma derived from veins. The method for measuring the AT activity value is not particularly limited, and the AT activity value can be measured, for example, by a chromogenic synthetic substrate method in which the AT activity value is determined from the residual amount of the active tenth factor.

Specifically, a blood sample is collected in a blood collection tube containing sodium citrate, and the blood sample is rapidly centrifuged to collect plasma. Next, heparin is added to the plasma to form an AT-heparin complex, and a constant excess amount of an active tenth factor is added to the AT-heparin complex. The active tenth factor is inactivated by forming an AT-heparin-active tenth factor complex according to the amount of AT-heparin complex in the sample. After the complex formation reaction, a matrix solution is added to carry out the reaction, and the colorimetric determination of the free paranitroaniline is carried out, whereby the remaining activity of the active tenth factor can be measured. Since the remaining activity of the active tenth factor reflects the AT activity in the sample, the AT activity value can be obtained by a standard curve prepared separately. For colorimetric determination of p-nitroaniline, an autoanalyzer BM1650 (manufactured by Nippon electronics Co., Ltd.) was used. Further, the standard curve can be made by using a standard substance.

The dose of the therapeutic and/or ameliorating agent for DIC containing thrombomodulin as an active ingredient of the present invention administered in 1 day varies depending on the age, body weight, degree of disease, route of administration and the like of a patient, and usually the upper limit is preferably 5mg/kg or less, more preferably 2mg/kg or less, further preferably 1mg/kg or less, and particularly preferably 0.8mg/kg or less, and the lower limit is preferably 0.005mg/kg or more, more preferably 0.01mg/kg or more, further preferably 0.02mg/kg or more, and particularly preferably 0.05mg/kg or more, based on the amount of thrombomodulin.

Administered 1 time every 1 day or several times as needed. The administration interval may be 1 time per day, or 1 time per 2 to 14 days, more preferably 1 time per 3 to 10 days, and still more preferably 1 time per 4 to 7 days.

Particularly in intravenous administration, 1 time per 1 day is preferable, but not limited thereto. In addition, in subcutaneous administration, preferably every 1 day of 1 or 1 week of 1 times of administration, can be according to the dose to adjust the dosing interval.

The method of administering the therapeutic and/or ameliorating agent for DIC comprising thrombomodulin as an active ingredient of the present invention may be carried out by a generally used administration method, that is, a non-oral administration method (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.). Intramuscular administration or subcutaneous administration is particularly preferable in terms of enabling the administration interval to be prolonged by maintaining the blood concentration for a long period of time. In addition, there are other ways in which intravenous administration is preferred. In addition, oral administration, intrarectal administration, intranasal administration, sublingual administration, and the like may also be employed.

For intravenous administration, a method of administering a desired amount at once or intravenous administration by drip may be mentioned.

From the viewpoint of short administration time, a method of administering a desired amount at once is preferred. In the case of single administration, the administration with a syringe usually requires a time, and the time required for the administration varies depending on the amount of liquid to be administered, but is usually 2 minutes or less, more preferably 1 minute or less, and still more preferably 30 seconds or less. The lower limit is not particularly limited, but is preferably 1 second or more, more preferably 5 seconds or more, and further preferably 10 seconds or more. The dose is not particularly limited, and may be any of the above-mentioned preferred doses.

The administration is preferably carried out intravenously by instillation in view of ease of keeping the blood level of thrombomodulin constant, and is not particularly limited as long as the above-mentioned preferred amount is given, but the upper limit of the amount administered per 1 day is preferably 1mg/kg or less, more preferably 0.5mg/kg or less, still more preferably 0.1mg/kg or less, particularly preferably 0.08mg/kg or less, and most preferably 0.06mg/kg or less, and the lower limit thereof is preferably 0.005mg/kg or more, more preferably 0.01mg/kg or more, further preferably 0.02mg/kg or more, and particularly preferably 0.04mg/kg or more.

The administration time in intravenous drip administration is preferably 4 hours or less, more preferably 3 hours or less, further preferably 2 hours or less, particularly preferably 1 hour or less, and most preferably 30 minutes or less as the upper limit, and preferably 10 minutes or more, more preferably 15 minutes or more, and further preferably 20 minutes or more as the lower limit.

When the lyophilized preparation is prepared, it can be administered to a patient after being dissolved in water, such as distilled water (or water for injection), physiological saline, etc., at the time of use.

Further, when a liquid preparation is prepared, it can be prepared by mixing an effective amount of thrombomodulin with a carrier that can be used for a medicament. That is, in order to treat the above-mentioned diseases, an effective amount of thrombomodulin may be mixed with a known appropriate amount of a carrier to prepare a preparation suitable for effective administration to a patient. For example, thickeners such as sucrose, glycerin, methylcellulose, carboxymethylcellulose, etc. may be added; various inorganic salt pH adjusters and the like are prepared as additives.

[ description of sequence Listing ]

Sequence number 1: amino acid sequence encoded by gene for production of TME456

Sequence number 2: base sequence encoding the amino acid sequence of SEQ ID NO. 1

Sequence number 3: amino acid sequence encoded by gene for production of TME456M

Sequence number 4: base sequence encoding the amino acid sequence of SEQ ID NO. 3

Sequence number 5: amino acid sequence encoded by a Gene for the production of TMD12

Sequence number 6: a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 5

Sequence number 7: amino acid sequence encoded by a Gene for the production of TMD12M

Sequence number 8: base sequence encoding the amino acid sequence of SEQ ID NO. 7

Sequence number 9: amino acid sequence encoded by a Gene for the production of TMD123

Sequence number 10: base sequence encoding the amino acid sequence of SEQ ID NO. 9

Sequence number 11: amino acid sequence encoded by a Gene for the production of TMD123M

Sequence number 12: a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 11

Sequence number 13: synthetic DNA for mutation used for site-specific mutagenesis

Examples

The present invention will be specifically described below by way of examples and test examples, but the present invention is not limited to these examples at all.

The thrombomodulin of the present invention used in the test examples was prepared according to the method of the above-mentioned Shanben et al (the method described in Japanese patent application laid-open No. Sho 64-6219). This production example will be explained below.

Production example 1

< obtaining of thrombomodulin >

A thrombomodulin was obtained by transfecting DNA encoding the amino acid sequence of SEQ ID NO. 9 (specifically, the base sequence of SEQ ID NO. 10) into Chinese Hamster Ovary (CHO) cells and obtaining a high-purity purified product in which an active fraction was recovered from a culture solution of the transformed cells in a 20mM phosphate buffer solution (pH 7.3) containing 50mM NaCl by the purification method of the conventional method described above. Further, the solution was subjected to ultrafiltration to obtain a thrombomodulin (hereinafter, sometimes abbreviated as TMD123) solution having a concentration of 11.2 mg/mL.

< preparation of additive solution >

480g of arginine hydrochloride (manufactured by Ajinomoto corporation) was weighed into a 10L stainless steel container, and 5L of water for injection was added thereto to dissolve the arginine hydrochloride. 1M sodium hydroxide solution was added to adjust the pH to 7.3.

< preparation of drug solution-filling >

The total amount of the additive solution was put into a 20L stainless steel vessel, and 2398mL of the above-obtained TMD123 solution (26.88 g based on the amount of soluble thrombomodulin protein, 12% excess thereof) was added thereto, followed by mixing and stirring. Further, water for injection was added to a total amount of 12L, and the mixture was uniformly mixed and stirred. The liquid medicine was filtered and sterilized by a filter (MCGL 10S made by Millipore) having a pore size of 0.22 μm. The filtrate was filled into vials (visual), 1mL per Vial, half stoppered with rubber stoppers.

< lyophilization >

The freeze-drying process was carried out in the order of freeze-drying → nitrogen gas filling → full-pressure rubber stopper → cap screwing under the following conditions to obtain a TMD 123-containing preparation containing 2mg of soluble thrombomodulin and 40mg of arginine hydrochloride in 1 container.

< conditions of lyophilization >

Pre-cooling (from room temperature to 15 ℃ C. in 15 minutes) → formal cooling (from 15 ℃ C. to-45 ℃ C. in 2 hours) → maintenance (-45 ℃ C. in 2 hours) → vacuum start (-45 ℃ C. in 18 hours) → warming (from-45 ℃ C. to 25 ℃ C. in 20 hours) → maintenance (25 ℃ C. in 15 hours) → warming (from 25 ℃ C. to 45 ℃ C. in 1 hour) → maintenance (45 ℃ C. for 5 hours) → room temperature (from 45 ℃ C. to 25 ℃ C. in 2 hours) → double-pressure nitrogen filling (to-100 mgHg) → full pressure plug → screw cap-up ℃. /)

Production example 2

A DNA encoding the amino acid sequence of SEQ ID NO. 11 (specifically, the base sequence of SEQ ID NO. 12) was transfected into Chinese Hamster Ovary (CHO) cells, a purified thrombomodulin (hereinafter, sometimes abbreviated as TMD123M) solution was obtained from the culture solution of the transformed cells by the purification method of the above-mentioned conventional method, and a lyophilized preparation of TMD123M was obtained by the same method as described above.

Production example 3

A DNA encoding the amino acid sequence of SEQ ID NO. 1 (specifically, the base sequence of SEQ ID NO. 2) was transfected into Chinese Hamster Ovary (CHO) cells, purified thrombomodulin (hereinafter, sometimes abbreviated as TME456) was obtained from the culture broth of the transformed cells by the purification method of the aforementioned conventional method, and a lyophilized preparation of TME456 was obtained by the same method as described above.

Production example 4

A DNA encoding the amino acid sequence of SEQ ID NO. 3 (specifically, the base sequence of SEQ ID NO. 4) was transfected into Chinese Hamster Ovary (CHO) cells, purified thrombomodulin (hereinafter, sometimes abbreviated as TME456M) was obtained from the culture broth of the transformed cells by the purification method of the aforementioned conventional method, and a lyophilized preparation of TME456M was obtained by the same method as described above.

Production example 5

A DNA encoding the amino acid sequence of SEQ ID NO. 5 (specifically, the base sequence of SEQ ID NO. 6) was transfected into Chinese Hamster Ovary (CHO) cells, purified thrombomodulin (hereinafter, sometimes abbreviated as TMD12) was obtained from the culture broth of the transformed cells by the purification method of the above-mentioned conventional method, and a lyophilized preparation of TMD12 was obtained by the same method as described above.

Production example 6

A DNA encoding the amino acid sequence of SEQ ID NO. 7 (specifically, the base sequence of SEQ ID NO. 8) was transfected into Chinese Hamster Ovary (CHO) cells, purified thrombomodulin (hereinafter, sometimes abbreviated as TMD12M) was obtained from the culture broth of the transformed cells by the purification method of the above-mentioned conventional method, and a lyophilized preparation of TMD12M was obtained by the same method as described above.

[ test example 1]

For patients with DIC whose symptoms are induced by hematopoietic malignancy or infection as diagnosed by DIC based on DIC diagnostic criteria of the general health and welfare province as shown below, the therapeutic and/or ameliorating agents for DIC of the present invention prepared by the following methods were administered, and the survival rate of patients on day 28 after the start of administration was examined.

It is to be noted that the AT preparation is not used during the administration period of the therapeutic and/or ameliorating agent for DIC of the present invention.

< preparation example 1>

To the lyophilized preparation of TMD123 obtained in production example 1, physiological saline was added to prepare a test solution of 0.5 mg/ml.

< measurement of AT Activity value >

4.5ml of a blood sample of a subject patient before administration on the day of starting administration of the therapeutic and/or improving agent for DIC of the present invention was collected in a blood collection tube containing 0.5ml of 3.8% sodium citrate, and after mixing and centrifugation by inversion, plasma was collected and stored by freezing. Heparin is then added to the plasma to form an AT-heparin complex, and an excess amount of factor Xa is further added to the AT-heparin complex. After the complex formation reaction, a substrate solution (Test-team S ATIII (CHROMOGENIX Co., Ltd)) was added to the reaction mixture to carry out a reaction, and the residual factor Xa activity was measured by measuring the absorbance at 410nm of free p-nitroaniline and carrying out colorimetric quantification (Test-team S ATIII, attached manual). Since the remaining factor Xa activity reflects the AT activity in the sample, the AT activity value was determined using a separately prepared calibration curve. For colorimetric quantification of p-nitroaniline, an automatic analyzer BM1650 (manufactured by Nippon electronics Co., Ltd.) was used. As normal plasma used for determining the AT activity value, a normal plasma agent "first" (first chemical) was used.

< dosage and administration method >

The specimen solution prepared in the preparation example was collected at a ratio of 0.12ml/kg (equivalent to 0.06mg/kg) from patients suffering from DIC, and added to physiological saline to prepare 100 ml. The physiological saline solution was intravenously instilled 1 time a day for 30 minutes. This operation was repeated for 6 days.

< results >

When the test solution was administered by the above administration method and death on day 28 after the start of administration was examined, the survival rate of the patient group having an AT activity value of 50% or more and less than 70% was 64.3%, and the survival rate of the patient group having an AT activity value of 70% or more was 80%. On the other hand, for the survival rate of the patient group having an AT activity value of less than 50%, only 1 case of the death cases, the survival rate was 92.3%, and the survival rate was superior for the above 2 groups. The results are shown in Table 1.

In addition, in patients (5 cases) in which the AT activity value was 40% or less, the survival rate was 100%.

[ Table 1]

[ test example 2]

The AT activity value in rat plasma was reduced to less than 60% by administering a rabbit anti-rat AT antibody (20mg/kg) to the tail vein of male Sprague-Dawley rats (body weight 180-240 g). After 1 hour of antibody administration, tissue factor (rabbit-derived thromboplastin; 90mg/kg) was continuously administered to the tail vein of rats for 1 hour under pentobarbital anesthesia. 0.1 to 10mg/kg of thrombomodulin of various production examples of the present invention was administered into the tail vein on the opposite side immediately before administration of the tissue factor. The effect of the present invention can be confirmed by collecting blood from the abdominal aorta after completion of administration of the tissue factor, measuring the number of platelets, fibrinogen or AT activity value, and examining the therapeutic and/or ameliorating effect of DIC.

Industrial applicability

By using the thrombomodulin-containing preparation of the present invention, it is possible to effectively treat and/or improve the symptoms of DIC patients having an AT activity value of less than 50% in plasma, preferably without using it in combination with AT.

Sequence listing

<110> Asahi Kasei Pharma corporation

<120> therapeutic and/or ameliorating agent for disseminated intravascular coagulation syndrome

<130>F107098-WO

<160>13

<210>1

<211>132

<212>PRT

<213> human

<400>1

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro

20 25 30

Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro

35 40 45

Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala

50 55 60

Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro

65 70 75 80

Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu

85 90 95

Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly

100 105 110

Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile

115 120 125

Gly Thr Asp Cys

130

<210>2

<211>396

<212>DNA

<213> human

<400>2

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgacccg 60

tgcttcagag ccaactgcga gtaccagtgc cagcccctga accaaactag ctacctctgc 120

gtctgcgccg agggcttcgc gcccattccc cacgagccgc acaggtgcca gatgttttgc 180

aaccagactg cctgtccagc cgactgcgac cccaacaccc aggctagctg tgagtgccct 240

gaaggctaca tcctggacga cggtttcatc tgcacggaca tcgacgagtg cgaaaacggc 300

ggcttctgct ccggggtgtg ccacaacctc cccggtacct tcgagtgcat ctgcgggccc 360

gactcggccc ttgtccgcca cattggcacc gactgt 396

<210>3

<211>132

<212>PRT

<213> human

<400>3

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro

20 25 30

Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro

35 40 45

Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala

50 55 60

Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro

65 70 75 80

Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu

85 90 95

Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly

100 105 110

Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile

115 120 125

Gly Thr Asp Cys

130

<210>4

<211>396

<212>DNA

<213> human

<400>4

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgacccg 60

tgcttcagag ccaactgcga gtaccagtgc cagcccctga accaaactag ctacctctgc 120

gtctgcgccg agggcttcgc gcccattccc cacgagccgc acaggtgcca gatgttttgc 180

aaccagactg cctgtccagc cgactgcgac cccaacaccc aggctagctg tgagtgccct 240

gaaggctaca tcctggacga cggtttcatc tgcacggaca tcgacgagtg cgaaaacggc 300

ggcttctgct ccggggtgtg ccacaacctc cccggtacct tcgagtgcat ctgcgggccc 360

gactcggccc ttgcccgcca cattggcacc gactgt 396

<210>5

<211>480

<212>PRT

<213> human

<400>5

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys

465 470 475 480

<210>6

<211>1440

<212>DNA

<213> human

<400>6

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgtcc gccacattgg caccgactgt 1440

<210>7

<211>480

<212>PRT

<213> human

<400>7

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys

465 470 475 480

<210>8

<211>1440

<212>DNA

<213> human

<400>8

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgccc gccacattgg caccgactgt 1440

<210>9

<211>516

<212>PRT

<213> human

<400>9

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys

465 470 475 480

Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro

485 490 495

Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu

500 505 510

Val His Ser Gly

515

<210>10

<211>1548

<212>DNA

<213> human

<400>10

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgtcc gccacattgg caccgactgt 1440

gactccggca aggtggacgg tggcgacagc ggctctggcg agcccccgcc cagcccgacg 1500

cccggctcca ccttgactcc tccggccgtg gggctcgtgc attcgggc 1548

<210>11

<211>516

<212>PRT

<213> human

<400>11

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys

465 470 475 480

Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro

485 490 495

Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu

500 505 510

Val His Ser Gly

515

<210>12

<211>1548

<212>DNA

<213> human

<400>12

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgccc gccacattgg caccgactgt 1440

gactccggca aggtggacgg tggcgacagc ggctctggcg agcccccgcc cagcccgacg 1500

cccggctcca ccttgactcc tccggccgtg gggctcgtgc attcgggc 1548

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of DNA

<400>13

aatgtggcgg gcaagggccg a 21

Claims (5)

1. Use of thrombomodulin in the preparation of a medicament for treating and/or improving disseminated intravascular coagulation syndrome in patients with disseminated intravascular coagulation syndrome having an antithrombin activity value in plasma of 40% or less, wherein the thrombomodulin is a peptide obtained from transformed cells prepared by transfecting host cells with a DNA encoding the amino acid sequence described in SEQ ID NO. 9 or SEQ ID NO. 11.

2. The use according to claim 1, wherein the thrombomodulin is soluble thrombomodulin.

3. The use according to claim 1 or 2, wherein the thrombomodulin is not administered in combination with antithrombin.

4. Use of thrombomodulin, which is a peptide obtained from transformed cells prepared by transfecting host cells with a DNA encoding the amino acid sequence recited in seq id No. 9 or 11, for the preparation of a medicament for reducing the probability of death of a human patient having disseminated intravascular coagulation syndrome with an antithrombin activity value in plasma of 40% or less.

5. The use according to claim 4, wherein the thrombomodulin is soluble thrombomodulin.