NZ624542B2 - Medicament for therapeutic treatment and/or improvement of sepsis - Google Patents

Abstract

Disclosed is the use of thrombomodulin in the manufacture of a medicament for therapeutic treatment and/or improvement of sepsis, wherein the sepsis is severe and is accompanied with one or more organ dysfunctions, and wherein a value of International Normalized Ratio (INR) of a plasma specimen is more than 1.4. Also disclosed is the use of thrombomodulin in the manufacture of a medicament for decreasing the mortality of patients with sepsis, wherein the sepsis is severe and is accompanied with one or more organ dysfunctions, and wherein a value of International Normalized Ratio (INR) of a plasma specimen is more than 1.4. ore than 1.4. Also disclosed is the use of thrombomodulin in the manufacture of a medicament for decreasing the mortality of patients with sepsis, wherein the sepsis is severe and is accompanied with one or more organ dysfunctions, and wherein a value of International Normalized Ratio (INR) of a plasma specimen is more than 1.4.

Description

SPECIFICATION Title of the Invention: Medicament for therapeutic treatment and/or improvement of sepsis Technical Field The present invention s to a medicament for therapeutic treatment and/or improvement of sepsis in a severe septic patient.

Background Art Sepsis is a systemic inflammatory response syndrome (SIRS) d by infection. Specifically, sepsis is defined as a ogical condition that meets, in addition to the presence of infection, two or more of the SIRS items ((1) body temperature > 38°C or < 36°C, (2) heart rate > 90/minute, (3) respiration rate > /minute, or PaCO2 < 32 torr, and (4) leucocyte count > 12,000/uL or < 4000/uL, or immature leucocytes > 10%). Although presence of bacteria in blood (bacteremia) has been significantly focused so far, bacteria—positive result of blood culture is not necessarily required according to the above definition. Among sepsis, a condition presenting organ dysfunction, organ hypoperfusion, or nsion is called severe sepsis. The organ hypoperfusion or abnormal perfusion includes lactic acidosis, oliguria, mental clouding, and the like. Among the severe sepsis, a condition uously ting hypotension despite of sufficient load of fluid therapy is called septic shock atent document 1). It is considered that the atory failure observed in these pathological conditions is caused by malfunction of the sympathetic nervous system or a mediator released from neutrophiles and the like, and the organ dysfunction is caused by tissue hypoxia (dysoxia). omodulin has been known as a substance that acts to specifically bind to in so as to inhibit the blood coagulation activity of thrombin, and at the same time, exerts anticoagulant activity so [as to significantly promote the ability of thrombin to activate Protein C. It has also been known that thrombomodulin exerts to prolong the clotting time by thrombin, or suppresses platelet ation by thrombin. n C is a vitamin K-dependent protein that plays an important role in a blood coagulation and fibrinolysis, and activated by the action ofthrombin to be ted as activated Protein C. It has been known that the activated Protein C inactivates activated blood coagulation factor V and activated blood coagulation factor VIII in Viva, and is involved in generation of a plasmjnogen activator having thrombolytic action (Non-patent document 2). Accordingly, it has been considered that thrombomodulin promotes the activation of n C by thrombin, and therefore is useful as an anticoagulant or a thrombolytic agent. It has also been reported that, in an animal ment, thrombomodulin is effective for therapy or prophylaxis of diseases associated with hypercoagulable state (Non-patent document 3).

Thrombomodulin was first discovered and obtained as a glycoprotein expressed on the vascular endothelial cells of various animal species including humans, and then successfully cloned. Specifically, a gene of a human thrombomodulin precursor ing a‘signal peptide was cloned from a human lung cDNA y by genetic engineering techniques and the entire gene sequence ofthrombomodulin was analyzed, and as a result, an amino acid sequence consisting of 575 es containing a signal peptide (in l, 18 amino acid residues are exemplified) was revealed (Patent document 1). It is known that a mature thrombomodulin, from which the signal peptide is cleaved, is composed of 5 regions, namely, an N-terminal region (amino acid residues 1 to 226, these positions are defined under an tion that the signal peptide consists of 18 amino acid residues, and the same shall apply to the following descriptions), a region having six ke structures (amino acid residues 227 to 462), an O'linked ylation region (amino acid residues 463 to 498), a transmembrane region (amino acid residues 499 to 521), and an cytoplasmic region (amino acid residues 522 to 557), from the N-terminal side of the mature peptide. It is also known that a partial protein having the same activity as that of the entire length thrombomodulin (i.e., a minimal active unit) is mainly consisting of the 4th, 5th, and 6th EGF'like structures from the N-terminal side in the region having six ke structures (Non-patent document 4).

The entire length thrombomodulin is hardly dissolved in the absence of a surfactant, and addition of a surfactant is essential for manufacturing an entire thrombomodulin preparation. A soluble omodulin is also available that can be fully dissolved even in the absence of a surfactant. The soluble thrombomodulin may be prepared by removing at least a part of the transmembrane region or the entire transmembrane region. For example, it has been confirmed that a soluble thrombomodulin consisting of only 3 regions, namely, the NTerminal region, the region having six EGF-like structures, and the O-ljnked glycosylation region (Le, a soluble omodulin having an amino acid sequence consisting of amino acid residues 19 to 516 of SEQ ID NO: 9) can be obtained by applying recombination techniques, and that the resulting recombinant soluble thrombomodulin has the same activity as that of an entire thrombomodulin (Patent document 1). Some other reports are also available ing soluble thrombomodulins (Patent nts 2 to 9). Ahuman urine- derived soluble thrombomodulin and the like are also exemplified as native thrombomodulins t documents 10 and 11).

As ized in many cases, as a result of spontaneous mutations or ons occurring at the time of obtainment, polymorphic mutations have been found in the human genes. At present, thrombomodulin proteins in which the amino acid at the position 473 of human thrombomodulin sor having the amino acid sequence consisting of 575 amino acid residues is converted to Val or Ala have been identified. In the nucleotide ce ng the amino acid sequence, this variation of amino acid residue corresponds to mutation to T or C at the position 1418 (Non~patent document 5). However, the two types of thrombomodulins are completely identical in terms of their activity and physicochemical properties, and it can be considered that they are substantially identical.

It has been reported that thrombomodulin is effective for a therapeutic treatment of disseminated intravascular coagulation (henceforth also referred to as DIC) (Non-patent document 6). As for use of thrombomodulin, in addition to the aforementioned use, thrombomodulin is ed to be used in therapeutic and prophylactic treatments of various diseases such as acute coronary syndrome (ACS), thrombosis, peripheral vessel obstruction, obstructive arteriosclerosis, vasculitis, functional disorder occurring after heart y, complication caused by organ transplantation, angina pectoris, ent ischemic attack, toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive disease, e.g., fulminant hepatitis, veno occlusive disease of liver occurring after bone marrow transplantation), and deep venous osis (DVT), and further, sepsis and adult respiratory ss me (ARDS) (Patent nt 12).

[Prior art references] [Patent documents] Patent document 1: Japanese Patent Unexamined ation (Kokai) No. 64-6219 (EP 312598A) Patent document 2: Japanese Patent Unexamined Publication No. 5-213998 (EP 474273A2) Patent document 3: Japanese Patent Unexamined Publication No. 2-255699 Patent document 4: Japanese Patent Unexamined Publication No. 80(EP 41284A) Patent document 5: Japanese Patent ined Publication No. 3-259084 (EP 445681A2) Patent document 6: Japanese Patent Unexamined Publication No. 4-210700 Patent document 7: WO92/00325 Patent document 8: WO92/03149 Patent document 9: 5755 Patent document 10: Japanese Patent Unexamined Publication No. 3-86900 (EP 356836A2) Patent document 11: Japanese Patent Unexamined Publication No. 3-218399 (EP 376251A2) Patent document 12: WO03/061687 Non-patent Documents Non-patent document 1: American College of Chest Physicians, 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, 028 (2005) Summary of the Invention Object to be Achieved by the ion An object of the present invention is to provide a medicament or method for effective therapeutic treatment or improvement of sepsis in a severe septic t, or to at least provide the public with a useful alternative.

Means for Achieving the s International ized Ratio (henceforth also abbreviated as "INR") in a plasma specimen of a patient with sepsis is known to mean coagulopathy. For example, in Congress of Critical Care Medicine (CCM) held in 2003, INR > 1.5 is reported as well as aPPT > 60 seconds as criteria of coagulopathy (Crit. Care Med., 31, pp.1250-1256 (2003)). r, the value of INR has not yet been authorized as a clear criterion, e the value has not been verified h clinical trials and the like. Actually, through Phase III clinical trial for treatment of patients with severe sepsis, Tifagofin as a tissue factor pathway inhibitor in a class of anticoagulants is ed to have achieved more favorable result in a group of patients with INR ≤ 1.2 than a group of patients with INR > 1.2 as a result of clinical test in which patients with INR > 1.2 were mainly targeted (JAMA, July 9, Vo. 290, No. 2, pp.238-247 (2003)).

Whilst as another result of the aforementioned clinical test, among the class of patients with INR > 1.2, the drug is reported to have achieved higher effect in patients with INR > 1.5 than in patients with INR > 1.2. Further, with Xigris, which is the only drug that has been verified to be effective against sepsis through clinical study, prolongation of prothrombin-time (PT) was observed in most of the patients in the study (93.4%).

As explained above, in the therapeutic treatment of patients with sepsis by an anticoagulant, high efficacy is expected by choosing a class of ts anied with coagulopathy in view of some of the clinical study results. However, it is considered that the definition of coagulopathy has not yet been authorized, because, for example, clinical results to the contrary were also obtained. In other words, how an excellent result can be ed by choosing target patients by means of the INR value has not been clarified, and there is no common technical dge that what level of the INR value of a patient with sepsis assures particular effectiveness of the drug.

With regard to correlation of the INR value with clinical effectiveness, it is considered that only a limited part of the correlation has been known as case-by-case basis as for some of individual drugs.

Under the circumstances, the ors d their attention to omodulin among anticoagulants, and conducted various ches on therapeutic and/or improving effect against sepsis. As a result, they unexpectedly found that sepsis can be therapeutically treated and/or improved more effectively in severe septic patients with one or more organ dysfunctions (except severe septic patient with organ dysfunction limited to the liver or kidney) than in severe septic ts without organ dysfunction when the INR value ofthe patients is more than 1.4, in other words, as for therapeutic treatment and/or ement of sepsis by thrombomodulin, the inventors found that there is a particular ation between severe septic patients with one or more organ dysfunctions, among the class of septic patients, and the INR value more than 1.4, which is unexpected by one of ordinary skill in the art. r surprisingly, the inventors found that, for severe septic patients with the INR value more than 1.4 and equal to or less than 1.6, particularly remarkable effect was achieved in that a difference in mortality rate between thrombomodulin group and placebo group was higher than 15%, and as a result, they accomplished the present invention. In view of the difference in mortality rate of about 6% between drug group and placebo group achieved by Xigris (N. Engl. J. Med, 344, No.10, March 8, pp.699"709 (2001)), which is a sole commercially ble drug in Europe for treatment of sepsis, the value of 15% as the difference in mortality rate is a remarkable value as high as about 2.5-fold, and therefore, it can be recognized that one of embodiments of the present invention achieves unexpectedly surprising effect.

Specifically, the present invention es the followings: [All A medicament for therapeutic treatment and/or improvement of sepsis which comprises thrombomodulin as an active ingredient, wherein said medicament is to administer to a patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

[Al-2] A medicament for therapeutic treatment and/or improvement of sepsis accompanied with coagulopathy which comprises thrombomodulin as an active ingredient, wherein said medicament is to administer to a t with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of ational Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

[A2] The medicament ing to [A1] or [Al-2] mentioned above, wherein said medicament is to administer to a patient with severe sepsis accompanied with one or more organ dysfunctions, n a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4 and equal to or less than 1.6.

[AB] The medicament according to any one of [A1] to [A2] mentioned above, wherein the patient with severe sepsis is a severe septic patient who is not a septic patient with organ dysfunction limited to the liver or .

When the referred item numbers are indicated with such a range as "[A1] to [A2]" mentioned above, and the range includes an item indicated with a number having a subnumber such as [Al-2], it is meant that the item indicated with the number having a subnumber such as [Al-2] is also cited. The same shall apply to the following ions.

[A4] The medicament according to any one of [A1] to [A3] ned above, wherein said medicament is to administer to a patient with severe sepsis accompanied with one or more organ dysfunctions selected from the group consisting of liver dysfunction, kidney dysfunction, respiratory organ dysfunction, and circulatory organ dysfunction.

[A5] The medicament according to any one of [A1] to [A4] mentioned above, wherein the thrombomodulin is a soluble thrombomodulin.

[AS-2] The medicament according to any one of [A1] to [A5] mentioned above, wherein the soluble thrombomodulin has the following properties (1) to (4)3 (1) an action of selectively binding to thrombin, (2) an action of promoting activation of Protein C by thrombin, (3) an action of extending in clotting time, and (4) an action of suppressing platelet aggregation caused by thrombin.

[A53] The medicament ing to any one of [A1] to [A5] mentioned above, wherein the e thrombomodulin has the following properties (1) to (5)1 (1) an action of selectively binding to thrombin, (2) an action of promoting activation of Protein C by thrombin, (3) an action of extending thrombin clotting time, (4) an action of suppressing et aggregation caused by in, and (5) anti-inflammatory action.

[AG] The medicament according to any one of [A1] to [A5'3] mentioned above, wherein the tbrombomodulin is a peptide able from a transformed cell prepared by transfecting a host cell with a DNA coding for the amino acid sequence of SEQ ID NO: 9 or SEQ ID N01 11.

[A7] The medicament according to any one of [A1] to [A6] mentioned above, wherein the soluble thrombomodulin is a peptide containing the amino acid ce of (i' 1) or (i-2) mentioned below, and said peptide is a peptide having the thrombomodulin activities; (i-l) the amino acid sequence of the positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or (i-2) the amino acid sequence of (i- 1) mentioned above, further including substitution, deletion or addition of one or more amino acid residues.

[A7'2] The medicament according to any one of [A1] to [A6] mentioned above, wherein the soluble thrombomodulin is a e containing: (i) the amino acid sequence of the positions 367 to 480 in the amino acid ce of SEQ ID N01 9 or 11, and the amino acid sequence of (ii'l) or (ii~2) mentioned below, and said peptide is a peptide having the thrombomodulin activities: (ii- 1) the amino acid sequence of the positions 19 to 244 in the amino acid sequence of SEQ ID N01 9 or 11, or (ii-2) the amino acid sequence of (ii-1) mentioned above, further including substitution, deletion or addition of one or more amino acid residues, [A8] The medicament according to any one of [A1] to [A7-2] mentioned above, wherein the thrombomodulin is administered at a dose of 0.005 to 1 mg/kg within 5 minutes by intravenous bolus stration.

[B 1] A method for therapeutic treatment and/or ement of sepsis, which comprises the step of administrating thrombomodulin to a patient with severe sepsis, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

[B 1-2] A method for therapeutic treatment and/or improvement of sepsis accompanied with coagulopathy, which comprises the step of administrating thrombomodulin to a patient with severe sepsis, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

[B2] The method according to [B 1] or [Bl-2] mentioned above, which comprises the step of administrating thrombomodulin to a patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International ized Ratio (INR) of a plasma specimen ed from said patient is more than 1.4 and equal to or less than 1.6.

[B3] The method according to any one of [B 1] to [B-2] ned above, wherein the patient with severe sepsis is a severe septic patient who is not a septic patient with organ dysfunction limited to the liver or kidney.

[B4] The method according to any one of [B 1] to [B3] mentioned above, which comprises the step of administrating thrombomodulin to a patient with severe sepsis accompanied with one or more organ dysfunctions selected from the group consisting of liver dysfunction, kidney dysfunction, respiratory organ ction, and circulatory organ ction.

[B5] The method according to any one of [B 1] to [B4] mentioned above, n the thrombomodulin is a soluble thrombomodulin.

[B6] The method according to any one of [B1] to [B5] mentioned above, wherein the thrombomodulin is a peptide able from a transformed cell ed by transfecting a host cell with a DNA coding for the amino acid sequence of SEQ ID NO: 9 or SEQ ID N03 11.

[B7] The method according to any one of [B1] to [B6] mentioned above, wherein the soluble thrombomodulin is a peptide containing the amino acid sequence of (i'l) or (i-2) mentioned below, and said peptide is a peptide having the thrombomodulin activities; (i- 1) the amino acid sequence of the positions 19 to 516 in the amino acid sequence of SEQ ID N02 9 or 11, or (i-2) the amino acid sequence of (i' 1) mentioned above, further including substitution, deletion or addition of one or more amino acid residues.

[B8] The method according to any one of [B1] to [B7] mentioned above, wherein the thrombomodulin is administered at a dose of 0.005 to 1 mg/kg within 5 minutes by intravenous bolus administration.

[BS-2] The method according to any one of [B 1] to [BS] mentioned above, wherein the thrombomodulin is a thrombomodulin having the characteristic features mentioned in M52], [A5'3] or [A7'2l mentioned above.

[B9] Use of thrombomodulin as a medicament for eutic treatment and/or improvement of sepsis, wherein said medicament is to administer to a t with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

[B92] The use ing to [B9] mentioned above, wherein the thrombomodulin is a thrombomodulin having the characteristic features mentioned in , [A5-3l or [A7- 2] mentioned above. [39-3] The use according to [B9] mentioned above, which has the characteristic features mentioned in any one of [All to [A8] mentioned above.

[C 1] A ment for therapeutic ent and/or improvement of inated intravascular coagulation, wherein said medicament is to administer to a patient with disseminated ascular coagulation accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is higher than 1.4.

[C2] The medicament according to [Cl] mentioned above, which has the characteristic features mentioned in any one of [All to [A8] mentioned above.

Effect of the Invention With the medicament of the present invention containing thrombomodulin, sepsis in a severe septic patient, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4, can be effectively treated and/or ed.

Modes for ng out the Invention Hereafter, several preferred embodiments of the present invention (preferred modes for carrying out the invention, henceforth also referred to as "embodiments" in the specification) will be specifically explained. However, the scope of the present invention is not limited to the specific ments explained below.

The thrombomodulin of this embodiment preferably is known to have an action of (1) selectively binding to thrombin (2) to promote activation of Protein C by thrombin. In addition, it is preferred that the omodulin is confirmed to generally have (3) an action of ing thrombin clotting time, (4) an action of suppressing platelet aggregation caused by thrombin, and/or (5) anti-inflammatory action. Such actions possessed by thrombomodulin may be referred to as thrombomodulin activities.

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

The action of thrombomodulin to bind with thrombin can be confirmed by the study s described in various known publications such as Thrombosis and Haemostasis, 70(3) 3418-422 (1993) and The Journal of Biological Chemistry, 264, 9, pp.4872-4876 (1989). As for the action ofpromoting activation of n C by thrombin, degree of the activity ofpromoting the tion of Protein C by thrombin or presence or absence of the action can be easily confirmed by the study methods clearly described in various known publications including, for example, Japanese Patent Unexamined Publication No. 64-6219. Further, the action of extending thrombin clotting time, and/or the action of suppressing platelet aggregation caused by thrombin can be similarly and easily confirmed. Furthermore, the anti-inflammatory action can also be ed by the study methods described in various known publications including, for example, Blood, 11213361'3670 (2008) and The l of Clinical Investigation, 115, 521267-1274 (2005).

The thrombomodulin used for the present invention is not particularly limited so far as having the thrombomodulin activities. The thrombomodulin is preferably a soluble thrombomodulin under the condition Without surfactants. The solubility of the soluble thrombomodulin in water such as led water used for injection (in the absence of a surfactant such as Triton X'lOO 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, 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.

For determining whether or not a soluble thrombomodulin is successfully dissolved in water, it is understood that clear appearance of a solution and the absence of apparently observable insoluble nces is served as simple criteria, after the soluble thrombomodulin is dissolved in water and the solution is observed by Visual inspection, for example, just under a white light at a on corresponding to an nation of approximately 1000 luxes. It is also possible to observe the presence or absence of any residue after filtration.

The molecular weight of the thrombomodulin is not limited so far that it has the thrombomodulin activities as described above. The molecular weight is ably 100,000 or smaller, more preferably 90,000 or smaller, still more preferably 80,000 or smaller, most preferably 70,000 or smaller, and the molecular weight is preferably 50,000 or larger, most preferably 60,000 or larger. The lar weight of the soluble omodulin can be easily measured by ordinary methods for measuring molecular weight of protein. Measurement by mass ometry is red, and MALDI-TOF- MS method is more preferred. For ing soluble thrombomodulin having a molecular weight Within a desired range, a soluble thrombomodulin, which is obtained by culturing a transformant cell prepared by transfecting a host cell with a DNA encoding soluble thrombomodulin using a vector, can be subjected to fractionation using column chromatography or the like as described later.

The thrombomodulin used for the present invention preferably comprises the amino acid sequence ting of the amino acid residues at the positions 19 to 132 of SEQ ID N01 1, which has been known as the central portion of the thrombomodulin activities of human thrombomodulin, and the thrombomodulin is not particularly limited, so long as the thrombomodulin comprises the amino acid sequence consisting of the amino acid es at the positions 19 to 132 of SEQ ID N01 1. The amino acid sequence ting of the amino acid es at the positions 19 to 132 of SEQ ID N01 1 may be naturally or artificially mutated, so long as the sequence has an action to promote the activation of Protein C by thrombin, namely, one of the thrombomodulin activities. Specifically, the sequence may se substitution, deletion, or addition of one or more amino acid residue in the amino acid sequence consisting of the amino acid residues at the positions 19 to 132 of SEQ ID N01 1. Acceptable level of the mutation is not particularly limited, so long as the amino acid sequence has the thrombomodulin activities. An e includes a gy 50% or more as amino acid sequences, and the homology is preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. Such mutated amino acid sequence including substitution, deletion or addition of one or more amino acid es is ed to as homologous mutation sequence. As described later, these mutated amino acid sequences can be easily produced by using ordinary gene manipulation techniques. The thrombomodulin is not particularly d so far that it has the aforementioned sequence and the action of selectively binding to thrombin to promote activation of Protein C by thrombin at least as the whole thrombomodulin, but the thrombomodulin preferably also has the nflammatory action.

The amino acid sequence of SEQ ID N03 3 comprises the mutation ofVal as the amino acid at the position 125 of the sequence of SEQ ID NO: 1 to Ala. The thrombomodulin used for the present invention also preferably comprises the amino acid sequence from the position 19 to 132 of SEQ ID N03 3.

As described above, although the thrombomodulin used for the present invention is not particularly limited so long that the thrombomodulin has at least the amino acid sequence from the on 19 to 132 of SEQ ID NO! 1 or 3, or a homologous mutation sequence thereof, and comprises at least a peptide sequence having the thrombomodulin activities, preferred examples of the omodulin include a peptide consisting of the sequence from the on 19 to 132 or 17 to 132 in either of SEQ ID NO! 1 or SEQ ID N01 3, and a e ting of a homologous mutation ce of the aforementioned sequence and having at least the thrombomodulin activities. Apeptide consisting of the sequence from the position 19 to 132 in either of SEQ ID N01 1 or SEQ ID N02 3 is more preferred. In another embodiment, a peptide consisting of a homologous mutation sequence of the sequence from the position 19 to 132 or 17 to 132 in either of SEQ ID NO: 1 or SEQ ID N01 3 and having at least the thrombomodulin activities is more preferred.

As another ment of the thrombomodulin according to the present invention, the thrombomodulin preferably comprises the amino acid sequence from the positions 19 to 480 of SEQ ID N01 5, which is not ularly limited so long as the thrombomodulin comprises the amino acid sequence from the position 19 to 480 of SEQ ID NO: 5. The amino acid sequence from the positions 19 to 480 of SEQ ID N01 5 may be a homologous mutation sequence thereof, so long as the sequence has an action to promote the tion of Protein C by thrombin, i.e., one of the thrombomodan activities.

The sequence of SEQ ID N01 7 comprises the on ofVal as the amino acid at the position 473 of the sequence ofSEQ ID NO: 5 to Ala. The thrombomodulin used for the present invention also preferably comprises the amino acid sequence from the position 19 to 480 of SEQ ID N01 7.

As described above, although the thrombomodulin used for the t invention is not particularly limited so long as the thrombomodulin has at least the sequence from the position 19 to 480 in either ofSEQ ID N03 5 or SEQ ID N03 7, or a homologous mutation sequence thereof, and comprises at least a peptide sequence having the thrombomodulin activities, red examples of the thrombomodulin include a peptide consisting of the sequence from the position 19 to 480 or 17 to 480 in either of SEQ ID N01 5 or SEQ ID NO: 7, and a peptide consisting of a homologous mutation sequence of the aforementioned sequence and having at least the thrombomodulin activities. de consisting of the sequence from the position 19 to 480 ofSEQ ID NO 5 or 7 is more preferred. In another embodiment, a peptide consisting of a homologous mutation sequence of the sequence from the position 19 to 480 or 17 to 480 in either of SEQ ID N03 5 or SEQ ID NO 7, and having the thrombomodulin activities is more red.

As r embodiment of the thrombomodulin according to the present invention, the thrombomodulin preferably comprises the amino acid sequence from the position 19 to 515 of SEQ ID NO! 9, which is not particularly limited so long as the thrombomodulin comprises the amino acid ce from the position 19 to 515 ofSEQ ID N01 9. The amino acid sequence from the on 19 to 515 of SEQ ID N01 9 may be a homologous mutation sequence thereof, so long as the sequence has an action to e the tion of Protein C by thrombin, i.e., the thrombomodulin activities.

The amino acid sequence of SEQ ID NO: 11 comprises the mutation ofVal as the amino acid at the position 473 of SEQ ID N01 9 to Ala. The thrombomodulin used for the t invention also preferably comprises the amino acid sequence from the position 19 to 515 of SEQ ID N01 11.

As described above, although the thrombomodulin used for the present invention is not particularly limited so long as the thrombomodulin has at least the sequence from the position 19 to 515 in either of SEQ ID N01 9 or SEQ ID N01 11, or a homologous mutation sequence thereof, and comprises a peptide sequence having at least the thrombomodulin activities, more preferred examples include a peptide having the sequence from position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in either of SEQ ID N01 9 or SEQ ID NO! 11, and a peptide consisting of a homologous mutation sequence of the aforementioned sequence and having at least the thrombomodulin activities. A e having the sequence from the position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQ ID N01 9 is particularly preferred. A mixture thereof is also a preferred example. In another ment, a e having the sequence from the position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQ ID NO: 11 is particularly preferred. A mixture thereof is also a preferred e. Further, a peptide consisting of a homologous mutation sequence thereof and having at least the thrombomodulin activities is also a preferred example. It is preferred that the soluble thrombomodulin also has the anti-inflammatory action.

Apeptide having a homologous on sequence is as described above, and means a peptide that may comprise substitution, deletion, or addition of at least one, namely, one or more, preferably several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3) amino acid residues, in the amino acid sequence ofthe subjected peptide. gh acceptable level of mutation is not particularly limited so long as the peptide has the thrombomodulin activities, an example ofthe able level of homology includes 50% or more as an amino acid sequences, and the homology may be preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

Preferred es of the thrombomodulin used for the present invention also include the peptide ting of the sequence of SEQ ID NO 14 (462 amino acid es), the peptide consisting of the sequence of SEQ ID N03 8 (272 amino acid residues), and the peptide ting of the sequence of SEQ ID N01 6 (236 amino acid residues) described in Japanese Patent Unexamined Publication No. 64-6219.

The thrombomodulin used for the present invention is not particularly limited so long as the omodulin has at least the amino acid sequence from the position 19 to 132 in either ofSEQ ID N01 1 or SEQ ID NO: 3. As such a omodulin, a peptide having at least the amino acid sequence fi'om the position 19 to 480 in either of SEQ ID N03 5 or SEQ ID N01 7 is preferred, and a peptide having at least the amino acid sequence from the position 19 to 515 in either of SEQ ID NO! 9 or SEQ ID N03 11 is more preferred. A more preferred example of the peptide having at least the amino acid sequence from the position 19 to 515 in either of SEQ ID N09 or SEQ ID N03 11 is a peptide having the sequence from the position 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 19 to 514 in either of SEQ ID NO! 9 or SEQ ID N01 11.

Furthermore, a mixture ofpeptides each consisting of the sequence from the position 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 19 to 514 in either of SEQ ID N01 9 or SEQ ID N03 11 is also a preferred example.

In the case of the aforementioned mixture, the mixing ratio of a peptide that starts from the position 17 and a peptide that starts from the position 19 for each of SEQ ID NOS: 9 and 11 is, for example, 30170 to 50350, preferably 35265 to 45155.

Further, the mixing ratio of a peptide that terminates at the position 514, a peptide that terminates at the position 515, and a peptide that terminates at the on 516 for each of SEQ ID NOS: 9 and 11 is, for example, 010100 to 0190110, or 0370330 to 1039010, or 1010190 to 20310170, ifdesired.

The mixing ratio of the peptides can be determined by an ordinary .

The sequence of the ons 19 to 132 in SEQ ID NO! 1 corresponds to the sequence of the positions 367 to 480 in SEQ ID N01 9, and the sequence of the positions 19 to 480 in SEQ ID N01 5 corresponds to the sequence of the positions 19 to 480 in SEQ ID N01 9. Further, the \Sequence of the positions 19 to 132 in SEQ ID NO: 3 corresponds to the sequence of the positions 867 to 480 in SEQ ID NO: 11, and the sequence ofthe positions 19 to 480 in SEQ ID N01 7 corresponds to the sequence of the positions 19 to 480 in SEQ ID NO: 11. Furthermore, all the sequences of the positions 1 to 18 in SEQ ID NOS! 1, 3, 5, 7, 9 and 11 are identical sequences.

As described below, these thrombomodulins according to the present invention can be ed from transformant cells prepared by transfecting host cells with a DNA encoding the peptide (specifically, the nucleotide sequences of SEQ ID NOS! 2, 4, 6, 8, , 12, and the like) by using a vector.

It is sufficient that these es only have the aforementioned amino acid ces, and a sugar chain may be attached or not attached, which not particularly limited. In gene manipulation techniques, a type of a sugar chain, a on to which a sugar chain is added, and a level of addition thereof differ depending on a type of host cells used, and any techniques may be used. As for binding position of a sugar chain and a type thereof, facts described in Japanese Patent ined Publication No. 11- 341990 are known, and the thrombomoduljns according to the t invention may be added with the same sugar chain at the same position. Two types of N'linked sugar chains, those of fucosyl biantennary type and l triantennary type, may bind to the thrombomodulin of this embodiment, and ratio thereof is, for example, 1000 to 60:40, ably 955 to 60340, more preferably 90310 to 70130. The ratio of these sugar chains can be measured on a two-dimensional sugar chain map bed in Biochemical Experimental Methods, Vol. 23, Methods of Researches on Glycoprotein Sugar Chains, Japan Scientific Societies Press (1990), and the like. Furthermore, when a sugar composition of the thrombomodulin of this ment is examined, neutral saccharides, aminosaccharides, and sialic acid are detected, ofwhich content may be, each independently for example, 1 to 30%, preferably 2 to 20%, more preferably 5 to 10%, in terms of weight ratio based on the protein content. The sugar contents 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 ridesi phenol-sulfuric acid method, aminosaccharidesi Elson-Morgan method, sialic acid: periodic acid-resorcinol method).

Although the method for obtaining thrombomodulin is not limited to obtaining it by genetic manipulation as described later, as a signal sequence that can be used for expression where the thrombomodulin is obtained by gene lation, a nucleotide sequence encoding the amino acid sequence of the positions 1 to 18 in SEQ ID N02 9, and a nucleotide sequence encoding the amino acid sequence of the positions 1 to 16 in SEQ ID N01 9 can be used, and other known signal sequences such as the signal sequence of human tissue plasminogen activator can also be used (International Publication W088/9811).

When a DNA sequence encoding thrombomodulin is introduced into a host cell, examples ofpreferred methods include a method of incorporating a DNA sequence encoding thrombomodulin into, preferably, a vector, more ably 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 is tuted With a promoter ce, a sequence for adding a ribosome binding site to mRNA, a DNA sequence ng a protein to be expressed, a ng signal, a terminator sequence for transcription termination, a replication origin sequence, and the like. Examples of preferred animal cell expression vector include pSVZ-X reported by Mulligan R.C. et al.

(Proc. Natl. Acad. Sci. USA, 78, 2072 (1981)); pBP69T (69-6) reported by Howley} PM. et a1. (Methods in Emzymology, 101, 387 (1983), Academic Press), and the like.

Further, there is also another red embodiment in which DNA is introduced into an expression vector expressible in a microorganism.

Examples of host cell that can be used in production of such peptides as mentioned above include animal cells. Examples ofthe animal cells e e hamster ovary (CHO) cells, COS'I cells, 0087 cells, VERO (ATCC COL-81) cells, BHK cells, canine kidney-derived MDCK cells, hamster AV-12~664 cells, and the like. In addition, examples of host cell derived from human e HeLa cells, W138 cells, human 293 cells, and PERCG cells. Ofthese cells, CHO cells are very common and preferred, and among the CHO cells, dihydrofolate reductase (DHFR)’deficient CHO cells are more preferred.

In a gene manipulation process or a peptide production process, microorganisms such as Escherichia 0011' are also often used. A host-vector system suitable for each process is ably used, and an appropriate vector system can also be selected for the aforementioned host cells. A thrombomodulin gene used in a genetic recombination technique has been cloned. Examples of production of thrombomodulinby such a gene recombination technique have been disclosed, and further, s for purifying thrombomodulin to obtain a purified product thereof are also known (Japanese Patent ined Publication Nos. 64-6219, 2255699, 5' 218998, 5310787, 7155176; and J. Biol. Chem., 2641035110353 (1989)). Therefore, the thrombomodulin used for the present invention can be produced by using the methods described in the aforementioned reports, or by similar methods. For example, Japanese Patent Unexamined Publication No. 64-6219 discloses the chia 001iK- 12 strain DH5 (ATCC Accession N0. 67283) containing a plasmid pSVZTMJ2 that contains a DNA encoding the full-length omodulin. This strain re'deposited at the former al Institute of Bioscience and Human-Technology (currently Independent Administrative Institution, National ute ofAdvanced Industrial Science and Technology, International Patent Organism Depositary) richia 001i DH5/pSV2TMJ2) (FERM BP-5570) can also be used. The thrombomodulin according to the present invention can be prepared by a known gene manipulation technique using a DNA encoding the full-length thrombomodulin as a starting al.

The thrombomodulin of this ment may be prepared by a conventionally known method or a similar method. For example, the aforementioned method of Yamamoto et al. (Japanese Patent Unexamined Publication No. 64-6219) or the method described in Japanese Patent Unexamined Publication No. 5213998 can be ed to.

Specifically, for example, a DNA encoding the amino acid sequence of SEQ ID NO! 9 is prepared from a human-derived thrombomodulin gene by a gene manipulation technique, and may be further d as required. For such modification, in order to obtain a DNA encoding the amino acid ce of SEQ ID NO! 11 (which specifically consists of the nucleotide sequence of SEQ ID N03 12), codons encoding the amino acid at the position 473 in the amino acid sequence of SEQ ID NO: 9 (in particular, the nucleotide at the position 1418 in SEQ ID NO 10) are mutated by site- directed mutagenesis according to the method described by Zoller M.J. et a1. (Method in Enzymology, 8-500 (1983), Academic Press). For example, by using a synthetic DNA for mutation having the nucleotide sequence of SEQ ID N01 13, the nucleotide T at the position 1418 in SEQ ID N01 10 may be converted to the nucleotide C to obtain a mutated DNA.

The DNA prepared as described above is incorporated into, for example, Chinese hamster ovary (CI-IO) cells to obtain transformant cells. Such cells are subjected to appropriate selection, and thrombomodulin d by a known method can be produced from a culture solution obtained by culturing a selected cell. As described above, the DNA (SEQ ID N03 10) encoding the amino acid sequence of SEQ ID NO! 9 is ably transfected into the aforementioned host cell.

The method for producing thrombomodulin of this embodiment is not limited to the entioned method. For example, it is also possible to extract and purify the thrombomodulin from urine, blood, other body fluids and the like, or extract and purify the omodulin from a tissue producing thrombomodulin or a culture of the aforementioned tissue and the like. Further, the thrombomodulin may be further subjected to a ge treatment using a protease, as required.

For the e of the aforementioned transformant cell, a medium used for ordinary cell culture may be used, and it is preferable to culture the transformant cell in various kinds of media in advance to choose an optimal medium. For example, a known medium such as MEM medium, DMEM medium, and 199 medium may be used as a base medium, and a further ed medium or a medium added with supplements for various media may be used. .Examples ofthe culture method include serum culture, in which culture is performed in a medium containing blood serum, and serum-free culture, in which culture is performed in a medium not ning blood serum. Although the culture method is not particularly limited, the serum-free culture is preferred.

When serum is added to a medium in the case ofthe serum culture, bovine serum is preferred. Examples ne serum include fetal bovine serum, neonate bovine serum, calf bovine serum, adult bovine serum, and the like, and any of these examples may be used so far that the serum is suitable for the 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 marketed, and for example, for the CHO cell, CD-CHO, CHO'S-SFMII and CHO-III-PFM are sold by Invitrogen, and IS CHO, IS CHO-CD medium, and the like are sold by Irvine Scientific. These media may be used Without any treatment, or they may be ed or added with supplements and used. Examples of the free medium further include the DMEM medium containing 5 mg/L each of insulin, transferrin, and selenious acid. As described above, the medium is not particularly limited so far that the medium can be used to produce the thrombomodulin of this embodiment. The culture method is not ularly limited, and any of batch culture, repetitive batch e, fed'batch culture, perfusion culture, and the like may be used.

When the thrombomodulin used for the present invention is prepared by the aforementionedvcell culture method, diversity may be observed in the inus amino acid due to anslational modification of the protein. For example, the amino acid ofthe position 17, 18, 19 or 22 in SEQ ID N01 9 may serve as the N- terminus amino acid. Further, for example, the N-terminus amino acid may be modified so that the glutamic acid at the position 22 is changed to pyroglutamic acid.

It is preferred that the amino acid of the position 17 or 19 serves as the N-terminus amino acid, and it is more preferred that the amino acid of the position 19 serves as the N-terminus amino acid. Further, there is also another ment in which the amino acid of the position 17 serves as the N'terminus amino acid, which is a red embodiment. As for the modification, diversity and the like mentioned above, similar examples can be mentioned for the ce of SEQ ID NO: 11.

Further, when the soluble thrombomodulin is prepared by using a DNA having the nucleotide sequence of SEQ ID N01 10, diversity of the C'terminus amino acid may be observed, and a peptide shorter by one amino acid residue may be produced.

Specifically, the C-terminus amino acid may be modified so that the amino acid of the position 515 serves as the C'terminus amino acid, and further the position 515 is amidated. Further, a peptide shorter by two amino acid residues may be produced.

Specifically, the amino acid of the position 514 may serve as the C'terminus amino acid.

Therefore, any of peptides having significant diversity of the N-terminus amino acid and C-terminus amino acid, or a mixture ofthem may be produced. It is red that the amino acid of the position 515 or the amino acid of the position 516 serves as the C-terminus amino acid, and it is more preferred that the amino acid of the position 516 serves as the inus amino acid. Further, there is also another embodiment in which the amino acid of the position 514 serves as the C-terminus amino acid, which is a preferred embodiment. Concerning the modification, ity and the like described above, the same shall apply to a DNA having the nucleotide sequence of SEQ ID NO: 12.

The thrombomodulin obtained by the method described above may be a mixture ofpeptides having diversity in the N'terminus and nus amino acids.

Specific examples include a e ofpeptides having the sequences of the 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 N03 9.

Then, isolation and purification mbomodulin from a culture supernatant or culture obtained as described above can be carried out by known methods [edited by Takeichi Horio, Tanpakusbitsu/Koso no K1290 Jikken H0 (Fundamental Experimental s for Proteins and Enzymes) (1981)]. For example, it is preferable to use ion exchange chromatography or adsorption chromatography, which utilizes an interaction between thrombomodan and a chromatographic carrier on which functional groups having a charge opposite to that of thrombomodulin are immobilized. Another preferred example is affinity chromatography utilizing specific affinity with thrombomodulin. red examples of adsorbent include thrombin that is a ligand of thrombomodulin and an anti- thrombomodulin dy. As the antibody, anti-thrombomodulin antibodies having appropriate properties or recognizing appropriate epitopes can be used. Examples include, for example, those described in Japanese Patent Publication u) No. 5' 42920, Japanese Patent Unexamined Publication Nos. 64'45398 and 6205692 and the like. Other examples include gel filtration chromatography and ultrafiltration, which utilize the molecular size of thrombomodulin. Other es further include hydrophobic chromatography that utilizes hobic bond n a chromatographic carrier on which hydrophobic groups are immobilized, and a hydrophobic portion of thrombomodulin. Furthermore, hydroxyapatite may be used as a carrier in adsorption chromatography, ofwhich examples include, for example, those described in se Patent Unexamined Publication No, 9" 110900. These means may be used in combination, as required. Although degree ofpurification can be selected depending on a purpose of use and the like, it is desirable to purify thrombomodulin until a single band is obtained as a result of electrophoresis, preferably SDS'PAGE, or a single peak is obtained as a result of gel filtration HPLC or reverse phase HPLC of the isolated and purified product. It should be understood that, when two or more types of thrombomodulins are used, it is preferred that only the bands of the thrombomodulins are substantially obtained, and it is not required to obtain one single band.

Specific es of the purification method used in the present invention include a purification method using the omodulin activities as a criterion, for example, a ation method comprising roughly purifying a e supernatant or a culture product with an ion exchange column Q-Sepharose Fast Flow to t a fraction having the thrombomodulin activities; then purifying the on with an affinity column, DIP-thrombin-agarose propylphosphorylthrombin agarose) column, as the main purification step to recover a fraction having pontent omodulin activities; then concentrating the recovered on and followed by gel filtration to obtain a thrombomodulin active fraction as a purified product (Gomi K. et al., Blood, 751 1396-1399 ). An example of the thrombomodulin activities used as the criterion is an activity of promoting the activation of Protein C by thrombin.

Other preferred examples of the purification method will be exemplified below.

An appropriate ion exchange resin having good adsorptive condition for omodulin is selected and purification by ion exchange tography is performed. Aparticularly preferred example is a method comprising the use of Q- ose Fast Flow equilibrated with a 0.02 mol/L Tris-H01 buffer (pl-I 7.4) ning 0.18 mol/L NaCl. After washing as required, elution can be performed with a 0.02 mol/L Tris~HCl buffer (pH 7.4) containing 0.3 mol/L NaCl, for example, to obtain thrombomodulin as a roughly purified product.

Then, for example, a substance having specific affinity to thrombomodulin can be immobilized on a resin to perform purification by affinity chromatography.

Preferred examples e a DIP-thrombin-agarose column and an anti- thrombomodulin monoclonal dy column. In the case of the DlP-thrombin- agarose column, the column is equilibrated beforehand with a 20 mmol/L Tris-HC1 buffer (pH 7.4) containing 100 mmol/L NaCl and 0.5 mmol/L calcium chloride, and the aforementioned roughly purified product is then charged on the column, washed as required, and then eluted with, for example, a 20 mmol/L Tris-H01 buffer (pH 7.4) ning 1.0 mol/L NaCl and 0.5 mmol/L calcium chloride to obtain thrombomodulin as a purified product. In the case of the anti-thrombomodulin monoclonal antibody column, an example of the method comprises: contacting an anti-thrombomodulin onal dy solution in a 0.1 mol/L NaHCOs buffer (pH 8.3) containing 0.5 mol/L NaCl with Sepharose 4FF (GE Health Care Biosciences) activated with CNBr beforehand to obtain the resin ose 4FF coupled with the anti-thrombomodulin monoclonal antibodies, equilibrating the resin filled in a column beforehand with, for example, a 20 mmol/L phosphate buffer (pH 7.3) containing 0.3 mol/L NaCl, washing as required, and then performing elution with a 100 mmol/L glycine-HCl buffer (pH 3.0) containing 0.3 mol/L NaCl. An effluent may be neutralized with an appropriate buffer to obtain a product as a purified product.

Subsequently, the purified product is adjusted to pH 3.5, and then charged on a cation exchanger, ably harose FF (GE Health Care Biosciences) as a strong cation exchanger, equilibrated with a 100 mmol/L e'HCl buffer (pH 3.5) ning 0.3 mol/L NaCl, and washing is performed with the same buffer to obtain a sorptive fraction. The resulting fraction is neutralized with an appropriate buffer to obtain a highly purified product. These products are preferably concentrated by ultrafiltration.

Further, it is also preferable to exchange the buffer by gel filtration. For example, a highly purified product concentrated by ultrafiltration can be d on a Sephacryl S'300 column or 8-200 column equilibrated with a 20 mmol/L phosphate buffer (pl-I 7.3) containing 50 mmol/L NaCl, and then developed for fractionation withva mmol/L phosphate buffer (pH 7.3) containing 50 mmol/L NaCl. The activity for promoting the activation of Protein C by thrombin can be confirmed to collect an active fraction and thereby obtain a buffer-exchanged highly purified t. In order to improve safety, a highly purified product obtained as described above is preferably filtered through an appropriate filter for eliminating Viruses such as Planova 15N (Asahi Kasei Medical Co., Ltd), and then the resultant can be concentrated by ultrafiltration to a desired concentration. Finally, the product is preferably filtered through an aseptic filtration filter.

According to the present invention, there is ed a medicament for therapeutic treatment and/or improvement of sepsis which comprises omodulin as an active ingredient, wherein said ment is to administer to a patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

That is, the medicament for therapeutic treatment and/or ement of sepsis ing to this embodiment is a medicament for therapeutic treatment and/or improvement of sepsis in a patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4. [007 ll There is also ed a medicament for decreasing mortality of a human patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4.

As for the therapeutic treatment and/or improvement of , es of preferred effects thereof e, for example, "prevention of death of a patient from sepsis". Examples also include "prevention of aggravation of general conditions of a patient by sepsis".

The sepsis referred to in this embodiment is known as a severe systemic infectious disease wherein microorganisms continuously or intermittently invade into blood from an infection focus, which disease is induced by a disease such as ious diseases, malignant tumors, hepatic cirrhosis, renal failure, diabetes, and dystocia, or a therapeutic treatment for injury or e such as use of ling catheter, infusion device, dialysis, and the like and tracheostomy. If the symptoms advance, a systemic shock is induced by septic shock, i.e., rapid decrease of blood pressure and peripheral circulatory failure, and lethality is provided by organ ctions of vital organs, such as lung, kidney, liver, heart, alimentary canal, and central nervous system. As a complication accompanying sepsis, there is induced adult respiratory distress syndrome (ARDS) characterized by edema of lung stroma, hemorrhage and acute respiratory failure due to lung capillary obstruction associated with D10 or tion of neutrophiles and migration and accumulation thereof in lung parenchyma, which s in extremely bad prognosis.

The sepsis referred to in this embodiment is the ic inflammatory ' response syndrome (SIRS) induced by infection. More specifically, it includes a pathological condition that meets, in addition to the presence of infection, two or more of the SIRS items ((1) body temperature > 38°C or < 36°C, (2) heart rate > 90/minute, (3) respiration rate > ute, or PaCOz < 32 torr, and (4) leucocyte count > /uL or < 4000/uL, or bademia > 10%), and sepsis can be basically diagnosed on the basis of such a pathological ion.

There are several methods for diagnosing sepsis, and they are summarized in Levy M. et al., Crit. Care. Med., 01256. For example, there are a method based on diagnosis performed by a medical practitioner, and a method of using test values and the like Examples of the latter include a method in which When two items are fulfilled among the four items of (1) body temperature > 38°C or < 36°C, (2) heart rate > 90/minute, (3) respiration rate > 20/minute, or necessity of artificial respiration, and (4) leucocyte count > 12,000/uL or < 4000/uL, or bandemia > 10%, diagnosis of SIRS is established, and SIRS for which a microorganism is identified or ted as the cause thereof is diagnosed as sepsis [LaRosa S., the homepage of The Cleveland Clinic].

Another method r to the above method 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).

Examples of the ms of sepsis include, for example, bacteriemia, septicemia, systemic inflammatory se syndrome (SIRS), sepsis (SIRS for which a microorganism is fied or suspected as the cause thereof), severe sepsis, septic shock, intractable septic shock, and multiple organ dysfunction me (henceforth 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 aforementioned conditions are exemplified as symptoms on which the medicament of the present invention for therapeutic ent and/or improvement is effective.

Although the sepsis is not particularly limited so long as a disease is diagnosed as sepsis on the basis of the aforementioned diagnosis criteria, it is preferably sepsis accompanied by abnormal coagulation (sepsis with coagulopathy).

Although the coagulopathy is not ularly limited so long as INR of a plasma specimen obtained from t is more than 1.2, it is preferably more than 1.3, more preferably more than 1.4.

Examples of the bacteriemia include a condition that presence of bacteria in blood is verified by a positive result of blood culture.

Examples of the septicemia e a condition that presence of microorganisms or other toxins in blood is confirmed.

Examples of the systemic inflammatory se syndrome (SIRS) e a condition of a preliminary stage of BIG, as described above.

Examples of the severe sepsis include sepsis accompanied by one or more symptoms ing organ dysfunction such as metabolic is, organ rfllsion, acute encephalopathy, oliguria, hypoxemia or disseminated intravascular coagulation, and hypotension. As sepsis, one presenting organ dysfunction, organ hypoperfusion, or hypotension is called severe sepsis. The organ rfusion or abnormal perfusion includes lactic acidosis, oliguria, mental clouding, and the like. Among the severe sepsis, a condition persistently presenting hypotension despite of sufficient load of fluid therapy is called as septic shock.

More specifically, the severe sepsis referred to in this embodiment is as follows.

Examples of the septic shock include a condition with hypotension (blood pressure of 90 mmHg or lower or lower than usual blood pressure by 40 mmHg or more), not ding to resuscitation by fluid replacement, and accompanied by organ failure.

Examples of the intractable septic shock include a condition with septic shock continuing over 1 hour or longer, and not responding to a hypertensor with fluid therapy.

Examples of the multiple organ dysfunction syndrome (MODS) include a condition with malfunction of one or more organs, and requiring l intervention for maintaining homeostasis.

INR referred to in this embodiment is an examination criterion that defines blood coagulopathy. INR means a prothrombin time forth also abbreviated as PT) normalized as for differences between manufacturing lots of thromboplastin preparations. INR is lly defined as follows: INR value = (Coagulation time (sec) of test specimen/Coagulation time (sec) of control value) specimen)"51 In the equation, coagulation time (sec) of test specimen represents PT of test plasma specimen of a subject to be examined, and ISI represents International Sensitivity Index.

Examples of the severe sepsis referred to in this embodiment include sepsis accompanied by one or more symptoms ing organ dysfunction such as metabolic acidosis, acute encephalopathy, ia, hypoxemia or disseminated intravascular coagulation, and hypotension, as described above. The term severe means that the disease is in a critical condition for life support. Examples of the severe sepsis include, in ular, sepsis accompanied by one or more organ dysfunctions. Although the organ dysfunction is not particularly limited so far that the organ dysfunction is induced by sepsis, the organ dysfunction preferably includes e of an organ that is essential for supporting life. Examples ofthe one or more organ ctions 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, atory organ dysfunction, and kidney ction, and more preferred examples include one or more organ dysfunctions selected from the group consisting of respiratory organ dysfunction, and circulatory organ dysfunction. Although number of the organ dysfunctions is not particularly limited so far that the number is one or more, the number may be preferably two or more. In ular, it is preferred that there are two kinds of organ dysfunctions of respiratory organ dysfunction and circulatory organ dysfunction.

The atory organ dysfunction is not particularly d so long as a generally known atory organ dysfunction, and examples include, for example, blood pressure decrease and shock.

The respiratory organ dysfunction is not particularly limited so long as a generally known respiratory organ dysfunction, and examples include, for example, hypoxemia, acute lung injury and dyspnea.

The kidney dysfunction is not particularly d so long as a generally known kidney dysfunction, and examples include, for example, renal function disorder, oliguria, and renal failure.

The liver dysfunction is not particularly limited so long as a generally knows liver dysfunction, and examples include, for example, hepatic function disorder, jaundice, hepatic failure, and the like.

These organ ctions are generally known as described in publications published before the application date of this application, for example, Funada H., "Elucidation and Treatment Strategy for Sepsis", lyaku l 00., Ltd., p.38" (2006)), "Surviving Sepsis gni international ines for management of severe sepsis and septic shock 2008" (Crit. Care Med., 2008 Jan;36(1)1296'327), and the like.

It is supposed that organ dysfunction may be induced by a factor other than sepsis, such as in the case of nduced organ dysfunction, and accordingly, it is desirable that ts with organ dysfunction d to the liver or kidney are excluded from the severe septic patients. It is also known that thrombocytopenia may be developed as a result of organ dysfunction. Although platelet count in patients to be administered with the medicament of this embodiment is not particularly limited so far that the platelet count is less than 300,000/uL, the count is preferably less than 200,000/uL, more preferably less than 150,000/uL.

In this embodiment, the value ofINR in a plasma specimen of a sepsis patient is not particularly limited so far that the value is more than 1.4, and when INR is more than 1.4, thrombomodulin is more effective for sepsis patients with one or more organ dysfunctions. The maximum INR may be, for example, 2.0 or lower, preferably 1.9 or lower, more preferably 1.8 or lower, still more preferably 1.7 or lower, most preferably 1.6 or lower. The value may also be preferably 1.5 or lower. It may also be preferred that a patient with an INR value of 1.7 is excluded.

The expression "INR more than 1.4" may also be indicated as "INR > 1.4".

In this embodiment, DIC is a e or syndrome whereby large quantities of blood coagulation-accelerating substances are generated as a result of tissue damage caused by various diseases, so that the function of a ation system is excessively accelerated, and small thrombuses are formed in lized blood vessel (microthrombus formation) and they clog small vessels, and at the same time, thrombocytes or coagulation factors ary for the control of bleeding are consumed, thereby causing clotting abnormality. Specifically, as a result of fibrin ion in vascular vessel, bleeding due to ption opathy or organ e due to microthrombus formation occurs. D10 is also referred to as disseminated intravascular coagulation syndrome or diffuse intravascular coagulation syndrome.

DIC has various types of clinical symptoms depending on the type of underlying pathogenic ion. In addition to observation of bleeding or organ ms, a preferred method for diagnosing an illness as DIC comprises keeping the score of DIC on the basis of several test values as described below and then diagnosing the illness as DIC when the DIC score has reached a certain level. Examples of such test values e the number of blood platelets, the concentration of fibrin/fibrinogen degradation products (hereinafter abbreviated as FDP, at times) decomposed by plasmin, a D'dimer concentration, a fibrinogen concentration, and a prothrombin time.

Moreover, it is also possible to diagnose a certain condition as preDIC based on a decrease in platelets, an increase in the D-dimer or FDP concentration, and the like Without keeping the DIC score (Masao Nakagawa, "Search report regarding use of criteria of disseminated intravascular coagulation (DIC)," Research Study Team of table Disease (Blood Coagulation ality), the Ministry of Health and Welfare, Study report 1999, 19992 65-72; Katsumi Deguchi, "Tentative plan regarding standards for initiation of early treatment of BIG," Research Study Team of Intractable Disease (Blood ation Abnormality), the Ministry of Health and Welfare, Study report 1999, 19991 73'77; and Katsumi Nakagawa & Hajime Tsuji, "Current diagnosis of DIC — Reports on s of inquiry survey" Clinical Blood. 1999, 40! 362-364).

In this embodiment, a sepsis patient with an INR value more than 1.2, preferably more than 1.3, more preferably more than 1.4, in a plasma specimen of the patient can be called a D10 patient in a broad sense, and the ment for therapeutic treatment and/or improvement of sepsis ing to this embodiment may be used as a medicament for therapeutic treatment and/or improvement of D10.

The medicament of this embodiment may also be used for DIC. Sepsis is also regarded as SIRS induced by critical clinical invasion from infection, and closely relates to DIC ofwhich causative disease is an infectious disease. DIC is often ped simultaneously with sepsis, and the medicament of this embodiment may also be used for such a sepsis patient simultaneously developing DIC. In other words, the medicament of this embodiment may be used for a patient suffering from or suspected to suffer from either one of DIC and sepsis, or the both.

In this ment, INR can be measured, for example, as follows.

Specifically, tissue thromboplastin and Ca2+ are added to plasma (test specimen) obtained by adding sodium citrate, time (PT) required for coagulation (precipitation of fibrin) is measured, and evaluation is established on the basis of ve ratio of the time in terms of second with respect to that of a control specimen ity ratio). The activity ratio can be obtained as "coagulation time (second) of test specimen/coagulation time (second) of control specimen", but the ratio may vary among laboratories in which the test is ented due to difference in sensitivity of used tissue thromboplastin.

The INR value was d in order to eliminate such ion, and by evaluating PT using the INR value corrected with an international sensitivity index (henceforth also abbreviated as ISI), ion caused by difference of tories can be eliminated to obtain a standard result. ISI represents difference from the international standard sample. ISI is determined for every tissue thromboplastin reagent, and is attached to the reagent. Examples of the thromboplastin reagent include Thromborel S (registered ark, Sysmex Corp .), Thromboplastin C+ (registered trademark, Sysmex Corp .), and the like, but not limited to these examples. Thromborel S (registered trademark) uses human ta thromboplastin (ISI value is around 1.0), and thromboplastin C+ (registered trademark) uses rabbit brain thromboplastin (ISI value is about 1.8).

ISI is attached to each tissue thromboplastin reagent, and the INR value is calculated in accordance with Equation 1 mentioned above.

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

As therapeutic treatments of sepsis, such basic ents as mentioned below are generally performed by referring to known publications ving Sepsis Campaigni International guidelines for management of severe sepsis and septic shock: Crit. Care Med, 2008, -327; Crit. Care Med, 32(3), 1250-56 (2003)), and thrombomodulin and another medicament may be used in combination. However, the other medicament used in combination is not limited to those mentioned below.

When hypotension ues in a septic shock patient even after the central venous pressure (CV?) rises to a desired value, dopamine may be administered in order to raise the average blood pressure to at least 60 mmHg. When the dopamine dose exceeds 20 ng/kg/minute, another essor (usually norepinephrine) may be additionally administered.

For therapeutic treatment against causative bacteria of sepsis, an antibiotic is generally used. For the selection of the otic, there is required well-grounded estimation based on suspected cause, clinical sign, knowledge concerning microorganisms and knowledge concerning pattern of sensitivity common to a specific hospital ward for inpatients, results of inary culture test, and the like.

Intensive normalization of blood sugar level in sepsis patients improves clinical outcome of the patients in critical conditions.

When an antibiotic is used, a specimen such as blood, body fluid or wound part can be investigated, and a drug effective for the causative bacterium can be chosen.

For example, in the case of septic shock of unknown cause, gentamycin or tobramycin and a third generation cephalosporin may be administered in combination. Further, when ion of resistant Staphylococcus or Enterococcus bacteria is suspected, vancomycin is additionally administered.

In general, the dose is ed to maintain the blood sugar level at 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 eutic treatment of sepsis, it may be stered at a supplemental dose.

To a t of high death risk (APACHE II score 2 25, multiple organ failure due to sepsis, ARDS due to septic shock or sepsis), a recombinant activated Protein C (rhAPC, drotrecogin 01) may be administered when there are no contraindications (hemorrhage and the like).

Although target patients are limited, packed red blood cell transfusion may be performed aiming at Hb 7.0 to 9.0 g/dL.

In the case of ed erythropoiesis in a sepsis t due to renal failure, rerythropoietin (EPO) may be administered.

In the case of severe sepsis, heparin may be administered at a low dose unfractionated heparin or low lar weight n may be administered for prevention of DVT.

The medicament of the present invention may contain a carrier. As the carrier usable in the present invention, a water-soluble r is preferred, and tonicity agent, buffering agent, viscosity enhancer, surfactant, preservative, antiseptic, soothing agent, pH modifier, or the like acceptable as pharmaceutical ves usually preferred. For example, the ment ofthe present invention can be prepared by adding sucrose, glycerin, pH modifier ting of an inorganic salt, or the like as additives. Further, if necessary, amino acids, salts, carbohydrates, surfactants, albumin, gelatin or the like may be added as disclosed in Japanese Patent Unexamined Publication Nos. 64-6219 and 6321805. Method for adding these ves is not particularly limited. However, in the case ofpreparing a lyophilized product, examples include, for example, a method ofmixing a solution containing at least one therapeutic agent selected from an immunosuppressant and a therapeutic agent for hematological malignancy, and a solution containing thrombomodulin, then adding additives to the e, and mixing the resulting mixture, and a method of mixing additives with at least one therapeutic agent selected from an immunosuppressant and a therapeutic agent for hematological malignancy dissolved in water, water for injection, or an appropriate buffer hand, adding a solution containing thrombomodulin to the mixture, mixing the resulting mixture to prepare a solution, and lyophilizing the solution, in manners as those commonly employed. When the ment of the present invention is a medicament comprising a ation ofthe components ofthe medicament, each component is preferably prepared by adding a carrier according to an appropriate preparation method. The ment ofthe present invention may be provided in the form of an injection, or in the form of a lyophilized preparation to be dissolved upon use.

As for preparation of the medicament of the present invention, an s solution for injection can be ed by filling a solution containing 0.1 to 10 mg of omodulin, water for injection, and additives in an ampoule or vial in a volume of, for example, 0.5 to 10 mL. Examples ofthe preparation method also include a method of freezing such a solution, and drying the frozen solution under reduced pressure to prepare a lyophilized preparation.

The medicament of the present invention is desirably administered by parenteral administration such as intravenous administration, intramuscular administration, and subcutaneous administration. The medicament may also be administered by oral administration, intrarectal stration, asal stration, sublingual administration or the like. When the medicament of the present invention is a medicament comprising a combination of multiple active ingredients, each active ingredient of the medicament is preferably administered by an administration method suitable for the ingredient.

Examples of method for the enous administration include a method of administering a desired dose of the medicament at one time (intravenous bolus administration), and intravenous administration by drip infiision. [0 102] The method of administering a desired dose of the medicament at one time (intravenous bolus administration) is preferred from the viewpoint that the method requires only a short time for administration. ally it is preferred in case of sepsis patients who need the urgent treatment. When the medicament is administered at one time, a period required for administration by using an injectable syringe may generally varies. In general, the period of time required for the administration is, example, 5 minutes or shorter, preferably 3 s or r, more preferably 2 minutes or shorter, still more preferably 1 minute or shorter, particularly preferably 30 seconds or shorter, although it depends on a volume to be administered. Although the minimum stration time is not particularly limited, the period is preferably 1 second or longer, more ably 5 seconds or longer, still more preferably 10 seconds or longer. The dose is not particularly limited so long that the dose is Within the aforementioned preferred dose. Intravenous administration by drip infusion is also preferred from a viewpoint that blood level of thrombomodulin can be easily kept A daily dose of the medicament ofthe present invention may vary depending on age, body weight of patients, severity of disease, administration route and the like.

In general, the maximum dose is preferably 20 mg/kg or less, more preferably 10 mg/kg or less, still 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 rug/kg or more, more preferably 0.005 mg/kg or more, still 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, in terms of the amount of thrombomodulin.

In the case of intravenous bolus stration, although the dose is not particularly limited so long as the dose is within the aforementioned red dose, the maximum daily doSe is ably 1 mg/kg or less, more preferably 0.5 mg/kg less, still more ably 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 ably 0.01 mg/kg or more, still more preferably 0.02 mg/kg or more, and particularly preferably 0.04 mg/kg or more.

When the medicament of the present invention is administered to a patient having a body weight exceeding 100 kg, it may be preferably administered at a fixed dose of 6 mg, since blood volume is not proportional to the body weight, and blood volume is relatively reduced with respect to the body weight in such a patient.

In the case of continuous enous infusion, although the dose is particularly limited so long as the dose is Within the aforementioned preferred dose, the maximum daily dose is preferably 1 rug/kg or less, more preferably 0.5 mg/kg or less, still 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, still more preferably 0.02 mg/kg or more, and particularly preferably 0.04 mg/kg or more.

When the medicament of the present invention is administered to a patient having a body weight exceeding 100 kg, it may be preferably stered at a fixed dose of 6 mg, since blood volume is not proportional to the body weight, and blood volume is relatively reduced with respect to the body weight in such a patient.

The medicament is administered once or several times a day as required. As for stration interval, the medicament may be administered once in 2 to 14 days, preferably once in 2 to 7 days, more preferably once in 3 to 5 days.

[Explanation of Sequence listing] SEQ ID N01 11 Amino acid ce encoded by the gene used in production of TME456 SEQ ID N03 2! Nucleotide ce encoding the amino acid ce of SEQ ID N01 1 SEQ ID N03 33 Amino acid sequence encoded by the gene used in tion of TME456M SEQ ID N01 41 Nucleotide sequence encoding the amino acid sequence of SEQ ID N03 3 SEQ ID N05 53 Amino acid sequence encoded by the gene used in production of TMD 12 SEQ ID N01 61 Nucleotide ce encoding the amino acid sequence of SEQ ID N01 5 SEQ ID N03 73 Amino acid sequence encoded by the gene used in production of TMD12M SEQ ID NO: 8! Nucleotide sequence encoding the amino acid ce of SEQ ID N02 7 SEQ ID NO! 93 Amino acid sequence encoded by the gene used in production of TMD123 SEQ ID N01 103 Nucleotide sequence encoding the amino acid sequence of SEQ ID N02 SEQ ID N01 111 Amino acid sequence encoded by the gene used in production of TMD123M SEQ ID NOI 121 Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: SEQ ID N01 131 Synthetic DNA for mutation used for carrying out site-directed mutagenesis Examples The t invention will be explained in detail with reference to examples and test examples. However, the present invention is not limited by these examples.

The thrombomodulin of the present invention used in the test examples was prepared according to the aforementioned method ofYamamoto et al. (the method described in Japanese Patent Unexamined Publication No. 64-6219). Preparation es thereof are described below. Safety of the thrombomodulins obtained in these preparation examples was ed by single and repetitive intravenous administration tests using rats and monkeys, mouse reproduction test, local irritation test, pharmacological safety test, virus inactivation test, and the like. ration Example 1] A highly purified product was obtained by the aforementioned method.

Specifically, Chinese hamster ovary (CHO) cells were transfected with a DNA ng the amino acid ce of SEQ ID N03 9 (which specifically consisted of the nucleotide sequence ofSEQ ID N02 10). From the culture of the above transformant cells, a highly purified product was obtained by collecting an active fraction with a 20 mmol/L phosphate buffer (pH 7.8) containing 50 mmol/L NaCl according to the aforementioned conventional purification method. The product was further concentrated by using an ltration membrane to obtain a thrombomodulin solution having a concentration of 11.0 mg/mL (henceforth also abbreviated as TMD123 in the specification). ration ofpolysorbate solution> Polysorbate 80 was weighed (0.39 g) in a glass beaker, added with water for injection (30 mL), and dissolved. The TMD 123 solution obtained above (2239 mL, corresponding to 24.63 g of soluble thrombomodulin protein, added in a 5% excess ) was put into a 5L stainless steel vessel. The polysolvate solution obtained above was further 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 1 mol/L hloric acid solution. Water for ion was further added to the mixture up to a total amount of 3940 g, and the mixture was uniformly mixed and stirred. This drug solution was subjected to filtration sterilization using a filter having a pore diameter of 0.22 pm (MCGLlOS, manufactured by Millipore). The filtrate was filled in ampoules in an amount of 1.1 g each to obtain a TMD123 preparation.

[Preparation Example 2] Chinese hamster ovary (CHO) cells are transfected with a DNA encoding the amino acid sequence of SEQ ID N03 11 (which specifically consists of the nucleotide sequence of SEQ ID. N01 12), a solution ofthrombomodulin purified from a culture of the above transformant cells (henceforth also abbreviated as TMD 123M in the specification) by the aforementioned conventional ation method is obtained, and a TMD123M preparation is obtained in the same manner as that described above.

[Preparation Example 3] Chinese hamster ovary (CHO) cells are transfected with a DNA encoding the amino acid sequence ofSEQ ID NO? 1 (which specifically consists ofthe nucleotide sequence of SEQ ID N03 2), thrombomodulin purified from a culture of the above transformant cells (henceforth also iated as TME456 in the specification) by the aforementioned conventional purification method is obtained, and a TME456 preparation is ed in the same manner as that described above.

[Preparation Example 4] Chinese hamster ovary (CHO) cells are transfected with a DNA encoding the amino acid sequence of SEQ ID NO! 8 (which cally consists of the nucleotide sequence of SEQ ID NO! 4), thrombomodulin purified from a e of the above transformant cells (henceforth also iated as M in the specification) the aforementioned conventional purification method is obtained, and a TME456M preparation is ed in the same manner as that described above.

[Preparation Example 5] Chinese hamster ovary (CHO) cells are transfected with a DNA encoding the amino acid sequence ofSEQ ID N05 5 (which specifically ts of the nucleotide sequence of SEQ ID N01 6), omodulin purified from a culture of the above transformant cells (henceforth also abbreviated as TMD 12 in the specification) by the aforementioned conventional purification method is obtained, and a TMD 12 preparation is obtained in the same manner as that described above.

[Preparation e 6] Chinese hamster ovary (CI-IO) cells are transfected with a DNA encoding the amino acid sequence of SEQ ID N01 7 (which specifically consists of the nucleotide sequence of SEQ ID N03 8), thrombomodulin d from a culture of the above transformant cells forth also abbreviated as TMD 12M in the specifiéation) by the aforementioned conventional purification method is obtained, and a TMD12M preparation is obtained in the same manner as that described above.

[Preparation Example 7] Preparation ofplacebo formulation Polysorbate 80 was weighed (0.4 g) in a‘glass beaker, added with water for injection (30 mL), and dissolved. Water for ion (2000 mL) was put into a 5L stainless steel . The polysolvate solution obtained above was further added. Water for ion was further added to the mixture up to a total amount of 4000 g, and the mixture was uniformly mixed and stirred. This drug solution was subjected to filtration sterilization using a filter having a pore diameter of 0.22 um (MCGLlOS, manufactured by Millipore). The filtrate was filled in ampoules in an amount of 1.1 g each to obtain a placebo preparation.

[Example 1] By using TMD-123 prepared according to Preparation Example 1 as thrombomodulin, a randomized double-blind o-controlled study was conducted for patients with sepsis and DIC. ed patient number is 750 in total, and among them, 741 patients were administered with investigational drug (TMD-123 for 370 patients and placebo for 371 patients). TMD'123 was stered once a day at 0.06 mg/kg for successive 6 days via intravenous bolus administration. As the placebo, the preparation manufactured according to Preparation Example 7 was used.

For patients over the body weight of 100 kg, a fix dose of 6 mg was evenly stered once a day for successive 6 days via intravenous bolus administration in order to suppress side s due to overdose.

A plasma INR value of a patient before the administration of the test drug measured by the above method described as Equation 1.

Severe septic patients with organ ction limited to the liver or kidney were excluded for the analysis. In patients with organ dysfunction limited to the liver or kidney, the organ dysfunction may possibly be occurred on the basis of causes not limited to sepsis such as drug-induced organ dysfunction.

Outcome after 28 days from the start of the administration was observed and a mortality rate lity) ofeach patent group was calculated.

Further, a difference in mortality rate between TMD-123 group and placebo group was calculated as "Difference". In the group of patient without organ dysfunction, patients with the INR value of INR > 1.5 in plasma before the administration of the test drug gave maximum difference (Differencei 6.1%) between the ity rate of the group administered with 3 and that of the group administered with placebo, and the group of patient with INR > 1.6 gave the second highest difference in mortality rate (4.5%). In the group of patient with INR > 1.4, no significantly high difference in mortality rate was absolutely or relatively observed, i.e., difference in mortality rate was about 1.7% (Table 1).

From Table 1, it is recognized that the maximum result can be found n 1.5 and 1.6 of the lower limits of INR, and when the lower limits of INR are outside the range of from 1.5 to 1.6, the difference in mortality rate was significantly decreased.

Whilst, in the group of severe septic patients having one or more organ dysfunctions selected from circulatory organ dysfunction, respiratory organ dysfunction, kidney dysfunction, and liver dysfunction, the group of patients with INR > 1.4, not the group with INR > 1.5, gave the maximum ence in mortality rate (9.7%), which is absolutely and relatively high difference in mortality rate as compared to the other INR lower limits (Table 2). The group of severe septic patients having one or more organ dysfunctions selected from circulatory organ dysfunction, respiratory organ dysfunction, kidney dysfunction, and liver dysfunction gave more significant difference in mortality rate between TMD'123 group and placebo group on the whole as compared to the group ofpatients without organ dysfunction (the former: 5.4%, the latter 11%).

Further, in the group of severe septic ts having one or more organ dysfunctions selected from atory organ dysfunction, respiratory organ dysfunction, kidney dysfunction, and liver dysfunction, the t with 1.4 < INR 5 1.6 gave absolutely remarkable difference in mortality rate as being 16.0% (Table 3). As understood from Table 3, any of the other INR upper limits gave values of the difference in mortality rate in a range of 10 to 12%, which indicates relatively recognizable remarkably high difference in mortality rate of the aforementioned group.

On the other hand, no tendency of relatively outstanding peak increase in n group was observed in the group ents without organ dysfunction. The difference in mortality rate obtained was found at utmost to be 7.1% (in patient group with 1.4 < INR S 1.7) (Table 4).

[Table 1] INR value of TMD-l23 Placebo plasma before Difference Number Mortality Number ity admmlstratmn_ . _ INR >1.4 5/7 1: INR >16 42 3/42: INR >1.7 29 3/29: INR >1.8 22 3/22: INR >1.9 14 2/14: INR >20 9 1/9= [Table 2] INR value of TAD-123 o plasma before Difference Number Mortality Number Mortality administration INR>1.2 184 44/184: 23.9% 186 55/186: 29.6%- 7.9% [Table 3] INR value of 3 Placebo plasma before Difference Number Mortality Number Mortality administration 56 10/56= 17.9% 59 16.0% 14/76: 18.4% 11.2% 92 18/92: 19.6% 11.1% 101 19/101: 18.8% — 12.0% 110 20/110: 18.2% 10.6% [Table 4] INR value of m plasma before Difference administration 1.6>= INR >1.4 1/26= 3.8% 1.8>= INR >1.4 5/49: 10.2% 1.9>= INR >1.4 5/57: 2.0>= INR >1.4H 5/62: Industrial Applicability The medicament of the t invention containing thrombomodulin is useful as a medicament enabling effective therapeutic treatment and/or improvement of ' sepsis in a severe septic patient, wherein a INR value of a plasma specimen obtained from said patient is more than 1.4.

Claims (33)

What is claimed is:

1. The use of thrombomodulin in the manufacture of a medicament for therapeutic treatment and/or improvement of sepsis, wherein the sepsis is severe and is accompanied with one or more organ dysfunctions, and wherein a value of ational Normalized Ratio (INR) of a plasma en is more than 1.4.

2. The use according to claim 1, n the value of International Normalized Ratio (INR) of the plasma specimen is more than 1.4 and equal to or less than 1.6.

3. The use according to claim 1 or 2, wherein the organ dysfunction is not limited to the liver or kidney.

4. The use according to any one of claims 1 to 3, wherein the one or more organ dysfunctions are selected from the group consisting of liver dysfunction, kidney dysfunction, respiratory organ dysfunction, and circulatory organ dysfunction.

5. The use according to any one of claims 1 to 3, wherein the one or more organ dysfunctions are selected from the group consisting of respiratory organ dysfunction and circulatory organ dysfunction.

6. The use according to any one of claims 1 to 5, wherein the thrombomodulin is a soluble omodulin.

7. The use according to any one of claims 1 to 6, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by ecting a host cell with a DNA coding for the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

8. The use ing to any one of claims 1 to 7, wherein the thrombomodulin is a peptide containing the amino acid sequence of (i-1) or (i-2) ned below, and the peptide has thrombomodulin activities; (i-1) the amino acid sequence of the positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or (i-2) the amino acid sequence having 90% or more identity with the amino acid sequence of (i-1) mentioned above.

9. The use according to any one of claims 1 to 8, wherein the thrombomodulin is ated for intravenous bolus administration at a dose of 0.005 to 1 mg/kg within 5 minutes.

10. The use according to any one of claims 1 to 9, wherein the sepsis is severe sepsis with coagulopathy.

11. The use ing to any one of claims 4 to 10, wherein the atory organ dysfunction is shock.

12. The use according to any one of claims 1 to 11, wherein the organ dysfunctions are induced by sepsis.

13. The use according to any one of claims 1 to 12, wherein an INR value of 1.7 is excluded.

14. The use according to any one of claims 1 to 13, wherein the thrombomodulin is formulated for intravenous administration.

15. The use according to any one of claims 1 to 14, wherein the thrombomodulin is formulated for stration at a dose of 0.04 - 0.08 mg/kg/day.

16. The use according to any one of claims 1 to 15, wherein the thrombomodulin is formulated for administration for a period of 6 days.

17. The use of thrombomodulin in the manufacture of a medicament for decreasing the ity of patients with sepsis, wherein the sepsis is severe and is accompanied with one or more organ dysfunctions, and wherein a value of International Normalized Ratio (INR) of a plasma specimen is more than 1.4.

18. The use according to claim 17, wherein the value of International Normalized Ratio (INR) of the plasma specimen is more than 1.4 and equal to or less than 1.6.

19. The use according to claim 17 or 18, wherein the organ dysfunction is not limited to the liver or kidney.

20. The use according to any one of claims 17 to 19, wherein the one or more organ dysfunctions is selected from the group ting of liver dysfunction, kidney dysfunction, respiratory organ dysfunction, and circulatory organ dysfunction.

21. The use according to any one of claims 17 to 20, wherein the one or more organ dysfunctions are ed from the group ting of respiratory organ dysfunction and circulatory organ dysfunction.

22. The use ing to any one of claims 17 to 21, wherein the thrombomodulin is a soluble thrombomodulin.

23. The use according to any one of claims 17 to 22, wherein the omodulin is a peptide ed from a transformed cell prepared by transfecting a host cell with a DNA coding for the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

24. The use according to any one of claims 17 to 23, wherein the thrombomodulin is a peptide containing the amino acid sequence of (i-1) or (i-2) mentioned below, and the peptide has thrombomodulin activities: (i-1) the amino acid sequence of the positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or (i-2) the amino acid sequence having 90% or more identity with the amino acid sequence of (i-1) mentioned above.

25. The use according to any one of claims 17 to 24, wherein the thrombomodulin is formulated for intravenous bolus administration at a dose of 0.005 to 1 mg/kg within 5 minutes.

26. The use according to any one of claims 17 to 25, n the sepsis is severe sepsis with coagulopathy.

27. The use according to any one of claims 20 to 26, wherein the circulatory organ dysfunction is shock.

28. The use according to any one of claims 17 to 27, wherein the organ dysfunctions are induced by sepsis.

29. The use according to any one of claims 17 to 28, wherein an INR value of 1.7 is excluded.

30. The use according to any one of claims 17 to 29, wherein the thrombomodulin is formulated for intravenous administration.

31. The use according to any one of claims 17 to 30, wherein the omodulin is formulated for administration at a dose of 0.04 - 0.08 mg/kg/day.

32. The use according to any one of claims 17 to 31, wherein the thrombomodulin is formulated for stration for a period of 6 days.

33. The use according to claim 1 or 17, substantially as herein described with reference to any one of the accompanying examples and/or ce listing.