RU2799764C2 - Medicinal product for the therapeutic treatment and/or improvement of the condition of sepsis associated with coagulopathy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method of therapeutic treatment and/or improvement of the condition of a patient with sepsis. A method of therapeutic treatment and/or improvement of the condition of a patient with sepsis involves administering a medicinal product containing thrombomodulin as an active ingredient, where the said medicinal product is administered to a patient whose International Normalized Ratio (INR) value is greater than 1.4 immediately prior to administration, where the term “immediately before administration” means a period of 1 hour, calculated retrospectively from the start of administration of a medicinal product containing thrombomodulin, where the medicinal product is administered to a patient whose platelet count is greater than 30,000/mm³ just before administration.

EFFECT: above invention provides a method of effective therapeutic treatment and/or improvement in sepsis accompanied by coagulopathy.

11 cl, 1 dwg, 4 tbl, 8 ex

Description

The field of technology to which the invention belongs

[0001]

The present invention relates to a medicament for the therapeutic treatment and/or amelioration of sepsis accompanied by coagulopathy.

Prerequisites for the creation of the invention

[0002]

Sepsis is a systemic inflammatory response syndrome (SIRS) induced by infection. Specifically, sepsis is defined as a pathological condition characterized, in addition to the presence of infection, by two or more of the following SIRS features ((1) body temperature > 38°C or < 36°C, (2) heart rate > 90/min, (3) respiratory rate > 20/min, or PaCO ₂ <32 torr, and (4) leukocyte count > 12,000/mcL or < 4,000/mcL, or immature leukocytes. citations > 10%). Although much attention has been paid to the presence of bacteria in the blood (bacteremia) until now, a bacteria-positive blood culture was not mandatory according to the above definition. With regard to sepsis, a condition in which there is organ dysfunction, organ hypoperfusion, or hypotension is called severe sepsis. Organ hypoperfusion or abnormal perfusion includes lactic acidosis, oliguria, confusion, and the like. Of the states of severe sepsis, the state of continuous hypotension despite a sufficient load of infusion therapy is called septic shock (Non-Patent Document 1). It is believed that the severe circulatory disorder observed in these pathological conditions is caused by a malfunction of the sympathetic nervous system or a mediator released from neutrophils, etc., and organ dysfunction is caused by tissue hypoxia (dysoxia).

[0003]

Thrombomodulin is known as a substance that specifically binds to thrombin, inhibiting the blood-clotting activity of thrombin, and at the same time exhibits anticoagulant activity, significantly increasing the ability of thrombin to activate protein C. Thrombomodulin is also known to increase prothrombin time or inhibit platelet aggregation caused 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. Activated protein C is known to inactivate activated blood coagulation factor V and activated blood coagulation factor VIII in vivo and participate in the formation of a plasminogen activator having a thrombolytic effect (Non-Patent Document 2). Accordingly, thrombomodulin is believed to promote the activation of protein C by thrombin and is therefore useful as an anticoagulant or thrombolytic agent. It has also been reported that, in an animal experiment, thrombomodulin is effective for treating or preventing diseases associated with a hypercoagulable state (Non-Patent Document 3).

[0004]

Thrombomodulin was first discovered and obtained as a glycoprotein expressed on vascular endothelial cells of various animal species, including humans, and then successfully cloned. Specifically, a human thrombomodulin precursor gene that includes a signal peptide was cloned from a human lung cDNA library by genetic engineering, and the entire thrombomodulin gene sequence was analyzed, and as a result, an amino acid sequence of 575 residues containing a signal peptide was obtained (generally, 18 amino acid residues are cited as an example) (Patent Document 1). It is known that mature thrombomodulin, which is formed by cleavage of the signal peptide, consists of 5 regions, namely, an N-terminal region (amino acid residues 1 to 226, these positions are determined based on the assumption that the signal peptide consists of 18 amino acid residues, and the same applies to the following descriptions), a region having six EGF-like structures (amino acid residues 227 to 462), an O -linked glycosylation (amino acid residues 463 to 498), transmembrane region (amino acid residues 499 to 521) and cytoplasmic region (amino acid residues 522 to 557) on the N-terminal side of the mature peptide. It is also known that a partial protein having the same activity as that of a full-length thrombomodulin (i.e., the minimum active unit) is mainly composed of 4th, 5th, and 6th EGF-like structures from the N-terminal side in a region having six EGF-like structures (Non-Patent Document 4).

[0005]

Full-length thrombomodulin is practically insoluble in the absence of a surfactant, and the addition of a surfactant is necessary to obtain a whole thrombomodulin preparation. In this case, soluble thrombomodulin is known, which can be completely dissolved even in the absence of a surfactant. Soluble thrombomodulin can be obtained by removing at least part of the transmembrane region or the entire transmembrane region. For example, it was confirmed that soluble thrombomodulin consisting of only 3 regions, namely, an N-terminal region, a region having six EGF-like structures, and an O-linked glycosylation region (i.e., soluble thrombomodulin having an amino acid sequence consisting of amino acid residues 19-516 of SEQ ID NO: 9), can be obtained using recombinant methods, and that the resulting recombinant soluble thrombomodulin has the same activity , as well as naturally occurring thrombomodulin activity (patent document 1). There are also some other reports regarding soluble thrombomodulins (patent documents 2-9). Soluble thrombomodulin derived from human urine, and the like. also described as native thrombomodulins (patent documents 10 and 11).

[0006]

As recognized in many cases, as a result of spontaneous mutations or mutations occurring at the time of production, polymorphic mutations have been found in human genes. Thrombomodulin proteins have now been identified in which the amino acid at position 473 of a human thrombomodulin precursor having an amino acid sequence of 575 amino acid residues is converted to Val or Ala. In the nucleotide sequence encoding the amino acid sequence, this amino acid residue change corresponds to a mutation in T or C at position 1418 (Non-Patent Document 5). However, these two types of thrombomodulin are completely identical in terms of their activity and physicochemical properties, and they can be considered almost identical.

[0007]

Thrombomodulin has been reported to be effective for the therapeutic treatment of disseminated intravascular coagulation (hereinafter also referred to as DIC) (Non-Patent Document 6). Regarding the use of thrombomodulin, in addition to the above use, thrombomodulin is expected to be used for the therapeutic and prophylactic treatment of various diseases, such as acute coronary syndrome (ACS), thrombosis, peripheral vascular obstruction, obstructive arteriosclerosis, vasculitis, post-cardiac functional disorder, organ transplant complication, angina pectoris, transient ischemic attack, toxemia of pregnancy, diabetes, VOD of the liver (veins occlusive liver disease such as fulminant hepatitis, veno-occlusive liver disease following bone marrow transplantation), deep vein thrombosis (DVT), sepsis, and adult respiratory distress syndrome (ARDS) (Patent Documents 12-14).

[0008]

As is known, the international normalized ratio (hereinafter also abbreviated "INR") in a plasma sample from a patient with sepsis indicates coagulopathy. For example, the 2003 Congress on Critical Care Medicine (CCM) listed INR > 1.5 as well as PPT > 60 seconds as criteria for coagulopathy (Crit. Care Med., 31, pp. 1250-1256 (2003)). However, the INR value has not yet been approved as a clear criterion because the value has not been confirmed by clinical trials and the like. In fact, in phase III clinical trials for the treatment of patients with severe sepsis, Tyfacogin, as a tissue factor pathway inhibitor in the class of anticoagulants, showed a more favorable result in the group of patients with INR ≤ 1.2 than in the group of patients with INR > 1.2 as a result of a clinical trial whose main target was patients with INR > 1.2 (JAMA, July 9, Vo. 290, No. 2, pp.238-247 (200 3)). In addition, as another result of a clinical trial for the treatment of sepsis using thrombomodulin, among patients with INR > 1.2, this drug showed a higher effect in patients with INR > 1.5 than in patients with INR > 1.2 (patent document 13).

[0009]

As explained above, in light of some of the results of clinical studies in the therapeutic treatment of patients with sepsis using an anticoagulant, high efficiency is expected when choosing a class of patients with concomitant coagulopathy. However, it is believed that the definition of coagulopathy has not yet been approved, because, for example, conflicting clinical results have been obtained. In other words, how an excellent result can be obtained by targeting patients based on the INR value has not been clarified, and there is no general technical knowledge about what level of INR value in a patient with sepsis provides a particular effectiveness of this drug. With regard to the correlation of the INR value with clinical efficacy, it is believed that only a limited part of the correlation is known as a case-by-case basis for some individual drugs.

[0010]

Under these circumstances, among the anticoagulants, thrombomodulin attracted the attention of the inventors of the present invention, and they carried out various studies on the therapeutic and/or improving effect against sepsis. As a result, it has surprisingly been found that sepsis can be therapeutically treated and/or alleviated more effectively in severe sepsis patients with one or more organ dysfunctions (excluding severe sepsis patients having organ dysfunction limited to the liver or kidneys) than in severe sepsis patients without organ dysfunction when the INR value of the patients is greater than 1.4, in other words, with regard to therapeutic treatment and/or amelioration of sepsis using thrombomodulin, the inventors found that there is a particular correlation between severely septic patients with one or more organ dysfunctions in the sepsis patient class and an INR value greater than 1.4, which is unexpected by those of ordinary skill in the art. In addition, the inventors surprisingly found that for patients with severe sepsis with an INR value of more than 1.4 and equal to or less than 1.6, a particularly remarkable effect was achieved in that the difference in mortality between the thrombomodulin group and the placebo group was above 15% (Patent Document 13). However, any mode of administration to achieve such a desired clinical effect has not yet been fully elucidated.

Reference documents of the prior art

Patent Documents

[0011]

Patent Document 1: Publication of Unexamined Japanese Patent Application (Kokai) No. 64-6219

Patent Document 2: Publication of Unexamined Japanese Patent Application No. 5-213998

Patent Document 3: Publication of Unexamined Japanese Patent Application No. 2-255699

Patent Document 4: Publication of Unexamined Japanese Patent Application No. 3-133380

Patent Document 5: Publication of Unexamined Japanese Patent Application No. 3-259084

Patent Document 6: Japanese Unexamined Patent Application Publication No. 4-210700

Patent Document 7: WO92/00325

Patent Document 8: WO92/03149

Patent Document 9: WO93/15755

Patent Document 10: Publication of Unexamined Japanese Patent Application No. 3-86900

Patent Document 11: Publication of Unexamined Japanese Patent Application No. 3-218399

Patent Document 12: WO03/061687

Patent Document 13: WO2013/073545

Non-Patent Documents

[0012]

Non-Patent Document 1: American College of Chest Physicians, CHEST/101/6-/JUNE, 1992:1481-1483

Non-Patent Document 2: Koji Suzuki, Igaku no Ayumi (Progress of Medicine), Vol. 125, 901 (1983)

Non-Patent Document 3: K. Gomi et al., Blood, 75, 1396-1399 (1990)

Non-Patent Document 4: M. Zushi et al., J. Biol. Chem., 246, 10351-10353 (1989)

Non-Patent Document 5: D.Z. Wen et al. Biochemistry 26 4350-4357 (1987)

Non-Patent Document 6: S.M. Bates et al., Br. J. Pharmacol., 144, 1017-1028 (2005)

Non-Patent Document 7: Crit. Care Med., 31, 1250-1256 (2003)

Non-Patent Document 8: JAMA, 290, 238-247 (2003)

Non-Patent Document 9: "Results of Third Phase Clinical Trials for ART-123 in Overseas (preliminary report)", https://www.asahi-kasei.co.jp/asahi/jp/news/2018/me180802.html (August 2, 2018, Asahi Kasei Pharma Corporation)

The essence of the invention

Purpose achieved by the present invention

[0013]

An object of the present invention is to provide a medicament for effective therapeutic treatment and/or amelioration of sepsis and the like accompanied by coagulopathy, and a method for effective therapeutic treatment and/or amelioration of sepsis and the like accompanied by coagulopathy.

Means to achieve the goal

[0014]

To achieve the above object, the inventors of the present invention have focused on thrombomodulin among anticoagulants and carried out various studies on its therapeutic and/or improving effect in sepsis. As a result, they found that sepsis in patients in which it is accompanied by coagulopathy, as confirmed immediately before the introduction of thrombomodulin, can be extremely effectively treated and/or improved with thrombomodulin. The present inventors have also found that by intravenously administering 0.005 to 1 mg/kg of thrombomodulin to patients with sepsis accompanied by coagulopathy once a day for at least four consecutive days, sepsis can be extremely effectively treated and/or improved.

[0015]

The present inventors have also found that sepsis in patients who are confirmed to have a certain number of types of organ dysfunction just prior to thrombomodulin administration can be treated and/or alleviated extremely effectively with thrombomodulin.

[0016]

In particular, the present invention includes the following:

[1] A medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose International Normalized Ratio (INR) value is greater than 1.4 just prior to administration.

[0017]

[2] The drug according to [1] above, which is intended to be administered to a patient whose platelet count is greater than 30,000/mm ³ immediately before administration.

[3] The drug according to [1] or [2] above, which is to be administered to a patient whose thrombin-antithrombin complex (TAT) level is 10 ng/ml or more just before administration.

[4] The drug according to any one of [1] to [3] above, which is intended to be administered to a patient whose platelet count is greater than 30,000/mm ³ immediately before administration and whose Sofa score is not greater than 11.

[0018]

[5] A medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose thrombin-antithrombin complex (TAT) complex level is not less than 10 ng/mL immediately prior to administration.

[6] A drug for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose prothrombin 1+2 (F1+2) fragment level is greater than 229 pmol/L immediately before administration.

[7] A drug for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose prothrombin 1+2 (F1+2) fragment level is not less than 250 pmol/L immediately before administration.

[0019]

[8] A drug for therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose protein C activity level is not more than 40% immediately before administration.

[9] A drug for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose antithrombin III (AT III) activity level is less than 70% immediately before administration.

[10] A drug for therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose microparticle (MP) size is greater than 10 nm immediately before administration.

[11] A medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose APACHE II score is less than 35 immediately prior to administration.

[0020]

[12] A drug for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who has one to three types of organ dysfunction immediately prior to administration.

[13] The drug according to [12], which is intended to be administered to a patient who is not a sepsis patient having organ dysfunction limited to the liver or kidneys.

[14] A drug according to [1] or [2], which is intended to be administered to a patient who has one to three types of organ dysfunction immediately before administration.

[15] The drug according to [14], which is intended to be administered to a patient who is not a sepsis patient having organ dysfunction limited to the liver or kidneys.

[16] A drug for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient whose D-dimer level is not less than 3500 ng/ml immediately before administration.

[17] A drug according to [1] or [2], which is intended to be administered to a patient whose D-dimer level is not less than 3500 ng/ml immediately before administration.

[0021]

[18] A drug according to [1] or [2], which is intended to be administered to a patient whose prothrombin 1+2 (F1+2) fragment level is greater than 229 pmol/L immediately before administration.

[19] A drug according to [1] or [2], which is intended to be administered to a patient whose prothrombin 1+2 (F1+2) fragment level is not less than 250 pmol/L immediately before administration.

[20] A drug according to [1] or [2], which is intended to be administered to a patient whose protein C activity level is not more than 40% immediately before administration.

[21] A drug according to [1] or [2], which is intended to be administered to a patient whose antithrombin III (AT III) activity level is less than 70% immediately before administration.

[22] A drug according to [1] or [2], which is intended to be administered to a patient whose microparticle size (MP) immediately before administration is greater than 10 nm.

[23] A drug according to [1] or [2], which is intended to be administered to a patient whose APACHE II score is less than 35 immediately prior to administration.

[24] The drug according to any one of [1] to [23] above, which is intended to be administered to a patient with severe sepsis having impaired cardiovascular function and/or impaired respiratory function.

[0022]

[25] The drug according to any one of [1] to [24] above, wherein the thrombomodulin is a soluble thrombomodulin.

[26] The drug according to any one of [1] to [25] above, wherein the thrombomodulin is a thrombomodulin having the following properties (1) to (4):

(1) the action of selective binding to thrombin,

(2) an action promoting activation of protein C by thrombin,

(3) an action that prolongs thrombin time, and

(4) an action that suppresses platelet aggregation caused by thrombin.

[0023]

[27] The drug according to any one of [1] to [26] above, wherein the thrombomodulin is a thrombomodulin having the following properties (1) to (5):

(1) the action of selective binding to thrombin,

(2) an action promoting activation of protein C by thrombin,

(3) an action that prolongs thrombin time,

(4) an action that suppresses platelet aggregation caused by thrombin, and

(5) anti-inflammatory action.

[28] The drug according to any of [1]-[27] above, wherein thrombomodulin is a peptide obtained from a transformed cell obtained by transfection of a host cell with DNA encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

[29] The drug according to any one of [1] to [28] above, wherein thrombomodulin is a peptide containing the amino acid sequence (i-1) or (i-2) below, and said peptide is a peptide having thrombomodulin activities:

(i-1) the amino acid sequence from position 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or

(i-2) the amino acid sequence (i-1) above, further comprising a substitution, deletion or addition of one or more amino acid residues.

[0024]

[30] The drug according to any one of [1]-[29] above, wherein the soluble thrombomodulin is a peptide containing:

(i) the amino acid sequence from position 367 to 480 in the amino acid sequence of SEQ ID NO: 9 or 11 and the amino acid sequence (ii-1) or (ii-2) below, and said peptide is a peptide having thrombomodulin activities:

(ii-1) the amino acid sequence from position 19 to 244 in the amino acid sequence of SEQ ID NO: 9 or 11, or

(ii-2) the amino acid sequence (ii-1) above, further comprising a substitution, deletion or addition of one or more amino acid residues.

[31] The drug according to [1] or [2] above, which is intended for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis, in which he is accompanied by coagulopathy, at a frequency of once a day for at least four consecutive days.

[32] The drug according to any one of [1]-[31] above, wherein the term “immediately before administration” means a period of 24 hours calculated retrospectively from the start of thrombomodulin administration.

[33] A drug for the therapeutic treatment and/or improvement of sepsis, comprising thrombomodulin as an active ingredient, which is intended for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis accompanied by coagulopathy at a frequency of once a day for at least four consecutive days.

[34] A method for therapeutically treating and/or ameliorating sepsis, which comprises the step of administering thrombomodulin to a sepsis patient whose international normalized ratio (INR) value is greater than 1.4.

[0025]

[35] The method according to [34] above, which includes the step of administering thrombomodulin to a patient whose platelet count is greater than 30,000/mm ³ immediately before administration.

[36] The method according to [34] or [35] above, which comprises the step of administering thrombomodulin to a patient whose thrombin-antithrombin complex (TAT) level is 10 ng/ml or more just before administration.

[37] The method according to any one of [34] to [36] above, which comprises the step of administering thrombomodulin to a patient whose platelet count is greater than 30,000/mm ³ immediately prior to administration and whose Sofa score is not greater than 11.

[0026]

[38] A method for therapeutically treating and/or ameliorating sepsis, which comprises the step of administering thrombomodulin to a patient with sepsis who has a thrombin-antithrombin complex (TAT) complex level of not less than 10 ng/mL immediately prior to administration.

[39] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a patient with sepsis who, immediately prior to administration, has a prothrombin 1+2 (F1+2) fragment level greater than 229 pmol/L.

[40] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a septic patient whose prothrombin 1+2 (F1+2) fragment level is not less than 250 pmol/L just prior to administration.

[41] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a sepsis patient whose protein C activity level is not greater than 40% immediately prior to administration.

[42] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a septic patient whose antithrombin III (AT III) activity level is less than 70% immediately prior to administration.

[0027]

[43] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a sepsis patient whose microparticle size (MP) is greater than 10 nm immediately prior to administration.

[44] A method for therapeutically treating and/or ameliorating sepsis, which comprises the step of administering thrombomodulin to a septic patient who has an APACHE II score of less than 35 just prior to administration.

[45] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a septic patient having one to three types of organ dysfunction immediately prior to administration.

[46] The method according to [45], which includes the step of administering thrombomodulin to a non-sepsis patient having organ dysfunction limited to liver or kidney.

[47] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient who has one to three types of organ dysfunction just prior to administration.

[48] The method according to [47], which includes the step of administering thrombomodulin to a non-sepsis patient having organ dysfunction limited to the liver or kidneys.

[0028]

[49] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering thrombomodulin to a septic patient whose D-dimer level is not less than 3500 ng/mL immediately prior to administration.

[50] The method according to [34] or [35], which comprises the step of administering thrombomodulin to a patient whose D-dimer level is not less than 3500 ng/ml immediately before administration.

[51] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient whose prothrombin 1+2 (F1+2) fragment level is greater than 229 pmol/L just before administration.

[52] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient whose prothrombin 1+2 (F1+2) fragment level is not less than 250 pmol/L immediately before administration.

[53] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient whose protein C activity level is not more than 40% immediately before administration.

[0029]

[54] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient whose antithrombin III (AT III) activity level is less than 70% just before administration.

[55] The method according to [34] or [35], which includes the step of administering thrombomodulin to a patient whose microparticle size (MP) just prior to administration is greater than 10 nm.

[56] The method according to [34] or [35], which comprises the step of administering thrombomodulin to a patient whose APACHE II score is less than 35 immediately prior to administration.

[57] The method according to [34] or [35], which comprises the step of administering 0.005-1 mg/kg of thrombomodulin intravenously to a sepsis patient accompanied by coagulopathy at a frequency of once a day for at least four consecutive days.

[58] The method according to any one of [34]-[57] above, wherein the term “immediately prior to administration” means a period of 24 hours calculated retrospectively from the start of administration of thrombomodulin.

[59] A method for therapeutic treatment and/or amelioration of sepsis, which comprises the step of administering 0.005-1 mg/kg of thrombomodulin intravenously to a patient with sepsis accompanied by coagulopathy at a frequency of once a day for at least four consecutive days.

[0030]

[60] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who, immediately prior to administration, has an International Normalized Ratio (INR) value in the patient's plasma sample greater than 1.4.

[61] Use according to [60] above, wherein the drug is a drug for administration to a patient whose platelet count is greater than 30,000/mm ³ immediately before administration.

[62] The use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose thrombin-antithrombin complex (TAT) level is 10 ng/mL or more just before administration.

[63] Use according to any one of [60] to [62] above, wherein the drug is a drug for administration to a patient whose platelet count is greater than 30,000/mm ³ immediately prior to administration and the Sofa score is not greater than 11.

[0031]

[64] Use according to [60] or [61] above, wherein the drug is a drug for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with concomitant coagulopathy at a frequency of once a day for at least four consecutive days.

[65] Use according to any one of [60]-[64] above, wherein the term "immediately prior to administration" means a period of 24 hours calculated retrospectively from the start of thrombomodulin administration.

[66] The use of thrombomodulin as a drug for the therapeutic treatment and/or improvement of sepsis, where the drug is a drug for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis accompanied by coagulopathy at a frequency of once a day for at least four consecutive days.

[67] The use of thrombomodulin for the preparation of a medicament for the therapeutic treatment and/or amelioration of sepsis, wherein the medicament is a medicament for administration to a patient with sepsis who, immediately prior to administration, has an International Normalized Ratio (INR) value in the patient's plasma sample greater than 1.4.

[68] Use according to [67] above, wherein the drug is a drug for administration to a patient whose platelet count is greater than 30,000/mm ³ immediately before administration.

[0032]

[69] Use according to [67] or [68] above, wherein the drug is a drug for administration to a patient whose thrombin-antithrombin complex (TAT) level is 10 ng/mL or more just before administration.

[70] Use according to any one of [67]-[69] above, wherein the drug is a drug for administration to a patient whose platelet count is greater than 30,000/mm ³ immediately prior to administration and the Sofa score is not greater than 11.

[71] Use according to [67] or [68] above, wherein the drug is a drug for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis, in which he is accompanied by coagulopathy, at a frequency of once a day for at least four consecutive days.

[72] The method according to any one of [67]-[71] above, wherein the term "immediately before administration" means a period of 24 hours calculated retrospectively from the start of administration of thrombomodulin.

[73] The use of thrombomodulin for the preparation of a drug for the therapeutic treatment and/or improvement of sepsis, wherein the drug is a drug for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis accompanied by coagulopathy at a frequency of once a day for at least four consecutive days.

[0033]

[74] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who immediately prior to administration has a thrombin-antithrombin complex (TAT) level of not less than 10 ng/mL.

[75] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who, immediately prior to administration, has a prothrombin 1+2 (F1+2) fragment level greater than 229 pmol/L.

[76] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who immediately prior to administration has a prothrombin 1+2 (F1+2) fragment level of not less than 250 pmol/L.

[77] The use of thrombomodulin as a drug for therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis whose protein C activity level is not more than 40% immediately prior to administration.

[0034]

[78] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who immediately prior to administration has an antithrombin III (AT III) activity level of less than 70%.

[79] The use of thrombomodulin as a drug for therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis whose microparticle size (MP) immediately prior to administration is greater than 10 nm.

[80] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who has an APACHE II score of less than 35 immediately prior to administration.

[0035]

[81] The use of thrombomodulin as a drug for therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis having one to three types of organ dysfunction immediately prior to administration.

[82] Use according to [81], wherein the drug is a drug for administration to a patient who is not a sepsis patient having organ dysfunction limited to the liver or kidneys.

[83] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient who has one to three types of organ dysfunction just before administration.

[84] Use according to [83], wherein the drug is a drug for administration to a patient who is not a sepsis patient having organ dysfunction limited to the liver or kidneys.

[0036]

[85] The use of thrombomodulin as a drug for the therapeutic treatment and/or amelioration of sepsis, wherein the drug is a drug for administration to a patient with sepsis who immediately prior to administration has a D-dimer level of not less than 3500 ng/mL.

[86] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose D-dimer level is not less than 3500 ng/ml immediately before administration.

[87] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose prothrombin 1+2 (F1+2) fragment level is greater than 229 pmol/L immediately before administration.

[88] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose prothrombin 1+2 (F1+2) fragment level is not less than 250 pmol/L immediately before administration.

[89] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose protein C activity level is not more than 40% immediately before administration.

[90] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose antithrombin III (AT III) activity level is less than 70% immediately before administration.

[91] Use according to [60] or [61] above, wherein the drug is a drug for administration to a patient whose microparticle size (MP) immediately before administration is greater than 10 nm.

[92] The use according to [60] or [61] above, wherein the drug is a drug for administration to a patient who immediately prior to administration has an APACHE II score of less than 35.

[93] Use according to any one of [60]-[92] above, wherein the term “immediately before administration” means a period of 24 hours calculated retrospectively from the start of thrombomodulin administration.

Invention Effect

[0037]

In accordance with the present invention, sepsis accompanied by coagulopathy can be effectively treated and/or improved.

Brief description of the drawings

[0038]

Fig. 1 shows the test protocol used in the examples, where a total of 816 cases, confirmed not later than 24 hours before the test as meeting the criteria for coagulopathy and organ dysfunction in Example 1, were randomized at a time of no more than 12 hours, the first administration was carried out no later than 4 hours from the time of randomization in principle, and platelet count, plasma INR value, etc. measured again at the time of the first administration as the time point immediately before administration (baseline). In the drawing, R indicates the point in time when randomization is completed.

Methods for carrying out the invention

[0039]

In the following, certain preferred embodiments of the present invention are specifically explained (the preferred modes of carrying out the invention are also referred to as "embodiments" hereinafter). However, the scope of the present invention is not limited to the specific embodiments explained below.

[0040]

Thrombomodulin of this embodiment is preferably known to have the action of (1) selectively binding to thrombin (2) to promote thrombin activation of protein C. It is further preferred when it is confirmed that thrombomodulin generally has (3) an action to prolong thrombin time, (4) an action to inhibit thrombin-induced platelet aggregation, and/or (5) an anti-inflammatory action. Such actions that thrombomodulin has can be referred to as thrombomodulin activities.

[0041]

As thrombomodulin activities, thrombomodulin preferably has the actions (1) and (2) above, and more preferably has the actions (1) to (4) above. As thrombomodulin activities, thrombomodulin more preferably has all of the actions (1) to (5) above.

[0042]

The thrombomodulin action of binding to thrombin can be confirmed by the test methods described in various well-known publications such as Thrombosis and Haemostasis, 70(3):418-422 (1993) and The Journal of Biological Chemistry, 264, 9, pp,4872-4876 (1989). As for the activity promoting thrombin activation of protein C, the degree of activity promoting activation of protein C, or the presence or absence of such an activity, can be easily confirmed by test methods that are detailed in various well-known publications, including, for example, Japanese Unexamined Patent Application Publication No. 64-6219. In addition, an action prolonging the thrombin time and/or an action suppressing platelet aggregation caused by thrombin can also be easily confirmed. In addition, the anti-inflammatory effect can also be confirmed by the research methods described in various well-known publications, including, for example, Blood, 112:3361-3670 (2008) and The Journal of Clinical Investigation, 115, 5:1267-1274 (2005).

[0043]

Thrombomodulin used for the present invention is not particularly limited as long as it has thrombomodulin activity. Thrombomodulin is preferably soluble thrombomodulin under surfactant-free conditions. The solubility of soluble thrombomodulin in water, such as distilled water used for injection (in the absence of a surfactant such as Triton X-100 or polidocanol, and generally around the neutral pH range) is preferably, for example, 1 mg/ml or more, or 10 mg/ml or more; preferably 15 mg/ml or more, or 17 mg/ml or more; more preferably 20 mg/ml or more, 25 mg/ml or more, or 30 mg/ml or more; particularly preferably 60 mg/ml or more. In some cases, the solubility is, for example, 80 mg/ml or more, or 100 mg/ml or more. It should be understood that to determine the successful dissolution of soluble thrombomodulin, simple criteria are the clear appearance of the solution and the absence of clearly observable insoluble substances after dissolution of soluble thrombomodulin and visual inspection of the solution, for example, directly under a white light in a position corresponding to an illumination of approximately 1000 lux. It is also possible to observe the presence or absence of any residue after filtration.

[0044]

The molecular weight of thrombomodulin is not limited as long as it has thrombomodulin activities as described above. The molecular weight is preferably 100,000 or less, more preferably 90,000 or less, even more preferably 80,000 or less, most preferably 70,000 or less, and the molecular weight is preferably 50,000 or more, most preferably 60,000 or more. The molecular weight of soluble thrombomodulin can easily be measured by conventional methods for measuring protein molecular weight. Measurement by mass spectrometry is preferred, and MALDI-TOF-MS is more preferred. To obtain soluble thrombomodulin having a molecular weight in the desired range, soluble thrombomodulin which is obtained by culturing a transformed cell obtained by transfecting a host cell with DNA encoding soluble thrombomodulin using a vector, can be fractionated using column chromatography or the like as described below.

[0045]

Thrombomodulin used for the present invention preferably includes an amino acid sequence consisting of amino acid residues at positions 19-132 of SEQ ID NO: 1, which is known to be a central part of the thrombomodulin activities of human thrombomodulin, and thrombomodulin is not specifically limited as long as thrombomodulin includes an amino acid sequence consisting of amino acid residues at positions 19-132 of SEQ ID NO: 1. An amino acid sequence consisting of amino acid residues at positions 19-132 of SEQ ID NO: 1 may be naturally or artificially mutated, provided that the sequence has the effect of promoting thrombin activation of protein C, ie. one of the activities of thrombomodulin. In particular, the sequence may include a substitution, deletion, or addition of one or more amino acid residues in the amino acid sequence consisting of amino acid residues at positions 19-132 of SEQ ID NO: 1. The acceptable level of mutation is not particularly limited, provided that the amino acid sequence has thrombomodulin activities. An example includes a homology of 50% or more with respect to amino acid sequences, and the homology is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. Such a mutated amino acid sequence, including a substitution, deletion or addition of one or more amino acid residues, is called a homologous mutated sequence. As described below, these mutated amino acid sequences can be easily obtained by conventional gene manipulation techniques. Thrombomodulin is not particularly limited as long as it has the above sequence and thrombin selective binding action to promote thrombin activation of protein C at least as thrombomodulin in general, but thrombomodulin preferably also has an anti-inflammatory effect.

[0046]

The amino acid sequence of SEQ ID NO: 3 includes the mutation Val, as the amino acid at position 125 of SEQ ID NO: 1, in Ala. Thrombomodulin used for the present invention also preferably comprises the amino acid sequence from position 19 to 132 of SEQ ID NO: 3.

[0047]

As described above, although thrombomodulin used for the present invention is not specifically limited as long as thrombomodulin has at least the amino acid sequence of position 19 to 132 of SEQ ID NO: 1 or 3, or a mutated sequence homologous thereto, and includes at least a peptide sequence having thrombomodulin activities, preferred examples of thrombomodulin include a peptide consisting of the sequence of position 19 to 132 or 17 to 1 32 in any of SEQ ID NO: 1 or SEQ ID NO: 3, and a peptide consisting of a mutated sequence homologous to the above sequence and having at least thrombomodulin activities. A peptide consisting of the sequence from position 19 to 132 in either SEQ ID NO: 1 or SEQ ID NO: 3 is more preferred. In another embodiment, a peptide consisting of a mutated sequence homologous to position 19 to 132 or 17 to 132 in any of SEQ ID NO: 1 or SEQ ID NO: 3 and having at least thrombomodulin activities is more preferred.

[0048]

In another embodiment of thrombomodulin according to the present invention, thrombomodulin preferably comprises an amino acid sequence from positions 19-480 of SEQ ID NO: 5, which is not specifically limited provided that thrombomodulin comprises an amino acid sequence from positions 19 to 480 of SEQ ID NO: 5. An amino acid sequence from positions 19-480 of SEQ ID NO: 5 may be a homologous mutated sequence thereto, provided that the sequence has the effect of promoting thrombin activation of protein C, ie. one of the activities of thrombomodulin.

[0049]

The sequence of SEQ ID NO: 7 includes the mutation Val, as the amino acid at position 473 of SEQ ID NO: 5, in Ala. Thrombomodulin used for the present invention also preferably comprises the amino acid sequence from position 19 to 480 of SEQ ID NO: 7.

[0050]

As described above, although thrombomodulin used for the present invention is not specifically limited as long as thrombomodulin has at least the sequence from position 19 to 480 in any of SEQ ID NO: 5 or SEQ ID NO: 7, or a mutated sequence homologous thereto, and includes at least a peptide sequence having thrombomodulin activities, preferred examples of thrombomodulin include a peptide consisting of the sequence from position 19 to 480 or from 17 to 480 in any of SEQ ID NO: 5 or SEQ ID NO: 7, and a peptide consisting of a mutated sequence homologous to the above sequence and having at least thrombomodulin activity. A peptide consisting of the sequence from position 19 to 480 of SEQ ID NO: 5 or 7 is more preferred. In another embodiment, a peptide consisting of a mutated sequence homologous to position 19 to 480 or 17 to 480 in any of SEQ ID NO: 5 or SEQ ID NO: 7 and having at least thrombomodulin activities is more preferred.

[0051]

In another embodiment of thrombomodulin according to the present invention, thrombomodulin preferably comprises the amino acid sequence from position 19 to 515 of SEQ ID NO: 9, which is not specifically limited provided that thrombomodulin comprises the amino acid sequence from position 19 to 515 of SEQ ID NO: 9. The amino acid sequence from position 19 to 515 of SEQ ID NO: 9 may be a homologous mutated sequence thereto, provided that the sequence has the effect of promoting thrombin activation of protein C, ie. thrombomodulin activity.

[0052]

The amino acid sequence of SEQ ID NO: 11 includes the mutation Val, as the amino acid at position 473 of SEQ ID NO: 9, in Ala. The thrombomodulin used for the present invention also preferably comprises the amino acid sequence from position 19 to 515 of SEQ ID NO: 11.

[0053]

As described above, although thrombomodulin used for the present invention is not specifically limited as long as thrombomodulin has at least the sequence from position 19 to 515 in any of SEQ ID NO: 9 or SEQ ID NO: 11, or a mutated sequence homologous thereto, and includes a peptide sequence having at least thrombomodulin activities, more preferred examples include a peptide having the sequence from position 19 to 516, from 19 to 515, 17 to 516, or 17 to 515 in any of SEQ ID NO: 9 or SEQ ID NO: 11, and a peptide consisting of a mutated sequence homologous to the above sequence and having at least thrombomodulin activity. A peptide having the sequence 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQ ID NO: 9 is particularly preferred. Their mixture is also a preferred example. In another embodiment, a peptide having the sequence 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQ ID NO: 11 is particularly preferred. Their mixture is also a preferred example. In addition, a peptide consisting of a homologous immutated sequence and having at least thrombomodulin activities is also a preferred example. Preferably, thrombomodulin also has an anti-inflammatory effect.

[0054]

A peptide having a homologous mutated sequence is as described above and means a peptide which may include substitution, deletion or addition of at least one, i.e. one or more, preferably several (eg 1-20, preferably 1-10, more preferably 1-5, particularly preferably 1-3) amino acid residues in the amino acid sequence of the peptide in question. Although the acceptable level of mutation is not particularly limited as long as the peptide has thrombomodulin activities, an example of an acceptable level of homology includes 50% or more with respect to amino acid sequences, and the homology can be preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

[0055]

Preferred examples of thrombomodulin used for the present invention also include a peptide consisting of the sequence of SEQ ID NO: 14 (462 amino acid residues), a peptide consisting of the sequence of SEQ ID NO: 8 (272 amino acid residues), and a peptide consisting of the sequence of SEQ ID NO: 6 (236 amino acid residues), which are described in Japanese Unexamined Patent Application Publication No. 64-6219.

[0056]

Thrombomodulin used for the present invention is not specifically limited as long as thrombomodulin has at least the amino acid sequence from position 19 to 132 in any of SEQ ID NO: 1 or SEQ ID NO: 3. As such thrombomodulin, a peptide having at least an amino acid sequence from position 19 to 480 in any of SEQ ID NO: 5 or SEQ ID NO: 7 is preferred, and more preferred is a peptide having at least least amino acid sequence from position 19 to 515 in any of SEQ ID NO: 9 or SEQ ID NO: 11. A more preferred example of a peptide having at least the amino acid sequence from position 19 to 515 in any of SEQ ID NO: 9 or SEQ ID NO: 11 is a peptide consisting of the sequence from position 19 to 516, 19 to 515, 19 to 514, 1 7 to 516, 17 to 515, or 19 to 514 in any of SEQ ID NO: 9 or SEQ ID NO: 11. In addition, a mixture of peptides, each consisting of the sequence of positions 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 17 to 514 in any from SEQ ID NO: 9 or SEQ ID NO: 11 is also a preferred example.

[0057]

For one embodiment, although thrombomodulin is not specifically limited as long as it is the thrombomodulin described above, an example includes soluble thrombomodulin. For another embodiment, the example includes human thrombomodulin. For another embodiment, soluble human thrombomodulin can be exemplified. For another embodiment, thrombomodulin alfa (gene recombination product) can be exemplified. Thrombomodulin alfa (gene recombination product) is the active ingredient of Recomodulin (registered trademark) which is approved as a pharmaceutical in Japan. Thrombomodulin alfa (gene recombination product) is also called ART-123.

[0058]

In the case of the above mixture, the mixture ratio of the peptide starting at position 17 and the peptide starting at position 19 for each of SEQ ID NOs: 9 and 11 is, for example, 30:70 to 50:50, preferably 35:65 to 45:55.

[0059]

In addition, the mixture ratio of the peptide that ends at position 514, the peptide that ends at position 515, and the peptide that ends at position 516 for each of SEQ ID NOs: 9 and 11 is, for example, 0:0:100 to 0:90:10, or 0:70:30 to 10:90:0, or 10:0:90 to 20:10:7 0 if needed.

The ratio of peptides in the mixture can be determined by the usual method.

[0060]

The sequence of positions 19-132 in SEQ ID NO: 1 corresponds to the sequence of positions 367-480 in SEQ ID NO: 9, and the sequence of positions 19-480 in SEQ ID NO: 5 corresponds to the sequence of positions 19-480 in SEQ ID NO: 9. In addition, the sequence of positions 19-132 in SEQ ID NO: 3 corresponds to the sequence of positions 367-480 in SEQ ID NO: : 11, and the sequence from positions 19-480 in SEQ ID NO: 7 corresponds to the sequence from positions 19-480 in SEQ ID NO: 11. In addition, all sequences from positions 1-18 in SEQ ID NO: 1, 3, 5, 7, 9 and 11 are identical sequences.

[0061]

As described below, these thrombomodulins in accordance with the present invention can be obtained from transformed cells obtained by transfection of host cells with DNA encoding each peptide (in particular, the nucleotide sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, etc.) using a vector.

[0062]

It is sufficient that these peptides have only the above amino acid sequences, and the sugar chain may or may not be attached, but is not particularly limited. In gene manipulation methods, the type of sugar chain, the position at which the sugar chain is added, and the level of addition differ depending on the type of host cells used, and any methods can be used. Regarding the binding position of the sugar chain and its type, the facts described in Japanese Unexamined Patent Publication No. 11-341990 are known, and the same sugar chains at the same position can be added to thrombomodulins according to the present invention. Two types of N-linked sugar chains, fucosyl biantennary type and fucosyl triantennary type, can bind to the thrombomodulin of this embodiment, and their ratio is, for example, 100:0 to 60:40, preferably 95:5 to 60:40, more preferably 90:10 to 70:30. The ratio of these sugar chains can be measured on the two-dimensional sugar chain map described in Biochemical Experimental Methods, Vol. 23, Methods of Researches on Glycoprotein Sugar Chains, Japan Scientific Societies Press (1990), etc. In addition, when examining the thrombomodulin sugar composition of this embodiment, neutral saccharides, aminosaccharides and sialic acid are found, the content of which can each be independently, for example, 1 to 30%, preferably 2 to 20%, more preferably 5 to 10% by weight based on protein content. The sugar content can be measured by the methods described in Lecture of New Biochemical Experiments, Vol. 3, Sugar I, Glycoprotein (Book 1), Tokyo Kagaku Dojin (1990) (neutral saccharides: phenol-sulfuric acid method, aminosaccharides: Elson-Morgan method, sialic acid: periodic acid-resorcinol method).

[0063]

Although the method for producing thrombomodulin is not limited to its production by genetic manipulation as described below, as a signal sequence that can be used for expression when thrombomodulin is produced by gene manipulation, a nucleotide sequence encoding the amino acid sequence of positions 1 to 18 of SEQ ID NO: 9 and a nucleotide sequence encoding the amino acid sequence of positions 1 to 16 of SEQ ID NO: 9 can be used, and others can also be used. known signal sequences such as the human tissue plasminogen activator signal sequence (International Publication WO88/9811).

[0064]

When a DNA sequence encoding thrombomodulin is introduced into a host cell, examples of preferred methods include a method of incorporating the DNA sequence encoding thrombomodulin, preferably into a vector, more preferably into an expression vector capable of being expressed in animal cells, and then introducing the DNA with the vector. An expression vector is a DNA molecule that consists of a promoter sequence, a sequence for adding a ribosome binding site to mRNA, a DNA sequence encoding a protein to be expressed, a splicing signal, a terminator sequence for terminating transcription, a replication origin sequence, and the like. Examples of a preferred vector for expression in animal cells include pSV2-X as described by Mulligan R.C. et al. (Proc. Natl. Acad. Sci. USA, 78, 2072 (1981)); pBP69T (69-6) described by Howley P.M. et al. (Methods in Emzymology, 101, 387 (1983), Academic Press), etc. In addition, there is also another preferred embodiment in which the DNA is introduced into an expression vector expressed in a microorganism.

[0065]

Examples of a host cell that can be used to produce such peptides as mentioned above include animal cells. Examples of animal cells include Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO cells (ATCC CCL-81), BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664 cells, and the like. In addition, examples of human-derived host cells include HeLa cells, WI38 cells, human 293 cells, and PER.C6 cells. Of these cells, CHO cells are very common and preferred, and among CHO cells, dihydrofolate reductase-deficient (DHFR) CHO cells are more preferred.

[0066]

Microorganisms such as Escherichia coli are also frequently used in the gene manipulation process or in the peptide production process. Preferably, a host vector system suitable for each method is used, and a suitable vector system can also be selected for the above host cells. The thrombomodulin gene used in the gene recombinant method has been cloned. Examples of the preparation of thrombomodulin by such a genetically recombinant method have been disclosed, and furthermore, methods for purifying thrombomodulin to obtain a purified product thereof have been disclosed (Japanese Unexamined Patent Application Publication Nos. 64-6219, 2-255699, 5-213998, 5-310787, 7-155176 and J. Biol. Chem., 264: 1035 1-10353 (1989)). Therefore, the thrombomodulin used in the present invention can be obtained using the methods described in the above reports or similar methods. For example, Japanese Patent Publication No. 64-6219 discloses Escherichia coli K-12 strain DH5 (ATCC accession number 67283) containing the pSV2TMJ2 plasmid, which contains DNA encoding full-length thrombomodulin. This strain, re-deposited with the former National Institute of Bioscience and Human-Technology (now the Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary) ( Escherichia coli DH5/pSV2TMJ2) (FERM BP-5570), can also be used. Thrombomodulin of the present invention can be obtained by a known gene manipulation method using DNA encoding a full-length thrombomodulin as a starting material.

[0067]

Thrombomodulin of this embodiment can be obtained by a conventional known method or a similar method. For example, you can refer to the above method of Yamamoto et al. (Japanese Unexamined Patent Application Publication No. 64-6219) or the method described in Japanese Unexamined Patent Application Publication No. 5-213998. Specifically, for example, the DNA encoding the amino acid sequence of SEQ ID NO: 9 is genetically engineered from the human thrombomodulin gene, and can be further modified as needed. For such modification, in order to obtain DNA encoding the amino acid sequence of SEQ ID NO: 11 (which in particular consists of the nucleotide sequence of SEQ ID NO: 12), codons encoding the amino acid at position 473 in the amino acid sequence of SEQ ID NO: 9 (in particular, the nucleotide at position 1418 in SEQ ID NO: 10) are mutated using site-directed mutagenesis according to the method described by Zoller M. J. et al. (Method in Enzymology, 100:468-500 (1983), Academic Press). For example, using synthetic DNA for mutation having the nucleotide sequence of SEQ ID NO: 13, the T nucleotide at position 1418 in SEQ ID NO: 10 can be converted to a C nucleotide to obtain mutated DNA.

[0068]

The DNA obtained as described above is included, for example, in Chinese Hamster Ovary (CHO) cells to obtain transformed cells. Such cells are subjected to appropriate selection, and thrombomodulin purified in a known manner can be obtained from the culture solution obtained by culturing the selected cell. As described above, DNA (SEQ ID NO: 10) encoding the amino acid sequence of SEQ ID NO: 9 is preferably transfected into the above host cell.

[0069]

The thrombomodulin production method of this embodiment is not limited to the above method. For example, it is also possible to extract and purify thrombomodulin from urine, blood, other body fluids, and the like. or extracting and purifying thrombomodulin from thrombomodulin-producing tissue or culture of said tissue, and the like. In addition, thrombomodulin, if necessary, can be further processed by cleavage using a protease.

[0070]

For culturing the above transformed cell, the medium used for conventional cell culture can be used, and it is preferable to culture the transformed cell in various media in advance to select the optimal medium. For example, known media such as MEM media, DMEM media, and 199 media can be used as the base media, and an even more improved media or media with additives for various media can also be used. Examples of the culturing method include culturing in a serum medium, where culture is carried out in a medium containing blood serum, and culturing without serum, where culturing is carried out in a medium containing no blood serum. Although the culture method is not particularly limited, serum-free culture is preferred.

[0071]

When serum is added to the medium in the case of culture in a serum medium, bovine serum is preferred. Examples of bovine serum include fetal bovine serum, neonatal bovine serum, calf serum, adult bovine serum, and the like, and any of these examples can be used as long as the serum is suitable for cell culture. As the serum-free medium used in the serum-free culture, commercially available media can be used. Serum-free media suitable for various cells are available on the market, and for example, for CHO cells, CD-CHO, CHO-S-SFMII and CHO-III-PFM are sold by Invitrogen, and media IS CHO, IS CHO-CD, etc. sold by Irvine Scientific. These media can be used without any processing, or they can be improved or supplemented and used. Examples of a serum-free medium also include DMEM containing 5 mg/l each of insulin, transferrin, and selenous acid. As described above, the medium is not specifically limited, as long as it can be used to produce the thrombomodulin of this embodiment. The culture method is not particularly limited, and any of batch culture, repeated batch culture, fed-batch culture, perfusion culture, and the like can be used.

[0072]

When the thrombomodulin used for the present invention is obtained by the above cell culture method, a difference in the N-terminal amino acid can be observed due to post-translational modification of the protein. For example, the amino acid at position 17, 18, 19 or 22 in SEQ ID NO: 9 can serve as the N-terminal amino acid. In addition, for example, the N-terminal amino acid can be modified to replace the glutamic acid at position 22 with pyroglutamic acid. Preferably, the amino acid at position 17 or 19 serves as the N-terminal amino acid, and more preferably, the amino acid at position 19 serves as the N-terminal amino acid. In addition, there is also another embodiment in which the amino acid at position 17 serves as the N-terminal amino acid, which is a preferred embodiment. With respect to the modification, difference, and the like above, similar examples can be given for the sequence of SEQ ID NO: 11.

[0073]

In addition, when soluble thrombomodulin is produced using DNA having the nucleotide sequence of SEQ ID NO: 10, a C-terminal amino acid difference can be observed, and a peptide shorter by one amino acid residue can be obtained. In particular, the C-terminal amino acid can be modified so that the amino acid at position 515 serves as the C-terminal amino acid, and, in addition, position 515 is amidated. In addition, a peptide shorter by two amino acid residues can be obtained. In particular, the amino acid at position 514 may serve as the C-terminal amino acid. Therefore, any of the peptides having a significant difference between the N-terminal amino acid and the C-terminal amino acid, or a mixture thereof, can be obtained. Preferably the amino acid at position 515 or the amino acid at position 516 serves as the C-terminal amino acid, and more preferably the amino acid at position 516 serves as the C-terminal amino acid. In addition, there is also another embodiment in which the amino acid at position 514 serves as the C-terminal amino acid, which is a preferred embodiment. As for the modification, difference, and the like described above, the same applies to DNA having the nucleotide sequence of SEQ ID NO: 12.

[0074]

Thrombomodulin obtained by the method described above may be a mixture of peptides that differ in N-terminal and C-terminal amino acids. Specific examples include a mixture of peptides having sequences including positions 19 to 516, positions 19 to 515, positions 19 to 514, positions 17 to 516, positions 17 to 515, and positions 17 to 514 in SEQ ID NO: 9.

[0075]

The isolation and purification of thrombomodulin can then be carried out from the culture supernatant or culture obtained as described above by known methods [edited by Takeichi Horio, Tanpakushitsu/Koso no Kiso Jikken Ho (Fundamental Experimental Methods for Proteins and Enzymes) (1981)]. For example, it is preferable to use ion exchange chromatography or adsorption chromatography, which uses the interaction between thrombomodulin and a chromatographic carrier on which functional groups having a charge opposite to that of thrombomodulin are immobilized. Another preferred example is affinity chromatography using specific affinity for thrombomodulin. Preferred examples of the adsorbent include thrombin, which is a thrombomodulin ligand, and an anti-thrombomodulin antibody. Anti-thrombomodulin antibodies having appropriate properties or recognizing appropriate epitopes can be used as the antibody. Examples include, for example, the antibodies described in Japanese Patent Publication (Kokoku) No. 5-42920, Japanese Unexamined Patent Application Publication Nos. 64-45398 and 6-205692, and the like. Other examples include size exclusion chromatography and ultrafiltration, which use the molecular size of thrombomodulin. Other examples further include hydrophobic chromatography, which uses a hydrophobic bond between a chromatographic carrier on which hydrophobic groups are immobilized and a hydrophobic moiety of thrombomodulin. In addition, hydroxyapatite can be used as a carrier in adsorption chromatography, examples of which include, for example, those described in Japanese Unexamined Patent Publication No. 9-110900. If necessary, these agents can be used in combination. Although the degree of purification may be selected depending on the purpose of use and the like, it is desirable to purify thrombomodulin until a single band is obtained by electrophoresis, preferably SDS-PAGE, or until a single peak of isolated and purified product is obtained by gel filtration HPLC or reverse phase HPLC. It should be understood that when two or more types of thrombomodulin are used, it is preferable to obtain substantially only thrombomodulin bands and not to obtain a single band.

[0076]

Specific examples of the purification method used in the present invention include a purification method using thrombomodulin activities as a criterion, for example, a purification method comprising coarse purification of the culture supernatant or culture product using a Q-Sepharose Fast Flow ion exchange column to collect a fraction having thrombomodulin activities; then purifying the fraction on an affinity column, a DIP-thrombin-agarose (diisopropylphosphorylthrombin agarose) column as the main purification step to isolate a fraction having strong thrombomodulin activities; then concentration of the recovered fraction followed by gel filtration to obtain the thrombomodulin active fraction as a purified product (Gomi K. et al., Blood, 75: 1396-1399 (1990)). An example of thrombomodulin activities used as a criterion is the promotion of thrombin activation of protein C. Other preferred purification method examples will be illustrated below.

[0077]

A suitable ion exchange resin having good conditions for thrombomodulin adsorption is selected, and purification is carried out by ion exchange chromatography. A particularly preferred example is a method involving the use of Q-Sepharose Fast Flow equilibrated with Tris-HCl buffer 0.02 mol/l (pH 7.4) containing 0.18 mol/l NaCl. After washing, if required, elution can be carried out, for example, using 0.02 mol/l Tris-HCl buffer (pH 7.4) containing 0.3 mol/l NaCl, to obtain thrombomodulin as a crude product.

[0078]

Then, for example, a substance having a specific affinity for thrombomodulin can be immobilized on a resin for purification by affinity chromatography. Preferred examples include a DIP-thrombin-agarose column and an anti-thrombomodulin monoclonal antibody column. In the case of a DIP-thrombin-agarose column, the column is pre-equilibrated with 20 mmol/L Tris-HCl buffer (pH 7.4) containing 100 mmol/L NaCl and 0.5 mmol/L calcium chloride, and the above prepurified product is then loaded onto the column, washed if necessary, and then eluted using, for example, 20 mmol/L Tris-HCl buffer (pH 7 ,4) containing 1.0 mol/l NaCl and 0.5 mmol/l calcium chloride to obtain thrombomodulin as a purified product. In the case of an anti-thrombomodulin monoclonal antibody column, an example method includes: contacting a solution of anti-thrombomodulin monoclonal antibodies in 0.1 mol/l NaHCO ₃ buffer (pH 8.3) containing 0.5 mol/l NaCl with Sepharose 4FF (GE Health Care Biosciences) pre-activated with CNBr-activated to obtain Sepharose 4FF resin coupled to anti-thrombomodulin monoclonal antibodies , pre-equilibrating the resin, which is filled in the column, for example, phosphate buffer 20 mmol/l (pH 7.3) containing 0.3 mol/l NaCl, washing if necessary, and then the implementation of the elution of glycine-HCl buffer 100 mmol/l (pH 3.0) containing 0.3 mol/l NaCl. The eluate can be neutralized with a suitable buffer to give the product as a purified product.

[0079]

The resulting purified product is then adjusted to pH 3.5 and then loaded into a cation exchanger, preferably SP-Sepharose FF (GE Health Care Biosciences) as a strong cation exchanger, equilibrated with 100 mmol/l glycine-HCl buffer (pH 3.5) containing 0.3 mol/l NaCl, and washing is carried out with the same buffer to obtain a non-adsorbed fraction. The resulting fraction is neutralized with a suitable buffer to obtain a highly purified product. These products are preferably concentrated by ultrafiltration.

[0080]

In addition, buffer exchange by gel filtration is also preferred. For example, a highly purified product concentrated by ultrafiltration can be loaded onto a Sephacryl S-300 column or an S-200 column equilibrated with 20 mmol/L phosphate buffer (pH 7.3) containing 50 mmol/L NaCl and then fractionated using 20 mmol/L phosphate buffer (pH 7.3) containing 50 mmol/L NaCl. Activity to promote thrombin activation of protein C can be confirmed to collect the active fraction and thus obtain a highly purified product by buffer exchange. To improve safety, the highly purified product obtained as described above is preferably filtered through an appropriate virus removal filter such as Planova 15N (Asahi Kasei Medical Co., Ltd.), and then the resulting product can be concentrated by ultrafiltration to the desired concentration. Finally, the product is preferably filtered through an aseptic filter.

[0081]

As one embodiment of the present invention, there is provided a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has an International Normalized Ratio (INR) value in a patient's plasma sample greater than 1.4.

[0082]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has an international normalized ratio (INR) value in the plasma sample of the patient greater than 1.4, and the platelet count in the sample obtained from the patient immediately prior to administration is greater than 30,000/mm ³ .

[0083]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of sepsis, comprising thrombomodulin as an active ingredient, which is intended for intravenous administration of 0.005-1 mg/kg of thrombomodulin to a patient with sepsis at a frequency of once a day for at least four consecutive days.

[0084]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has an international normalized ratio (INR) value in a plasma sample of the patient greater than 1.4, and a thrombin-antithrombin complex (TAT) complex level in the sample obtained from the patient immediately prior to administration of 10 ng/ml or more.

[0085]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who is not a patient whose site of infection is not properly controlled.

[0086]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has an International Normalized Ratio (INR) value in a plasma sample of the patient greater than 1.4, a platelet count in the sample obtained from the patient immediately prior to administration is greater than 30,000/mm ³ , and a Sofa score immediately prior to administration is not greater than than 11.

[0087]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has a thrombin-antithrombin complex (TAT) complex level in a patient's plasma sample of at least 10 ng/mL.

[0088]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has a prothrombin 1+2 (F1+2) fragment level in a patient plasma sample greater than 229 pmol/L.

[0089]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has a prothrombin 1+2 (F1+2) fragment level in a patient's plasma sample of at least 250 pmol/L.

[0090]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has a protein C activity level in the patient's plasma sample of not more than 40%.

[0091]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has an antithrombin III (AT III) activity level in a patient's plasma sample of less than 70%.

[0092]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who, immediately prior to administration, has a microparticle (MP) size in a patient plasma sample greater than 10 nm.

[0093]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who has an APACHE II score of less than 35 immediately prior to administration.

[0094]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who has one to three types of organ dysfunctions immediately prior to administration.

[0095]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or improvement of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient, who is not a patient with sepsis, having organ dysfunction limited to the liver or kidneys, and who has one to three types of organ dysfunction immediately prior to administration.

[0096]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who has an International Normalized Ratio (INR) value greater than 1.4 immediately prior to administration and a platelet count immediately prior to administration greater than 30,000/mm ³ , and who has one to three types of organ dysfunction immediately prior to administration.

[0097]

In another embodiment, the present invention also provides a medicament for the therapeutic treatment and/or amelioration of a patient with sepsis, comprising thrombomodulin as an active ingredient, which is intended to be administered to a patient who has an International Normalized Ratio (INR) value greater than 1.4 immediately prior to administration and a platelet count immediately prior to administration greater than 30,000/mm ³ , and who is not a septic patient having organ dysfunction limited to the liver or kidneys, and has just before the introduction of one to three types of organ dysfunction.

[0098]

The term just before administration as used in the definitions of the embodiments of the present invention means a period of 24 hours, preferably 12 hours, more preferably 8 hours, even more preferably 4 hours, especially preferably 2 hours, calculated retrospectively from the time of initiation of thrombomodulin administration. However, when it is confirmed that the patient satisfies the above conditions, and the patient's plasma sample is obtained in a period of 24-48 hours, calculated retrospectively from the start of administration, high efficiency in the patient can also be achieved.

[0099]

With regard to therapeutic treatment and/or improvement in sepsis, examples of preferred effects include, for example, "preventing the death of a patient from sepsis". Examples also include "prevention of deterioration in the general condition of the patient associated with sepsis".

With regard to therapeutic treatment and/or improvement in sepsis, examples of preferred effects also include, for example, "reducing the mortality rate of patients with sepsis".

[0100]

An increase in the level of D-dimer in the blood, which is a marker of blood coagulation, may indicate developing fibrinolysis (secondary fibrinolysis or fibrinolysis), etc., and also indicate the formation of fibrin clots, which is a sign of other conditions accompanying thromboembolism and hypercoagulability. Preferably, a decrease in the concentration of D-dimer in the blood or suppression of its increase is observed when the drug of the present invention is administered to a patient, compared with when the drug is not administered.

[0101]

The thrombin-antithrombin complex (TAT), which is a blood coagulation marker, is a complex consisting of thrombin and its typical inhibitor, antithrombin, bonding together in a 1:1 ratio. Typically, upon activation of coagulation, a portion of thrombin rapidly binds to antithrombin to form TAT. The concentration of TAT in the blood is usually less than about 3-4 ng/ml. By measuring the concentration of TAT in the blood, one can know the degree of activation of clotting during blood collection. Preferably, there is a decrease in the concentration of TAT in the blood or suppression of its increase when the drug of the present invention is administered to the patient, compared with when the drug is not administered.

[0102]

The prothrombin 1+2 (F1+2) fragment is also known as a coagulation activation marker, like TAT, and is a peptide released from prothrombin during the conversion of prothrombin to thrombin. Preferably, there is a decrease in the concentration of the prothrombin 1+2 fragment in the blood or suppression of its increase when the drug of the present invention is administered to the patient, compared with when the drug is not administered.

[0103]

Preferably, when the drug of the present invention is administered to a patient, an increase in platelet count or suppression of its decrease is observed compared to when the drug is not administered.

[0104]

Antithrombin III (AT III) is one of the physiological anticoagulants, and it binds to a blood clotting factor such as activating factor X and activating factor II (thrombin) in a 1:1 ratio to inhibit its action and thus exhibit an anticoagulant effect.

[0105]

Microparticles (MPs) are membrane vesicles formed from various cells such as platelets, monocytes and vascular endothelial cells and are known to have a blood-clotting action and an inflammatory reaction-promoting action.

[0106]

Sepsis referred to in this embodiment is known as a severe systemic infectious disease in which microorganisms continuously or intermittently enter the blood from the site of infection, and this disease is caused by a disease such as infectious diseases, malignant tumors, cirrhosis of the liver, renal failure, diabetes, and dystocia, or by therapeutic treatment of injury or disease, such as the use of an indwelling catheter, infusion device, dialysis, and the like. and tracheostomy. If symptoms worsen, systemic shock is induced by septic shock, ie. a rapid decrease in blood pressure and peripheral circulatory failure, and lethality leads to dysfunction of vital organs such as the lungs, kidneys, liver, heart, digestive tract and central nervous system. As a complication accompanying sepsis, induced adult respiratory distress syndrome (ARDS) can be indicated, characterized by pulmonary stromal edema, hemorrhage and acute respiratory failure due to obstruction of the pulmonary capillaries associated with DIC or neutrophil activation and their migration and accumulation in the lung parenchyma, which leads to a very poor prognosis.

[0107]

The sepsis referred to in this embodiment is an infection-induced systemic inflammatory response syndrome (SIRS). More specifically, it includes a pathological condition that corresponds, in addition to the presence of infection, to two or more features of SIRS ((1) body temperature >38°C or <36°C, (2) heart rate > 90/min, (3) respiratory rate > 20/min, or PaCO ₂ <32 torr, and (4) white blood cell count >12,000/µl or <4,000/µl, or bandemia > 10%), and sepsis can be mainly diagnose on the basis of such a pathological condition.

[0108]

There are several methods for diagnosing sepsis and they are summarized in Levy M. et al., Crit. Care Med. 31:1250-1256. For example, there are methods based on a practitioner's diagnosis and methods using test values and the like. Examples of the latter include a method that matches two of the four features: (1) body temperature >38°C or <36°C, (2) heart rate >90/min, (3) respiratory rate >20/min, or artificial respiration is required, and (4) white blood cell count >12,000/mcL or <4,000/mcL, or bandemia >10%, SIRS diagnosed, and SIRS for which a microorganism is confirmed or suspected as its causes are diagnosed as sepsis [LaRosa S., the homepage of The Cleveland Clinic]. Another method similar to that described above is described in Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Crit. Care Med., 20, 864-874 (1992).

[0109]

Examples of symptoms of sepsis include, for example, bacteremia, septicemia, systemic inflammatory response syndrome (SIRS), sepsis (SIRS for which a microorganism is confirmed or suspected to be the cause), severe sepsis, septic shock, fatal septic shock, and multiple organ dysfunction syndrome (hereinafter also referred to as MODS) (Harrison's Principles of Internal Medicine, 15th edition of original work, Section 124, pp, 828-833, Medical Science International, Ltd.). The above conditions are examples of symptoms that are effectively affected by the drug of the present invention for therapeutic treatment and/or amelioration.

[0110]

Although sepsis is not specifically limited as long as the disease is diagnosed as sepsis based on the above diagnostic criteria, the use of the medicament of the present invention is sepsis accompanied by abnormal coagulation (coagulopathy sepsis). Examples of a coagulopathy score include, for example, INR. Although coagulopathy is not specifically limited, as long as the INR in the plasma sample obtained from the patient is greater than 1.2, it is preferably greater than 1.3, more preferably greater than 1.4.

[0111]

In addition, an abnormal decrease in the number of platelets (eg, thrombocytopenia) instead of or in addition to INR can also be indicated as an indicator of coagulopathy in sepsis accompanied by coagulopathy. The minimum standard value is, for example, 20,000/mm ³ , 25,000/mm ³ or 30,000/mm ³ , and the maximum standard value is, for example, 200,000/mm ³ , 180,000/mm ³ or 150,000/mm ³ . A preferred range indicative of an abnormal decrease in platelet count may be a range of platelet count of not less than 30,000/mm ³ and less than 150,000/mm ³ . A decrease in platelet count of more than 30% in 24 hours can also be the standard for an abnormal decrease in platelet count.

[0112]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal increase in the level of the thrombin-antithrombin complex (TAT) can also be indicated instead of or in addition to INR. The minimum standard value is, for example, 4 ng/mL, 8 ng/mL, 10 ng/mL, or 12 ng/mL, and the maximum standard value is, for example, 2000 ng/mL, 5000 ng/mL or 10000 ng/mL.

[0113]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal increase in the level of the prothrombin 1+2 (F1+2) fragment can also be indicated instead of or in addition to INR. The minimum standard value is, for example, 200 pmol/l, 229 pmol/l, 230 pmol/l or 250 pmol/l, and the maximum standard value is, for example, 10000 pmol/l, 20000 pmol/l or 40000 pmol/l.

[0114]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal decrease in the level of protein C can also be indicated instead of or in addition to INR. The maximum standard value is, for example, 40%, 50%, or 60%, and the minimum standard value is, for example, 5%, 8%, or 10%.

[0115]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal increase in microparticle size (MP) can also be indicated instead of or in addition to INR. The minimum standard value is, for example, 5 nm, 8 nm, or 10 nm, and the maximum standard value is, for example, 350 nm, 370 nm, or 400 nm.

[0116]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal decrease in the level of antithrombin III (AT III) can also be indicated instead of or in addition to INR. The maximum standard value is, for example, 45%, 60%, or 70%, and the minimum standard value is, for example, 5%, 8%, or 10%.

[0117]

As an indicator of coagulopathy in sepsis accompanied by coagulopathy, an abnormal increase in the level of D-dimer can also be indicated instead of or in addition to INR. The minimum standard value is, for example, 2000 ng/ml, 3000 ng/ml, or 3500 ng/ml, and the maximum standard value is, for example, 50,000 ng/ml, 60,000 ng/ml or 70,000 ng/ml.

[0118]

Although the time from 1) diagnosis, determination or confirmation whether a sepsis patient is a sepsis patient with coagulopathy (time 1) to 2) the start of therapeutic treatment and/or improvement of the patient using the drug of the present invention (time 2) is not specifically limited, unless it deviates from the time defined for the subject of the present invention, the time from time 1 to time 2 may be, for example, 24 hours.

[0119]

Examples of bacteremia include a condition in which the presence of bacteria in the blood is confirmed by a positive blood culture.

Examples of septicemia include a condition in which the presence of microorganisms or other toxins in the blood is confirmed, but it may be a pathological condition that corresponds, in addition to the presence of infection, to two or more of the signs of SIRS ((1) body temperature > 38°C or < 36°C, (2) heart rate > 90/min, (3) respiratory rate > 20/min, or PaCO ₂ <32 Torr, and (4) white blood cell count > 1200 0/mcL or < 4000/mcL, or immature leukocytes > 10%), and a positive blood culture is not required.

Examples of systemic inflammatory response syndrome (SIRS) include a pre-DIC condition as described above.

[0120]

Examples of severe sepsis include sepsis accompanied by one or more symptoms, including organ dysfunction such as metabolic acidosis, organ hypoperfusion, acute encephalopathy, oliguria, hypoxemia or disseminated intravascular coagulation, and hypotension. Sepsis with organ dysfunction, organ hypoperfusion, or hypotension is called severe sepsis. Organ hypoperfusion or abnormal perfusion includes lactic acidosis, oliguria, confusion, and the like. The state of severe sepsis with persistent hypotension despite sufficient fluid loading is termed septic shock.

[0121]

More specifically, the severe sepsis referred to in this embodiment is described below.

Examples of septic shock include a condition with hypotension (blood pressure of 90 mm Hg or less, or less than normal blood pressure of 40 mm Hg or more) that does not respond to resuscitation by fluid replacement and is accompanied by organ failure.

Examples of irreversible septic shock include a state of septic shock lasting for 1 hour or longer and not responding to a blood pressure agent with fluid therapy.

Examples of multiple organ dysfunction syndrome (MODS) include a condition with dysfunction of one or more organs, and requiring medical intervention to maintain homeostasis.

[0122]

The Sofa score (Sequential organ failure assessment) is an indicator of the degree of dysfunction of vital organs, represented by a numerical value, and the degree of organ dysfunction is assessed in five steps from zero to four points on six items: respiratory organs, blood coagulation system, liver function, cardiovascular system, central nervous system and kidney function. It is believed that when the score is greater than 5, the mortality reaches the level of about 20%. This score is useful in determining the severity of sepsis. In addition to the Sofa score, the Apache score (Acute physiology and chronic health evaluation) II is also known, and it is a score that predicts mortality and the like using 12 measures relating to respiration, circulation, and the like.

[0123]

Although the infectious disease leading to sepsis is not specifically limited, examples include pneumonia (acute lung infection), urinary tract infection, intestinal infection, bloodstream infection, and the like. The sepsis-producing infectious disease to be treated with the drug of the present invention is preferably an infectious disease other than a urinary tract infection (acute lung infection, intestinal infection, bloodstream infection, and the like).

[0124]

The INR referred to in this embodiment is one of the diagnostic criteria that defines a bleeding disorder. INR stands for prothrombin time (hereinafter abbreviated as PT) normalized for differences between manufacturing batches of thromboplastin preparations. INR is usually defined as follows:

INR value = (Coagulation time (sec) of the test sample / Coagulation time (sec) of the control sample) ^{(ISI value)}

In the equation, the coagulation time (sec) of the test sample is the PT of the test sample of the plasma of the subject to be tested, and ISI stands for International Sensitivity Index.

[0125]

Examples of severe sepsis referred to in this embodiment include sepsis accompanied by one or more symptoms including organ dysfunction such as metabolic acidosis, organ hypoperfusion, acute encephalopathy, oliguria, hypoxemia or disseminated intravascular coagulation, and hypotension as described above. The term "severe" means that the disease is in a life-sustaining condition. Examples of severe sepsis include, in particular, sepsis accompanied by dysfunction of one or more organs. Although the organ dysfunction is not specifically limited as long as the organ dysfunction is induced by sepsis, the organ dysfunction preferably includes failure of an organ that is essential to sustain life. Examples of one or more organ dysfunctions include one or more organ dysfunctions selected from the group consisting of circulatory organ dysfunction, respiratory organ dysfunction, kidney dysfunction and liver dysfunction, preferred examples include one or more organ dysfunctions selected from the group consisting of respiratory organ dysfunction, circulatory organ dysfunction and kidney dysfunction, and more preferred examples include one or more organ dysfunctions selected from the group consisting of respiratory organ dysfunction and dysfunction of the circulatory system. Although the number of organ dysfunctions is not specifically limited as long as the number is one or more, the number may preferably be two or more. It is particularly preferred when two types of organ dysfunctions are present, such as respiratory organ dysfunction and circulatory organ dysfunction. Respiratory organ dysfunction and circulatory organ dysfunction can also be referred to as respiratory dysfunction and cardiovascular dysfunction, respectively.

[0126]

In this embodiment, it is preferred that the patient has one or more organ dysfunction and three or less. It may also be preferable that the patient has at least a circulatory organ dysfunction or a respiratory organ dysfunction as an organ dysfunction.

[0127]

The circulatory organ dysfunction is not particularly limited as long as it is a well-known circulatory organ dysfunction, and examples include, for example, hypotension and shock. Specific examples include conditions observed after taking vasopressor drugs. Another example is a condition with an arterial lactate level > 2 mmol/l.

The respiratory organ dysfunction is not particularly limited as long as it is a well-known respiratory organ dysfunction, and examples include, for example, hypoxemia, acute lung injury, and dyspnea. Specific examples include conditions in which it is desirable or necessary to use a ventilator.

The renal dysfunction is not particularly limited as long as it is a well-known renal dysfunction, and examples include, for example, impaired renal function, oliguria, and renal insufficiency. Specific examples include a condition with a creatinine level ≥ 2.0 mg/dl.

Liver dysfunction is not particularly limited as long as it is a well-known liver dysfunction, and examples include, for example, liver dysfunction, jaundice, liver failure, and the like. Specific examples include a condition with a bilirubin level ≥ 2.0 mg/dl.

[0128]

These organ dysfunctions are widely known as described in publications published prior to the filing date of this application, for example, Funada H., "Elucidation and Treatment Strategy for Sepsis", Iyaku Journal Co., Ltd., p.38-(2006)), "Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock 2008" (Crit. Care Med., 2008 Jan; 36(1): 2 96-327), etc.

[0129]

It is contemplated that organ dysfunction may be induced by a factor other than sepsis, such as in the case of drug-induced organ dysfunction, and accordingly it is desirable that patients with organ dysfunction limited to the liver and kidneys be excluded from severe sepsis. It is also known that thrombocytopenia can develop as a result of organ dysfunction. Although the platelet count of patients to be administered the drug of this embodiment is not particularly limited as long as the platelet count is less than 300,000/µl, it is preferably less than 200,000/µl, more preferably less than 150,000/µl.

[0130]

In this embodiment, it is preferable that the patient's Apache (Acute physiology and chronic health evaluation) II score is less than 35. It may also be, for example, 25 or more.

[0131]

In this embodiment, the INR value in the plasma sample of the sepsis patient is not particularly limited as long as the value is greater than 1.4, and when the INR is greater than 1.4, thrombomodulin is more effective for septic patients having one or more organ dysfunctions. The maximum INR value, for example, may be 2.0 or less, preferably 1.9 or less, more preferably 1.8 or less, even more preferably 1.7 or less, most preferably 1.6 or less. It may also be preferably 1.5 or lower. It is also preferable to exclude a patient with an INR value of 1.7.

The expression "INR greater than 1.4" can also be specified as "INR > 1.4".

[0132]

In this embodiment, DIC is a disease or syndrome in which large amounts of blood clotting accelerators are generated as a result of tissue damage caused by various diseases, thus the function of the coagulation system is excessively accelerated, small clots (microthrombi formation) are formed in the vascular system and clog small vessels, and at the same time, platelets or clotting factors necessary to control bleeding are used up, thus causing a violation of blood clotting. In particular, as a result of the formation of fibrin in the blood vessels, bleeding occurs due to the exhaustion of the blood coagulation system or organ failure as a result of the formation of microclots. DIC is also called disseminated intravascular coagulation syndrome or diffuse intravascular coagulation syndrome.

[0133]

DIC has different types of clinical symptoms depending on the type of underlying pathogenic condition. In addition to observing bleeding or organ symptoms, the preferred method for diagnosing a disease as a DIC syndrome involves continuing to score DIC based on multiple scores as described below, and then diagnosing the disease as DIC when the DIC score reaches a certain level. Examples of such measured parameters include blood platelet count, concentration of fibrin/fibrinogen degradation products (hereinafter abbreviated as FDP, in some cases) formed by plasmin degradation, D-dimer concentration, fibrinogen concentration, prothrombin time, and the like. In addition, it is also possible to diagnose a certain condition as pre-DIC based on a decrease in platelets, an increase in the concentration of D-dimer or FDP, and the like. without the need for further evaluation of DIC (Masao Nakagawa, “Search report regarding use of criteria of disseminated intravascular coagulation (DIC),” Research Study Study Team of Intractable Disease (Blood Coagulation Abnormality), the Ministry of Health and Welfare, Study report 1999, 1999: 65-72; Katsumi Deguchi, “Tentative plan regarding standards for initiation of early treatment of DIC,” Research Study Team of Intractable Disease (Bloo d Coagulation Abnormality), the Ministry of Health and Welfare, Study report 1999, 1999: 73-77; and Katsumi Nakagawa & Hajime Tsuji, “Current diagnosis of DIC - Reports on results of inquiry survey” Clinical Blood. 1999, 40: 362-364).

[0134]

In this embodiment, a sepsis patient with an INR value in the patient's plasma sample greater than 1.2, preferably greater than 1.3, more preferably greater than 1.4, may be referred to as a DIC patient in a broad sense, and a therapeutic treatment and/or amelioration drug for sepsis according to this embodiment may be used as a therapeutic treatment and/or amelioration drug for a DIC with an INR value greater than 1.4.

[0135]

The drug of this embodiment can also be used to treat DIC. Sepsis is also considered as SIRS caused by critical clinical invasion resulting from infection and is closely related to DIC caused by an infectious disease. DIC often develops concomitantly with sepsis, and the drug of this embodiment can also be used for such a sepsis patient who co-develops DIC. In other words, the drug of this embodiment can be used for a patient diagnosed or suspected of DIC or sepsis, or both.

[0136]

In this embodiment, INR can be measured, for example, as follows. Specifically, tissue thromboplastin and Ca ²⁺ are added to plasma (test sample) obtained by adding sodium citrate, the time (PT) required for coagulation (fibrin precipitation) is measured, and an estimate is determined based on the relative time in seconds relative to the control sample (activity index). The activity index can be obtained as “coagulation time (seconds) of the test sample/coagulation time (seconds) of the control sample”, but the ratio may vary in different laboratories in which the test is performed, due to the difference in sensitivity of the tissue thromboplastin used. The INR value was designed to eliminate such bias, and by estimating the PT using the INR value adjusted using the International Sensitivity Index (hereinafter also abbreviated as ISI), bias due to laboratory differences can be eliminated to obtain a standard result. ISI represents a difference from the International Standard. The ISI is determined for each tissue thromboplastin reagent and is attached to the reagent. Examples of the thromboplastin reagent include, but are not limited to, Thromborel S (registered trademark, Sysmex Corp.), Thromboplastin C+ (registered trademark, Sysmex Corp.) and the like. Thromborel S (registered trademark) uses human placental thromboplastin (ISI value is about 1.0) and Thromboplastin C+ (registered trademark) uses rabbit brain thromboplastin (ISI value is about 1.8)

[0137]

The ISI is attached to each tissue thromboplastin reagent and the INR value is calculated according to the equation above.

Although the control sample is not particularly limited as long as the sample is a commercially available pooled human normal plasma, commercially available pooled citrate (Na citrate) normal human plasma and the like, available from Kojin-Bio Co., Ltd., for example, can be used. or International Bioscience Inc.

[0138]

With regard to the therapeutic treatment of sepsis, such basic treatments are usually used as listed below, with reference to known publications (Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock: Crit. Care Med., 2008, 36:296-327; Crit. Care Med., 32(3), 1250-56 (2003)), and thrombomodulin and other drug can be use in combination. However, the other drug used in combination is not limited to the following.

[0139]

When hypotension continues in a patient with septic shock even after central venous pressure (CVP) has risen to the desired value, dopamine may be administered to raise mean blood pressure to at least 60 mmHg. When the dose of dopamine exceeds 20 mcg/kg/min, another vasopressor (usually norepinephrine) may be added.

[0140]

Antibiotics are commonly used for therapeutic treatment against bacteria that cause sepsis. The choice of antibiotic requires a well-weighted assessment based on the suspected cause, clinical signs, knowledge of microorganisms and knowledge of the sensitivity pattern common to a particular hospital department for inpatients, results of preliminary microflora analysis, etc. Intensive normalization of blood sugar levels in patients with sepsis improves the clinical outcome of critically ill patients.

When an antibiotic is used, a sample, such as blood, body fluid, or part of a wound, can be examined and a drug effective against the bacterial pathogen can be selected. For example, in the case of septic shock of unknown cause, gentamicin or tobramycin and a third generation cephalosporin may be administered in combination. In addition, if infection with resistant Staphylococcus or Enterococcus bacteria is suspected, vancomycin is additionally administered.

[0141]

Typically, the dose is adjusted to maintain blood sugar levels of 80 to 110 mg/dL (4.4 to 6.1 mmol/L) by continuous intravenous injection of insulin.

Since corticosteroid therapy is effective for the therapeutic treatment of sepsis, it can be used as an additional dose.

[0142]

In a patient with a high risk of death (APACHE II score ≥ 25, multiple organ failure due to sepsis, ARDS due to septic shock or sepsis), recombinant activated protein C (rhAPC, drotrecogin α) can be prescribed in the absence of contraindications (hemorrhage, etc.).

Although the number of targeted patients is limited, RBC transfusions can be performed to achieve an Hb of 7.0 to 9.0 g/dL.

[0143]

Erythropoietin (EPO) can be administered if erythropoiesis is impaired in a patient with sepsis due to renal insufficiency.

In severe sepsis, low-dose unfractionated heparin or low molecular weight heparin may be given to prevent DVT.

[0144]

The drug of the present invention may contain a carrier. As the carrier used in the present invention, a water-soluble carrier is preferred, and a tonicity regulator, a buffering agent, a viscosity enhancer, a surfactant, a preservative, an antiseptic, a soothing agent, a pH modifier, or the like acceptable as pharmaceutical additives, are generally preferred. For example, the drug of the present invention can be prepared by adding sucrose, glycerol, a pH modifier consisting of an inorganic salt, or the like. as additives. In addition, if necessary, amino acids, salts, carbohydrates, surfactants, albumin, gelatin, and the like can be added as disclosed in Japanese Unexamined Patent Application Publications Nos. 64-6219 and 6-321805. The method of adding such additives is not particularly limited. However, in the case of obtaining a lyophilized product, examples include, for example, a method of mixing a solution containing at least one therapeutic agent selected from an immunosuppressant and a hematological malignant agent and a solution containing thrombomodulin, and then adding to the mixture of additives, and mixing the resulting mixture, as well as a method of mixing the additives with at least one therapeutic agent selected from an immunosuppressant and a hematological malignant agent previously dissolved in water, water for injection or an appropriate buffer, adding a solution containing thrombomodulin to the mixture, mixing the resulting mixture to obtain a solution and lyophilization of the solution in a commonly used manner. When the drug of the present invention is a drug including a combination of drug components, each component is preferably prepared by adding a carrier according to a suitable production method. The medicament of the present invention may be presented in the form of an injection or in the form of a lyophilized preparation for dissolution upon use.

[0145]

With regard to the preparation of the drug of the present invention, an aqueous solution for injection can be obtained by filling an ampoule or vial, for example, with a solution containing 0.1 to 10 mg of thrombomodulin, water for injection and additives, in a volume of 0.5 to 10 ml. Examples of the preparation method also include a method of freezing such a solution and drying the frozen solution under reduced pressure to obtain a lyophilized preparation.

[0146]

The medicament of the present invention is desirably administered parenterally, eg intravenously, intramuscularly and subcutaneously. The drug can also be administered by oral, intrarectal, intranasal, sublingual routes, and the like. When the drug of the present invention is a drug comprising a combination of several active ingredients, each active drug ingredient is preferably administered by an administration method suitable for that ingredient.

Examples of the method of intravenous administration include the method of administering the desired dose of the drug at one time (intravenous bolus administration) and intravenous administration by drip infusion.

[0147]

The method of administering the desired dose of the drug at one time (intravenous bolus administration) is preferred in that the method requires a short period of time for administration. Especially in the case of patients with sepsis, who often require urgent treatment, it may be preferable to administer in a short time. When the drug is administered at one time, the period required for administration using an injection syringe can usually vary. Typically, the period of time required for administration is, for example, 5 minutes or less, preferably 3 minutes or less, more preferably 2 minutes or less, even more preferably 1 minute or less, particularly preferably 30 seconds or less, although this depends on the volume administered. Although the minimum administration time is not particularly limited, the period is preferably 1 second or longer, more preferably 5 seconds or longer, even more preferably 10 seconds or longer. The dose is not particularly limited as long as the dose is within the above preferred dose. Intravenous administration by drip infusion is also preferred in that the blood thrombomodulin level can be easily maintained constant.

[0148]

The daily dose of the drug of the present invention may vary depending on the age, body weight of the patient, the severity of the disease, the route of administration, and the like. In general, the maximum dose is preferably 20 mg/kg or less, more preferably 10 mg/kg or less, even more preferably 5 mg/kg or less, particularly preferably 2 mg/kg or less, and most preferably 1 mg/kg or less, and the minimum dose is preferably 0.001 mg/kg or more, more preferably 0.005 mg/kg or more, even more preferably 0.01 mg/kg or more, particularly preferably 0.02 mg/kg or more, and most preferably 0.05 mg/kg or more, based on the amount of thrombomodulin.

[0149]

In the case of intravenous bolus administration, although the dose is not specifically limited as long as the dose is within the above preferred dose, the maximum daily dose is preferably 1 mg/kg or less, more preferably 0.5 mg/kg or less, even more preferably 0.1 mg/kg or less, particularly preferably 0.08 mg/kg or less, and most preferably 0.06 mg/kg or less, and the minimum dose is preferably 0.005 mg/kg or more, more preferably 0.01 mg/kg or more, even more preferably 0.02 mg/kg or more, and particularly preferably 0.04 mg/kg or more.

[0150]

When the drug of the present invention is administered to a patient having a body weight of more than 100 kg, it can preferably be administered at a fixed dose of 6 mg because the blood volume is not proportional to the body weight, and the blood volume is relatively reduced relative to the body weight of such a patient.

[0151]

In the case of continuous intravenous infusion, although the dose is not particularly limited as long as the dose is within the above preferred dose, the maximum daily dose is preferably 1 mg/kg or less, more preferably 0.5 mg/kg or less, even more preferably 0.1 mg/kg or less, particularly preferably 0.08 mg/kg or less, and most preferably 0.06 mg/kg or less, and the minimum dose is preferably 0.005 mg/kg or more, more preferably 0.01 mg/kg or more, even more preferably 0.02 mg/kg or more, and particularly preferably 0.04 mg/kg or more.

[0152]

When the drug of the present invention is administered to a patient having a body weight of more than 100 kg, it can preferably be administered at a fixed dose of 6 mg because the blood volume is not proportional to the body weight, and the blood volume is relatively reduced relative to the body weight of such a patient.

The drug is administered one or more times a day, as needed. As for the administration interval, the drug can be administered once every 2-14 days, preferably once every 2-7 days, more preferably once every 3-5 days.

[0153]

[Sequence Listing Explanation]

SEQ ID NO: 1: Amino acid sequence encoded by the gene used to produce TME456

SEQ ID NO: 2: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1

SEQ ID NO: 3: Amino acid sequence encoded by the gene used to produce TME456M

SEQ ID NO: 4: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3

SEQ ID NO: 5: Amino acid sequence encoded by the gene used for the production of TMD12

SEQ ID NO: 6: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5

SEQ ID NO: 7: Amino acid sequence encoded by the gene used for the production of TMD12M

SEQ ID NO: 8: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7

SEQ ID NO: 9: Amino acid sequence encoded by the gene used for the production of TMD123

SEQ ID NO: 10: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9

SEQ ID NO: 11: Amino acid sequence encoded by the gene used to produce TMD123M

SEQ ID NO: 12: Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11

SEQ ID NO: 13: Synthetic DNA for mutation used to carry out site-directed mutagenesis

Examples

[0154]

The present invention is further explained in detail with reference to examples and test examples. However, the present invention is not limited to these examples.

[0155]

Thrombomodulin of the present invention used in the test examples was obtained in accordance with the above method of Yamamoto et al. (the method described in Japanese Unexamined Patent Publication No. 64-6219). Examples of how to obtain it are described below. The safety of the thrombomodulins produced in these preparations was confirmed in the single and repeated intravenous administration tests using rats and monkeys, in the test for possible impairment of reproductive function in mice, in the local irritation test, in the pharmacological safety test, in the viral inactivation test, and the like.

[0156]

[Receiving Example 1]

<Receiving Thrombomodulin>

A highly purified product was obtained by the above method. Specifically, Chinese Hamster Ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 9 (which specifically consisted of the nucleotide sequence of SEQ ID NO: 10). From the culture of the above transformed cells, a highly purified product was obtained by collecting the active fraction using phosphate buffer 20 mmol/l (pH 7.3) containing 50 mmol/l NaCl, according to the indicated traditional purification method. The product was then concentrated using an ultrafiltration membrane to obtain a thrombomodulin solution at a concentration of 11.0 mg/ml (hereinafter also abbreviated as TMD123)

[0156]

[0157]

<Obtaining a Polysorbate Solution>

Polysorbate 80 was weighed (0.39 g) into a glass beaker, water for injection (30 ml) was added and dissolved.

[0158]

<Receipt and packaging of drug solution>

The TMD123 solution prepared as described above (2239 ml, corresponding to 24.63 g of soluble thrombomodulin protein added in a 5% excess) was placed in a 5 L stainless steel vessel. The previously prepared polysorbate solution was then added and sodium chloride (27.9 g) was added. Water for injection (600 ml) was added and the mixture was stirred. The mixture was adjusted to pH 6.0 by adding a solution of 1 mol/l hydrochloric acid. Again, water for injection was added to the mixture to a total of 3940 g, and the mixture was uniformly mixed and stirred. This drug solution was filter sterilized using a filter having a pore diameter of 0.22 μm (MCGL10S, manufactured by Millipore). The filtrate was loaded into ampoules of 1.1 g each to obtain the drug TMD123.

[0159]

[Receiving Example 2]

Chinese hamster ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 11 (which specifically consists of the nucleotide sequence of SEQ ID NO: 12), a thrombomodulin solution purified from the culture of the above transformed cells (hereinafter abbreviated as TMD123M) was obtained by the above conventional purification method, and a TMD123M preparation was obtained in the same manner as described above.

[0160]

[Receiving Example 3]

Chinese hamster ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 1 (which specifically consists of the nucleotide sequence of SEQ ID NO: 2), thrombomodulin purified from the culture of the above transformed cells (hereinafter abbreviated as TME456) by the above conventional purification method, and TME456 preparation was obtained in the same manner as described above.

[0161]

[Receiving Example 4]

Chinese hamster ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 3 (which specifically consists of the nucleotide sequence of SEQ ID NO: 4), thrombomodulin purified from the culture of the above transformed cells (hereinafter abbreviated as TME456M) by the above conventional purification method, and a TME456M preparation was obtained in the same manner as described above.

[0162]

[Receiving Example 5]

Chinese hamster ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 5 (which specifically consists of the nucleotide sequence of SEQ ID NO: 6), thrombomodulin purified from the culture of the above transformed cells (hereinafter abbreviated as TMD12) was obtained by the above conventional purification method, and a TMD12 preparation was obtained in the same manner as described above.

[0163]

[Receiving Example 6]

Chinese hamster ovary (CHO) cells were transfected with DNA encoding the amino acid sequence of SEQ ID NO: 7 (which specifically consists of the nucleotide sequence of SEQ ID NO: 8), thrombomodulin purified from the culture of the above transformed cells (hereinafter abbreviated as TMD12M) by the above conventional purification method, and a TMD12M preparation was obtained in the same manner as described above.

[0164]

[Receiving Example 7]

Receiving a placebo drug

<Obtaining a Polysorbate Solution>

Polysorbate 80 was weighed (0.4 g) into a glass beaker, water for injection (30 ml) was added and dissolved.

[0165]

<Receipt and packaging of drug solution>

Water for injection (2000 ml) was placed in a 5 L stainless steel vessel. Then, the previously prepared polysorbate solution was added. Again, water for injection was added to the mixture to a total of 4000 g, and the mixture was uniformly mixed and stirred. This drug solution was filter sterilized using a filter having a pore diameter of 0.22 μm (MCGL10S, manufactured by Millipore). The filtrate was loaded into ampoules of 1.1 g each to obtain a placebo preparation.

[0166]

[Example 1]

<Method for experiment>

Using TMD-123 prepared according to Production Example 1 as thrombomodulin, a randomized, double-blind, placebo-controlled trial was performed in patients with severe sepsis accompanied by coagulopathy. A total of 816 patients were scheduled. Of these, 402 patients were allocated to the TMD-123 administration group and 414 patients to the placebo administration group. The experiment involved 800 patients who were given the following administration (TMD-123 received 395 patients and placebo received 405 patients). TMD-123 was administered once a day at a dose of 0.06 mg/kg for no more than 6 consecutive days by intravenous bolus injection. The preparation prepared according to Preparation Example 7 was used as a placebo. Patients weighing more than 100 kg were evenly administered a fixed dose of 6 mg once a day for 6 consecutive days by intravenous bolus injection to suppress side effects caused by overdose.

[0167]

In this experiment, “accompanied by coagulopathy” meant 1) INR > 1.4, and 2) platelet count greater than 30,000/mm ³ and less than 150,000/mm ³ , or a decrease in platelets greater than 30% in 24 hours.

[0168]

Plasma INR values of patients prior to administration of the test drug were measured in the manner described above using the equation. The platelet count of the patients was measured in a manner that is traditionally used as such.

[0169]

In this experiment, the target patients were patients having the following organ dysfunction a and/or organ dysfunction b associated with sepsis.

1) Organ dysfunction a : impairment of cardiovascular function requiring both fluid therapy and a vasopressor drug sufficient to maintain a mean arterial pressure of 65 mmHg. or higher.

2) Organ dysfunction b : impaired respiratory function with an urgent need for artificial respiration and with a PaO ₂ /FiO ₂ ratio of less than 250 (or less than 200 when the infection is in the lung).

Respiratory dysfunction

The above violation of cardiovascular function can be called dysfunction of the circulatory organs. The above violation of the respiratory function can be called respiratory dysfunction.

[0170]

In this experiment, a total of 816 patients who met the above criteria for coagulopathy and the above criteria for organ dysfunction within no more than 24 hours were randomized for no more than 12 hours and received the first administration within 4 hours in principle from the time of randomization. During the first administration, the platelet count, plasma INR value, etc. at the time point immediately before the introduction was re-measured (baseline) (Fig. 1).

[0171]

The above expression "patients who confirmed meeting the above criteria for coagulopathy and the above criteria for organ dysfunction within no more than 24 hours" means that the period from the time when patients first met any of the criteria for INR value, platelet count and organ dysfunction, until the time when it was confirmed that patients meet all other criteria, is 24 hours or less. The above expression "randomized within no more than 12 hours" means that the period from the time that patients were confirmed to meet the above criteria for coagulopathy and the above criteria for organ dysfunction, until the completion of obtaining consent from the patient to participate in the clinical trial (informed consent, IC), confirmation of the patient's eligibility for participation in the clinical trial at the clinical coordinating center (CCC), registration of the clinical trial and randomization is 12 hours or less (Fig. 1).

The overall results were confirmed on the 28th day from the start of administration, and the mortality rates (mortality) in the patient groups were calculated as the main indicator in evaluating the effectiveness. Differences between mortality rates in the TMD-123 and placebo groups were calculated as Difference.

[0173]

Serious adverse events (SAE), major significant bleeding (MBE), and anti-drug antibodies (ADA) were observed as primary indicators for safety.

[0174]

<Experimental Results>

The above 800 cases were defined as the maximum analysis group (full analysis sample, FAS), the effectiveness for the complete analysis sample and the effectiveness for its subgroups, defined from different points of view, were analyzed (Table 1).

Table 1 No. Group Number of cases Mortality in the placebo group (a) % Mortality in the TMD-123 administration group (b) % Difference in efficacy between groups (a-b) % 1 Full sample for analysis 800 29.383 26.835 2.55 2 BL INR >1.4 654 32.537 28.527 4.01 BL INR ≤1.4 146 14.286 19.737 -5.45 3 BL INR >1.4, and
PLT >30000 (mm ³ ) 634 32.110 26.710 5.40 BL INR ≤1.4, and/or
PLT ≤30000 (mm ³ ) 166 17.949 27.273 -9.32 4 TAT ≥10 (ng/ml) 489 35.685 29.032 6.65 TAT <10 (ng/ml) 286 18.301 23.308 -5.01 5 Introduction four times or more 640 20.807 16.352 4.46 6 BL INR >1.4 and
administration 4 times or more 512 22.901 16,000 6.90 7 BL INR >1.4,
administration 4 times or more, and
PLT >30000 (mm ³ ) 503 22.568 15.447 7.12 8 BL INR >1.4,
TAT ≥10(ng/mL) and
PLT >30000 (mm ³ ) 393 38.384 29.231 9.15 9 BL INR >1.4,
PLT >30000 (mm ³ ),
And
Sofa score ≤11 573 31.164 24.555 6.61 10 BL INR >1.4,
PLT >30000 (mm ³ ),
And
Apache BL score ≤30 475 31.474 23.661 7.81 eleven BL F1+2 ≥250 (pmol/l) 508 32.937 28.906 4.03 12 BL protein C ≤40% 510 32.830 26.531 6.30 BL protein C >40% 215 15.179 19.417 -4.24 13 BL AT III <70% 657 29.552 25.466 4.09 14 BL MP >10 (nm) 626 30.313 24.837 5.48 15 BL APACHE II score ≥25 283 36.064 37,500 -1.45 BL APACHE II score <25 439 24.201 19.545 4.66 BL APACHE II score ≥35 51 30.435 39.286 -8.85 BL APACHE II score <35 671 28.863 25.305 3.56 16 BL number of organ dysfunctions=1 187 18.824 12.745 6.08 BL number of organ dysfunctions=2 347 27.273 27.485 -0.21 BL number of organ dysfunctions=3 210 42.727 36,000 6.73 BL number of organ dysfunctions=4 47 25,000 52.632 -27.63 1 ≤ BL number of organ dysfunctions ≤ 3 744 29.919 25.737 4.18 17 BL INR >1.4,
PLT >30000 (mm ³ )
And
1 ≤ BL number of organ dysfunctions ≤ 3 596 32.248 24.913 7.33 18 BL arterial lactate ≤6 (mmol/L) 530 26.354 24.901 1.45 BL arterial lactate >6 (mmol/L) 107 51.852 54.717 -2.87 19 BL plasma creatinine <2 (mg/dl) 528 26.996 24.528 2.47 BL plasma creatinine ≥2 (mg/dl) 262 32.847 32,000 0.85 20 Diabetic Patients 187 32.990 23.333 9.66 Non-diabetic patients 613 28.247 27.869 0.38 21 Patients using heparin 416 27,700 28.571 -0.87 Patients not using heparin 384 31.250 25,000 6.25 22 BL INR >1.4,
PLT >30000 (mm ³ )
And
BL F1.2 ≥250 (pmol/l) 408 35.407 27.638 7.77 23 BLINR > 1.4,
PLT >30000 (mm ³ )
AND
BL protein C ≤40% 410 36.073 26.702 9.37 24 BL INR >1.4,
PLT >30000 (mm ³ )
And
BL AT III <70% 518 32.714 25.703 7.01 25 BL INR >1.4,
PLT >30000 (mm ³ )
And
BL MP >10 (nm) 497 33.591 23,950 9.64 26 BL level of D-dimer ≥3500 (ng/ml) 332 29.448 23.669 5.78 27 BL INR >1.4,
PLT >30000 (mm ³ )
And
BL level of D-dimer ≥3500 (ng/ml) 268 31.579 22.963 8.62 28 BLINR> 1.4,
PLT >30000 (mm ³ )
And
Apache BL <35 524 31.636 24,900 6.74 29 BL F1+2 >229 (pmol/l) 536 33.088 28.788 4.30 thirty BLINR > 1.4,
PLT >30000 (mm ³ )
And
BL F1+2 >229 (pmol/l) 428 36.036 27.670 8.37

[0175]

In the above table, "BL INR >1.4" refers to the group of patients who showed an INR value greater than 1.4 at the time just before administration (baseline (BL)), from the full analysis sample.

In the above table, "BL INR ≤1.4" means a group of patients who showed an INR value of not more than 1.4 at the time point immediately before administration (baseline), from the complete analysis sample.

In the above table, "BL INR >1.4 and PLT >30,000 (mm ³ )" means a group of patients who showed an INR value greater than 1.4 at the time immediately before administration (baseline) and a platelet count greater than 30,000 (mm ³ ) at the time immediately before administration (baseline), from the complete sample for analysis.

[0176]

In the above table, "BL INR ≤1.4 and/or PLT ≤30,000 (mm ³ )" means a group of patients who showed an INR value of not more than 1.4 at the time immediately before administration (baseline) and/or a platelet count of not more than 30,000 (mm ³ ) at the time immediately before administration (baseline), from the complete set for analysis.

In the above table, "TAT ≥10 (ng/mL)" refers to a group of patients who showed a thrombin-antithrombin complex (TAT) level of at least 10 ng/mL from the full sample for analysis.

[0177]

Clearly, "TAT" in the above table means the measured level of the thrombin-antithrombin complex (TAT) at the time point immediately before administration (baseline).

[0178]

In the above table, "BL F1+2 ≥250 (pmol/L)" refers to the group of patients who showed a prothrombin 1+2 (F1+2) fragment level of not less than 250 pmol/L just before administration (baseline), from the complete sample for analysis.

In the above table, "BL F1+2 >229 (pmol/L)" refers to the group of patients who showed a prothrombin 1+2 (F1+2) fragment level greater than 229 pmol/L immediately before administration (baseline), from the full sample for analysis.

"BL AT III <70%" means a group of patients who showed an antithrombin III (ATIII) level of less than 70% immediately before administration (baseline), from the full sample for analysis.

[0179]

In the table above, "BL MP >10 (nm)" refers to a group of patients who showed a microparticle size greater than 10 nm just prior to administration (baseline), from the full sample for analysis.

In the above table, "BL protein C ≤40%" means a group of patients who showed a protein C level of no more than 40% at the time immediately before administration (baseline), from the complete sample for analysis.

[0180]

In the above table, "BL protein C >40%" means a group of patients who showed a protein C level greater than 40% at the time immediately before administration (baseline), from the complete sample for analysis.

In the above table, "BL APACHE II score ≥25" means a group of patients who showed an APACHE II score of at least 25 at the time immediately before administration (baseline), from the full analysis sample.

[0181]

In the above table, "BL APACHE II score <25" means a group of patients who showed an APACHE II score of less than 25 at the time immediately before administration (baseline), from the full analysis sample.

In the above table, "BL APACHE II score ≥35" means a group of patients who showed an APACHE II score of at least 35 at the time point immediately before administration (baseline), from the complete analysis sample.

[0182]

In the table above, "BL APACHE II score <35" means a group of patients who showed an APACHE II score of less than 35 at the time just before administration (baseline), from the full analysis sample.

In the above table, "BL number of organ dysfunctions" means the number of organ dysfunctions in a patient from the full sample for analysis at the time point immediately before administration (baseline).

[0183]

In the above table, "1 ≤ BL number of organ dysfunctions ≤3" means the group of patients who showed the number of organ dysfunctions of no less than 1 and no more than 3 at the time immediately before administration (baseline), from the full sample for analysis.

In the above table, "BL INR >1.4, PLT >30,000 (mm ³ ), and 1 ≤ BL number of organ dysfunctions ≤ 3" means a group of patients who showed an INR value greater than 1.4 at the time immediately before administration (baseline), a platelet count greater than 30,000 (mm ³ ) at the time immediately before administration (baseline), and the number of organ dysfunctions is not less than 1 and not more than 3 at the time point immediately before administration (baseline), from the full analysis sample.

[0184]

In the above table, "BL arterial lactate level ≤6 (mmol/l)" means a group of patients who showed an arterial lactate level of not more than 6 mmol/l just before administration (baseline), from the full sample for analysis.

In the above table, "BL arterial lactate level >6 (mmol/l)" means the group of patients who showed an arterial lactate level greater than 6 mmol/l just before administration (baseline), from the full sample for analysis.

[0185]

In the table above, "BL serum creatinine <2 (mg/dl)" means the group of patients who showed a serum creatinine level of less than 2 mg/dl immediately before administration (baseline), from the complete analysis sample.

In the above table, "BL serum creatinine ≥2 (mg/dl)" means the group of patients who showed a serum creatinine level of not less than 2 mg/dl just before administration (baseline), from the complete analysis sample.

[0186]

In the above table, "diabetic patients" refers to the group of patients who had a diabetic complication just prior to administration (baseline) from the full analysis sample.

In the table above, "non-diabetic patients" means the group of patients who did not have a diabetic complication immediately prior to administration (baseline), from the full sample for analysis.

[0187]

In the above table, "patients using heparin" refers to the group of patients who used heparin at a low dose just before administration (baseline), from the full sample for analysis.

In the table above, "heparin-free patients" refers to the group of patients who did not use low-dose heparin immediately prior to administration (baseline), from the full analysis sample.

[0188]

In the table above, “four or more administrations” refers to a group of patients who received 4 or more administrations (i.e., 4 to 6 administrations) of the study drug (TMD-123 or placebo) from the full analysis sample.

Needless to say, "administering 4 times or more" as indicated in the above table means "administering for at least 4 consecutive days".

[0189]

In the above table, "BL INR >1.4 and administration 4 times or more" means a group of patients who showed an INR value greater than 1.4 at the time point immediately before administration (baseline) and received administration of the investigational drug (TMD-123 or placebo) 4 or more times (i.e., 4 to 6 times), from the full set for analysis.

[0190]

In the above table, "BL INR >1.4, administration 4 times or more and PLT >30,000 (mm ³ )" means a group of patients who showed an INR value greater than 1.4 at the time immediately before administration (baseline), received administration of study drug (TMD-123 or placebo) 4 or more times (i.e., 4 to 6 times) and showed a platelet count greater than 30,000 (mm ³ ) per time point just prior to administration (baseline), from the full analysis sample.

[0191]

In the above table, "BL D-dimer level ≥3500 (ng/mL)" means a group of patients who showed a D-dimer level of not less than 3500 ng/mL immediately before administration (baseline), from the complete analysis sample.

[0192]

With respect to "BL INR >1.4, and PLT >30,000 (mm ³ )" above (634 cases), the following table (Table 2) shows the time from the time just before administration (baseline) to the first administration (leftmost column of Table 2), the efficacy observed for the patient groups administered with study drug during this time period (rightmost column of Table 2), etc.

[0193]

table 2 Elapsed time period Administration group TMD-123 Placebo administration group Difference in efficacy between groups (b-a) % Number of cases Mortality (a) % Number of cases Mortality (b) % Not more than 0.5 hours 136 26.471 164 32.927 6.46 Not more than 1.0 hour 194 25.258 228 32.456 7.20 No more than 2.0 hours 233 26.609 253 32.411 5.80 No more than 4.0 hours 259 26.255 276 31.159 4.90 No more than 8.0 hours 286 26.224 300 32,000 5.78 No more than 12.0 hours 300 27,000 318 32.390 5.39 No more than 24.0 hours 306 26.797 327 32.110 5.31 More than 24.0 hours 307 26.710 327 32.110 5.40

[0194]

With respect to the full sample for analysis (800 cases), the effect of the time period elapsed from the time when the criteria for coagulopathy and criteria for organ dysfunction could be confirmed (at the time of the first measurement) to the time just before administration (baseline) (at the time of re-measurement) within 24 hours on the INR value in patients' plasma is presented in the following table (Table 3).

[0195]

Table 3 The period of time elapsed from the first measurement to the time immediately before the introduction INR during the first measurement INR at the time immediately before administration Total Not more than 1.4 More than 1.4 Number of cases % Number of cases % Number of cases 0 hour
(only first measurement) Not more than 1.4 1 100.0 0 0 1 More than 1.4 0 0 138 100.0 138 No more than 4 hours Not more than 1.4 1 100.0 0 0 1 More than 1.4 9 15.0 51 85.0 60 More than 4 hours but not more than 6 hours Not more than 1.4 1 100.0 0 0 1 More than 1.4 19 18.8 82 81.2 101 More than 6 hours but not more than 8 hours Not more than 1.4 1 100.0 0 0 1 More than 1.4 28 22.4 97 77.6 125 More than 8 hours but not more than 12 hours More than 1.4 39 22.7 133 77.3 172 More than 12 hours but not more than 24 hours More than 1.4 35 19.9 141 80.1 176 More than 24 hours More than 1.4 12 50.0 12 50.0 24 Total Not more than 1.4 4 100.0 0 0 4 More than 1.4 142 17.8 654 82.2 796

[0196]

With respect to the full sample for analysis (800 cases), the effect of the time period elapsed from the time when the criteria for coagulopathy and criteria for organ dysfunction could be confirmed (at the time of the first measurement) to the time just before administration (baseline) (at the time of re-measurement) within 24 hours on the platelet count of patients is presented in the following table (Table 4).

[0197]

Table 4 The period of time elapsed from the first measurement to the time immediately before the introduction Platelet count during the first measurement Platelet count at the time immediately before administration Total Not more than 30000 (mm ³ ) More than 30000 (mm ³ ) Number of cases % Number of cases % Number of cases 0 hour
(only first measurement) Not more than 30000 (mm ³ ) 1 100.0 0 0 1 More than 30000 (mm ³ ) 0 0 122 100.0 122 No more than 12 hours Not more than 30000 (mm ³ ) 1 50.0 1 50.0 2 More than 30000 (mm ³ ) 14 3.0 459 97.0 473 More than 12 hours but not more than 24 hours Not more than 30000 (mm ³ ) 0 0 1 100.0 1 More than 30000 (mm ³ ) 6 3.6 163 96.4 169 More than 24 hours More than 30000 (mm ³ ) 2 6.5 29 93.5 31 Total Not more than 30000 (mm ³ ) 2 50.0 2 50.0 4 More than 30000 (mm ³ ) 22 2.8 773 97.2 795

[0198]

No significant change in the number of platelets was observed within 24 hours from the time of measurement, with regard to coagulopathy. It was also found that 70% or more of patients who showed an INR >1.4 maintained an INR value >1.4 (maintenance of coagulopathy) within 24 hours of measurement.

[0199]

It is believed that the beginning of administration is no later than 24 hours after confirming that the measured values of the number of platelets and the value of Inr in plasma in patients satisfy the above conditions (INR> 1.4 and the number of platelets> 30000 (mm ³ )) are preferable for obtaining high efficiency, and the results shown in the two of the above tables, it is assumed that as one of the reasons for the above. Coagulopathy should usually be preserved for 24 hours.

[0200]

In addition, there was no significant difference in the incidence of major bleeding between all 800 patients treated with study drug and patients who showed "BL INR >1.4 and PLT >30,000 (mm ³ )" as noted above (634 cases), and no significant difference was observed compared to the placebo group.

The Sofa score, determined in the present invention, is not one that is calculated in accordance with the usual method for calculating the Sofa assessment, but is an assessment calculated on the basis of the estimates of the five internal organs (respiratory organ, blood circulation, the liver, kidneys and coagulation), with the exception of the Glasgow Coma Scale scales, and the range includes estimates from 0 to 20. In addition, preliminary values obtained in accordance with the following criteria were used as assessments of the respiratory and blood circulation on the Sofa scale.

Respiratory Organ: For patients in whom the P/F ratio was not measured, the score was 3 for the resuscitated patient and 0 for the non-resuscitated patient.

Circulatory organ: The <1> score for a patient who meets the circulatory organ selection criteria established in the clinical trial was 3 points, and the <2> score for a patient administered "dobutamine" or "dopamine not listed as >5 μg/kg/min" was 2 points. Evaluation <3> of the patient who did not satisfy the conditions <1> and <2>, but showed an average blood pressure of less than 70 mm Hg. during the match test was 1 point, and the score of a patient who did not match any of the patients from <1> to <3> was 0 points.

[0202]

The APACHE (Acute Physiology and Chronic Health Evaluation) II score defined in the present invention corresponds to the system for assessing the severity of the pathology of a patient admitted to the intensive care unit, and is obtained as the sum of points that are assigned according to 12 physiological parameters, age and results of the assessment of complicated chronic diseases. A higher score is considered to indicate a higher severity, and the range is from 0 to 71 points.

[0202]

Industrial Applicability

[0203]

The thrombomodulin-containing drug of the present invention is useful as a drug that enables effective therapeutic treatment and/or reduction of the severity of sepsis accompanied by coagulopathy.

--->

SEQUENCE LIST

<110> Asahi Kasei Pharma Corporation

<120> Medicinal product for therapeutic treatment and/or

improvement in sepsis

<130> 181522WO01

<150> US 62/748706

<151> 2018-10-22

<150> US 62/771628

<151> 2018-11-27

<150> US 62/813352

<151> 2019-03-04

<160> 13

<210> 1

<211> 132

<212> Protein

<213> person

<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> person

<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> Protein

<213> person

<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> person

<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> Protein

<213> person

<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> person

<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> Protein

<213> person

<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> person

<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> Protein

<213> person

<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> person

<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> Protein

<213> person

<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> person

<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: Synthetic DNA

<400> 13

aatgtggcgg gcaagggccg a 21

<---

Claims (12)

1. A method for therapeutic treatment and/or improvement of the condition of a patient with sepsis, where the method includes administering a drug containing thrombomodulin as an active ingredient, where the specified drug is administered to a patient who, immediately before administration, has an international normalized ratio (INR) value greater than 1.4, where the term "immediately before administration" means a period of 1 hour, calculated retrospectively from the start of administration of a drug containing thrombomodulin, where the drug is administered to a patient who immediately before administration has a platelet count greater than 30,000/mm ³ . 2. The method of claim 1, wherein the drug is administered to a patient whose thrombin-antithrombin complex (TAT) complex level is 10 ng/mL or more immediately prior to administration. 3. The method according to claim 1 or 2, wherein the drug is administered to a patient whose platelet count is greater than 30,000/mm ³ immediately prior to administration and the Sofa score is not greater than 11. 4. The method according to any one of paragraphs. 1-3, wherein said drug is administered to a patient who has one to three types of organ dysfunction immediately prior to administration. 5. The method according to claim 1 or 2, wherein said drug is administered to a patient whose D-dimer level is not less than 3500 ng/ml immediately prior to administration. 6. The method according to claim 1 or 2, wherein said drug is administered to a patient whose prothrombin 1+2 (F1+2) fragment level is greater than 229 pmol/L immediately prior to administration. 7. The method according to claim 1 or 2, wherein said drug is administered to a patient whose protein C activity level is not greater than 40% immediately prior to administration. 8. The method according to claim 1 or 2, wherein said drug is administered to a patient whose antithrombin III (AT III) activity level is less than 70% immediately prior to administration. 9. The method according to claim 1 or 2, wherein said drug is administered to a patient whose microparticle size (MP) is greater than 10 nm immediately prior to administration. 10. The method of claim 1 or 2, wherein said drug is administered to a patient who, immediately prior to administration, has an APACHE II score of less than 35. 11. The method according to claim 1 or 2, where the drug containing 0.005-1 mg/kg of thrombomodulin is intravenously administered to a patient with sepsis, in which he is accompanied by coagulopathy, with a frequency of once a day for at least four consecutive days.

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