HK1196289B

HK1196289B - Medicine for treatment and/or improvement of sepsis

Info

Publication number: HK1196289B
Application number: HK14109850.4A
Authority: HK
Inventors: 鹤田一寿; 青木喜和; 大泽宽; I.考尔
Original assignee: 旭化成制药株式会社
Priority date: 2011-11-15
Filing date: 2012-11-14
Publication date: 2017-05-12

Abstract

A medicine for the effective treatment and/or improvement of sepsis in patients suffering from severe sepsis who have damage to one or more organs and the International Normalized Ratio (INR) value of a blood sample of whom is higher than 1.4, comprising thrombomodulin as active ingredient.

Description

Drug for treatment and/or amelioration of sepsis

Technical Field

The present invention relates to a drug for treating and/or ameliorating sepsis in a severe sepsis patient.

Background

Sepsis is Systemic Inflammatory Response Syndrome (SIRS) caused by infection. That is, it is defined as a disease state that satisfies 2 or more of the SIRS items ((1) body temperature >38 ℃ or <36 ℃, ((2) heart rate > 90/min, (3) respiratory rate > 20/min or PaCO2<32torr, (4) white blood cell count >12,000/μ L or <4000/μ L or immature white blood cells > 10%) in addition to the presence of infection. Although the presence of bacterial cells in blood has been emphasized in the past (bacteremia), this definition does not necessarily require blood culture positivity. In sepsis, a state in which organ dysfunction, organ hypoperfusion, or hypotension is present is called severe sepsis (severesepsilon). Organ hypoperfusion or perfusion abnormalities include lactic acidosis, oliguria, confusion, and the like. In severe sepsis, hypotension continues even when a sufficient infusion load is applied, and is called septic shock (non-patent document 1). The circulatory failure found in these pathological conditions is thought to be caused by dysfunction of the sympathetic nervous system or mediators released from neutrophils and the like, and organ dysfunction is thought to be caused by hypoxia (dysoxia) of tissues.

On the other hand, thrombomodulin is known to be a substance that specifically binds to thrombin, has a function of inhibiting the blood coagulation activity of thrombin and remarkably promoting the protein C activation ability of thrombin, and is known to have a potent blood coagulation inhibitory action. Thrombomodulin is known to prolong the time of coagulation by thrombin and to inhibit platelet aggregation by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation and fibrinolysis system, and is activated by the action of thrombin to become activated protein C. It is known that the activated protein C inactivates factor V and factor VIII, which are active forms of coagulation factors, in vivo, and that the activated protein C is involved in the production of plasminogen activator having thrombolytic activity (non-patent document 2). Therefore, thrombomodulin promotes activation of protein C by this thrombin, and is useful as an anticoagulant or thrombolytic agent, and there has been a report on animal experiments explaining that thrombomodulin is effective for treatment and prevention of diseases accompanied by hypercoagulability (non-patent document 3).

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

In contrast to the fact that the full-length thrombomodulin is difficult to dissolve unless it is present in a surfactant, and a surfactant must be added as a preparation, it is known that there is a soluble thrombomodulin that can be smoothly dissolved even in the absence of a surfactant. It has been confirmed that soluble thrombomodulin formed only of 3 regions, i.e., the N-terminal region, the 6 EGF-like structures and the O-type sugar chain addition region (i.e., formed of the amino acid sequences at positions 19 to 516 of SEQ ID NO. 9) can be obtained by applying recombinant technology, and that the recombinant soluble thrombomodulin has the activity of natural thrombomodulin (patent document 1). Some other examples of soluble thrombomodulin have been reported (patent documents 2to 9). In addition, examples of natural forms include soluble thrombomodulin derived from human urine and the like (patent documents 10 and 11).

Incidentally, in the gene, as has been confirmed in many cases, a variety of variations are found in humans by natural variations or acquired variations, and a sequence in which the amino acid at the 473 rd position of the human thrombomodulin precursor composed of the above-mentioned 575-residue amino acid sequence is Val and a sequence in which the amino acid is Ala have been confirmed. In the nucleotide sequence encoding this amino acid, the nucleotide sequence has a mutation at position 1418 into T and C, respectively (non-patent document 5). However, there was no difference in activity and physical properties at all, and it was judged that both were substantially the same.

It has been reported that thrombomodulin is effective in the treatment of disseminated (generalized) intravascular coagulation syndrome (hereinafter sometimes referred to as DIC) (non-patent document 6). In addition to the above, the use of thrombomodulin is expected to be used for the treatment and prevention of diseases such as Acute Coronary Syndrome (ACS), thrombosis, peripheral vascular occlusion, arteriosclerosis obliterans, vasculitis, functional disorders secondary to cardiac surgery, complications of organ transplantation, angina pectoris, transient ischemic attack, gestational toxicosis, diabetes, liver VOD (liver no-occlusive disease; hepatic venous occlusion after fulminant hepatitis or bone marrow transplantation), deep vein thrombosis (DVT; deep venous thrombosis), and sepsis or Adult Respiratory Distress Syndrome (ARDS) (patent document 12).

Documents of the prior art

Patent document

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

Patent document 2: japanese patent laid-open publication No. 5-213998

Patent document 3: japanese laid-open patent publication No. 2-255699

Patent document 4: japanese laid-open patent publication No. 3-133380

Patent document 5: japanese laid-open patent publication No. 3-259084

Patent document 6: japanese laid-open patent publication No. 4-210700

Patent document 7: international publication No. WO92/00325

Patent document 8: international publication No. WO92/03149

Patent document 9: international publication No. WO93/15755

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

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

Patent document 12: international publication No. WO2003/061687

Non-patent document

Non-patent document 1: american colledgeof chestPhysicans, CHEST/101/6/JUNE,1992:1481-1483

Non-patent document 2: suzuki hong zhi, medical competence あゆみ (medical progress) 1983; 125:901

Non-patent document 3: good, Blood 1990; 75:1396-1399

Non-patent document 4: zushimetal, JBiolChem 1989; 246:10351-10353

Non-patent document 5: wend zetal, Biochemistry 1987; 26:4350-4357

Non-patent document 6: s.m.batesetal, br.j.of pharmacol, 2005; 144:1017-1028

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a drug for effectively treating and/or ameliorating sepsis in a critically ill sepsis patient, or a method for same.

Means for solving the problems

It is known that the international normalized ratio (international normalized ratio, hereinafter sometimes simply referred to as "INR") of a plasma sample of a sepsis patient means blood coagulation disorder (Coagulopathy). For example, as indices of blood coagulation disorders reported in the International Emergency medical society (CCM) of 2003, aPPT >60 seconds and INR >1.5(Crit. CareMed., 2003; 31:1250-1256) have been suggested. However, the INR value is not verified by clinical trials or the like, and has not been determined as a clear result. The following are reported in the literature: in fact, in phase III clinical trials with patients with severe sepsis as an anticoagulant, the tissue factor pathway inhibitor Tifacogin (ティファコジン), the patients with INR ≦ 1.2 were better than the patients with INR >1.2 (JAMA, july9,2003, vol.290.no.2, P238-247), as a result of the clinical trials with patients with INR >1.2 as the main target. On the other hand, it is reported that: in the results of this clinical trial, patients with INR >1.5 tended to be more effective than patients with INR >1.2 in the patient group of INR > 1.2. Furthermore, prolongation of Prothrombin Time (PT) was also confirmed in most of the subject patients (93.4%) for the unique cageli (Xigris) that showed efficacy in clinical trials of sepsis.

In this way, in the treatment of sepsis with anticoagulants, high effects can be expected from some test results by targeting patients with blood coagulation disorders, but adverse results are obtained, and it is difficult to say that the definition of blood coagulation disorders has been established. That is, the proposition of how to prescribe a target patient using an INR value and obtain a good result is not clear, and there is no technical common knowledge about what INR value a drug is particularly effective for a sepsis patient. The relationship between INR values and therapeutic effects is, at best, understood in part as an individual case for each agent.

Under such circumstances, the present inventors have intensively studied the therapeutic and/or ameliorating effect on sepsis, focusing on thrombomodulin in anticoagulants. As a result, it was found that: when a sepsis patient with severe sepsis having 1 or more kinds of organ dysfunction among sepsis patients (but excluding a sepsis patient with organ dysfunction of only liver or kidney) is targeted, sepsis can be treated and/or improved more effectively if INR of the patient is greater than 1.4 unexpectedly, that is, sepsis treatment and/or improvement with thrombomodulin has a special relationship between a severe sepsis patient with 1 or more kinds of organ dysfunction among sepsis patients and INR >1.4, which cannot be predicted by those skilled in the art, compared to a patient without organ dysfunction. Further, surprisingly, the present inventors have found that a difference in mortality rate between thrombomodulin and placebo of more than 15% is particularly significant in patients with severe sepsis in which INR is greater than 1.4 and has a value of 1.6 or less, and thus completed the present invention. Considering that the drug-placebo mortality difference of about 6% is possessed by chegri (Xigris) currently marketed in europe as the only sepsis therapeutic agent (n.engl.j.med., vol.344, No.10, march8,2001, P699-709), and that this value of 15% mortality difference is a significant value of about 2.5 times its size, it is understood that one mode of the present invention has a very surprising effect.

Specifically, the present invention includes the following embodiments.

[ A1 ] A drug for use in the treatment and/or amelioration of sepsis, which comprises thrombomodulin as an active ingredient, and which is to be administered to a patient with severe sepsis, wherein the patient has 1 or more kinds of organ dysfunction and the value of the International Normalized Ratio (INR) of a plasma sample of the patient is a value greater than 1.4.

[ A1-2 ] A drug for the treatment and/or amelioration of sepsis associated with blood coagulation disorders, which comprises thrombomodulin as an active ingredient, and which is to be administered to a critically ill sepsis patient having 1 or more kinds of organ dysfunction, wherein the value of the International Normalized Ratio (INR) of a plasma sample of the patient is a value greater than 1.4.

[ A2 ] the drug according to the above [ A1 ] or [ A1-2 ], which is used for administration to a patient with severe sepsis having 1 or more organ dysfunctions, wherein the value of the International Normalized Ratio (INR) of a plasma sample of the patient is a value of 1.4 or more and 1.6 or less.

[ A3 ] the drug according to any one of [ A1 ] to [ A2 ] above, wherein the critically ill sepsis patient is a critically ill sepsis patient excluding a sepsis patient having only organ dysfunction of the liver or the kidney.

Note that, when the item numbers cited as [ a 1] to [ a 2] are shown as ranges, and items having branch numbers such as [ a1-2 ] are arranged in the ranges, this means that items having branch numbers such as [ a1-2 ] are also cited. The same applies hereinafter.

[ A4 ] the drug according to any one of [ A1 ] to [ A3 ] above, which is used for administration to a patient with severe sepsis having 1 or more organ dysfunctions selected from the group consisting of the liver, the kidney, the respiratory organ, and the circulatory organ.

[ A5 ] the pharmaceutical agent according to any one of [ A1 ] to [ A4 ] above, wherein the thrombomodulin is soluble thrombomodulin.

[ A5-2 ] the medicament according to any one of [ A1 ] to [ A5 ] above, wherein the thrombomodulin has the following properties (1) to (4),

(1) an effect of selectively binding to thrombin;

(2) promoting the activation of thrombin-based protein C;

(3) the effect of prolonging thrombin-based clotting time; and

(4) inhibiting the effects of thrombin-based platelet aggregation.

[ A5-3 ] the medicament according to any one of [ A1 ] to [ A5 ] above, wherein the thrombomodulin is a soluble thrombomodulin having the following properties (1) to (5),

(1) an effect of selectively binding to thrombin;

(2) promoting the activation of thrombin-based protein C;

(3) the effect of prolonging thrombin-based clotting time;

(4) inhibition of thrombin-based platelet aggregation; and

(5) has anti-inflammatory effect.

[ A6 ] the drug according to any one of [ A1 ] to [ A5-3 ] above, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO.9 or SEQ ID NO. 11.

[ A7 ] the pharmaceutical agent according to any one of [ A1 ] to [ A6 ] above, wherein the thrombomodulin is a peptide having an amino acid sequence of any one of (i-1) or (i-2) below, which has thrombomodulin activity,

(i-1) the amino acid sequence at positions 19 to 516 in the amino acid sequence of any one of SEQ ID NO.9 or SEQ ID NO. 11; or

(i-2) an amino acid sequence obtained by substituting, deleting or adding 1 or 2 or more amino acids in the amino acid sequence of (i-1).

[ A7-2 ] the pharmaceutical agent according to any one of [ A1 ] to [ A6 ], wherein the soluble thrombomodulin is:

(i) a peptide comprising the amino acid sequence at positions 367 to 480 in the amino acid sequence of any one of SEQ ID NO.9 or SEQ ID NO. 11, and comprising the amino acid sequence of any one of (ii-1) or (ii-2), wherein the peptide has thrombomodulin activity,

(ii-1) an amino acid sequence at positions 19 to 244 in the amino acid sequence of any one of SEQ ID NO.9 or SEQ ID NO. 11; or

(ii-2) an amino acid sequence obtained by substituting, deleting or adding 1 or 2 or more amino acids in the amino acid sequence of (ii-1).

[ A8 ] the medicament according to any one of [ A1 ] to [ A7-2 ] above, wherein the thrombomodulin is administered in an amount of 0.005mg/kg to 1mg/kg by intravenous bolus injection over 5 minutes.

[ B1 ] A method for treating and/or ameliorating sepsis, which comprises a step of administering thrombomodulin to a critically ill sepsis patient whose plasma sample has an International Normalized Ratio (INR) value of more than 1.4.

[ B1-2 ] A method for treating and/or ameliorating sepsis associated with blood coagulation disorders, which comprises a step of administering thrombomodulin to a critically ill sepsis patient whose plasma sample has an International Normalized Ratio (INR) value of more than 1.4.

[ B2 ] the method according to [ B1 ] or [ B1-2 ] above, which comprises the step of administering the drug to a patient with severe sepsis, wherein the patient has 1 or more kinds of organ dysfunction and the value of the International Normalized Ratio (INR) of a plasma sample of the patient is 1.4 or more and 1.6 or less.

[ B3 ] the method according to any one of [ B1 ] to [ B2 ] above, wherein the critically ill sepsis patient is a critically ill sepsis patient excluding a sepsis patient having only organ dysfunction of the liver or the kidney.

[ B4 ] the method according to any one of [ B1 ] to [ B3 ] above, which comprises a step of administering the drug to a patient with severe sepsis, wherein the patient with severe sepsis has 1 or more kinds of organ dysfunction selected from the group consisting of a liver, a kidney, a respiratory organ, and a circulatory organ.

[ B5 ] the method according to any one of [ B1 ] to [ B4 ] above, wherein the thrombomodulin is soluble thrombomodulin.

[ B6 ] the method according to any one of [ B1 ] to [ B5 ] above, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence represented by SEQ ID NO.9 or SEQ ID NO. 11.

[ B7 ] the method according to any one of [ B1 ] to [ B6 ] above, wherein the thrombomodulin is a peptide having an amino acid sequence of any one of (i-1) or (i-2) below, which is a peptide having thrombomodulin activity,

[ B8 ] the method according to any one of [ B1 ] to [ B7 ] above, wherein the thrombomodulin is administered in an amount of 0.005mg/kg to 1mg/kg by intravenous bolus injection over 5 minutes.

[ B8-2 ] the method according to any one of [ B1 ] to [ B8 ] above, wherein the thrombomodulin is a thrombomodulin having a characteristic described in any one of [ A5-2 ], [ A5-3 ] or [ A7-2 ] above.

[ B9 ] use of thrombomodulin as a medicament for the treatment and/or amelioration of sepsis, wherein the medicament is for administration to a critically ill sepsis patient having 1 or more organ dysfunctions, and the value of the International Normalized Ratio (INR) of a plasma sample of the patient is a value greater than 1.4.

[ B9-2 ] the use according to [ B9 ] above, wherein the thrombomodulin is a thrombomodulin having any one of the characteristics described in [ A5-2 ], [ A5-3 ] or [ A7-2 ] above.

[ B9-3 ] the use according to [ B9 ] above, which has the features described in any one of [ A1 ] to [ A8 ] above.

[ C1 ] A drug for the treatment and/or amelioration of disseminated intravascular coagulation syndrome, which drug comprises thrombomodulin as an active ingredient, and which drug is to be administered to patients with disseminated intravascular coagulation syndrome who have 1 or more organ dysfunctions, the value of the International Normalized Ratio (INR) of the patient's plasma sample being a value greater than 1.4.

[ C2 ] the drug according to [ C1 ] above, which has any one of the features of [ A1 ] to [ A8 ] above.

ADVANTAGEOUS EFFECTS OF INVENTION

The medicament containing thrombomodulin of the present invention is effective for treating and/or improving sepsis in patients with severe sepsis in which INR of a plasma sample of the patient is a value greater than 1.4.

Detailed Description

The present invention will be specifically described below with respect to some preferred embodiments (preferred embodiments for carrying out the present invention, which may be hereinafter simply referred to as "embodiments" in the present specification), but the scope of the present invention is not limited to the specific embodiments described below.

Thrombomodulin in the present embodiment is known to have (1) an action of selectively binding to thrombin and (2) an action of promoting activation of thrombin-based protein C. In addition, it is preferable that: (3) the effect of prolonging thrombin-based clotting time; (4) inhibition of thrombin-based platelet aggregation; and/or (5) anti-inflammatory effects. The action of these thrombomodulins is sometimes referred to as thrombomodulin activity.

The thrombomodulin activity has the effects of (1) and (2) above, and preferably further has the effects of (1) to (4) above. Further, it is more preferable that the thrombomodulin activity has all of the effects (1) to (5).

The binding of thrombomodulin to thrombin can be confirmed by, for example, an assay method described in various publicly known documents, such as Thrombosisandhhaemstasis 199370(3):418-422 or the Journarof biologicalchemichemistry 1989Vol.264, No. 9pp.4872-4876. The amount of activity or the presence or absence of activity that promotes the activation of protein C by thrombin can be easily confirmed by, for example, a test method described in various publicly known documents typified by Japanese patent application laid-open No. Sho 64-6219. In addition, the effect of prolonging the thrombin-based coagulation time and/or the effect of inhibiting the thrombin-based platelet aggregation can also be easily confirmed. Further, the anti-inflammatory effect can be confirmed by, for example, a test method described in various publicly known documents such as blood2008112:3361-3670 and the journal clinical investigation20051155: 1267-1274.

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

As described above, the molecular weight of thrombomodulin is not limited as long as it has thrombomodulin activity, and the upper limit of the molecular weight is preferably 100,000 or less, more preferably 90,000 or less, further preferably 80,000 or less, particularly preferably 70,000 or less, and the lower limit of the molecular weight is preferably 50,000 or more, particularly preferably 60,000 or more. The molecular weight of the soluble thrombomodulin can be easily determined by a conventional method for determining the molecular weight of a protein, preferably by mass analysis, more preferably by MALDI-TOF-MS method. In order to obtain soluble thrombomodulin of a molecular weight in a target range, as described later, it can be obtained as follows: a soluble thrombomodulin having a molecular weight in the above-mentioned target range can be obtained by preparing transformed cells by transfecting a host cell with a DNA encoding the soluble thrombomodulin using a vector, culturing the transformed cells to obtain a soluble thrombomodulin, and fractionating the soluble thrombomodulin by column chromatography or the like.

The thrombomodulin in the present invention is preferably one that contains the amino acid sequences at the 19 th to 132 th positions of SEQ ID NO.1, which are known as the central part of thrombomodulin activity in human thrombomodulin, and is not particularly limited as long as it contains the amino acid sequences at the 19 th to 132 th positions of SEQ ID NO. 1. The amino acid sequences 19 to 132 of SEQ ID NO.1 may be naturally or artificially mutated, and one or more amino acids may be substituted, deleted, or added to the amino acid sequences 19 to 132 of SEQ ID NO.1, as long as they have an action of promoting the activation of thrombin-based protein C, i.e., thrombomodulin activity. The allowable degree of variation is not particularly limited as long as it has thrombomodulin activity, and for example, as the amino acid sequence, 50% or more homology, preferably 70% or more homology, more preferably 80% or more homology, further preferably 90% or more homology, particularly preferably 95% or more homology, and most preferably 98% or more homology may be shown. The amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to such amino acid sequences are referred to as the same variant sequences. These variants can be easily obtained by using a general gene manipulation technique as described later. The thrombomodulin is not particularly limited as long as it has the above-mentioned sequence and at least has an action of selectively binding to thrombin as a whole to promote the activation of thrombin-based protein C, and preferably has an anti-inflammatory action together therewith.

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

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

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

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

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

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

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

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

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

Furthermore, as the thrombomodulin of the present invention, there may be mentioned, as preferred examples, a peptide of sequence No. 14(462 amino acid residues), a peptide of sequence No. 8(272 amino acid residues), or a peptide of sequence No. 6(236 amino acid residues) in Japanese patent laid-open No. 64-6219.

The thrombomodulin in the present invention is not particularly limited as long as it has at least the amino acid sequence of the 19 th to 132 th positions of SEQ ID NO.1 or SEQ ID NO. 3, and among them, a peptide having at least the amino acid sequence of the 19 th to 480 th positions of SEQ ID NO. 5 or SEQ ID NO. 7 is preferable, and a peptide having at least the amino acid sequence of the 19 th to 515 th positions of SEQ ID NO.9 or SEQ ID NO. 11 is more preferable. As the peptide having at least the 19 th to 515 th amino acid sequences of SEQ ID NO.9 or 11, there may be mentioned, as a more preferred example, peptides having the 19 th to 516 th, 19 th to 515 th, 19 th to 514 th, 17 th to 516 th, 17 th to 515 th or 17 th to 514 th amino acid sequences of SEQ ID NO.9 or 11, respectively. Further, a mixture of peptides of the sequence No.9 or 11, each of which is composed of the sequence at the 19 th to 516 th, 19 th to 515 th, 19 th to 514 th, 17 th to 516 th, 17 th to 515 th, or 17 th to 514 th positions in the sequence No.9 or 11, is more preferable.

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

The mixing ratios of peptides ending at 514, 515 and 516 positions in SEQ ID NO.9 or 11 are shown as (0:0:100) to (0:90:10), and in some cases as (0:70:30) to (10:90:0) and (10:0:90) to (20:10: 70).

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

The 19 th to 132 th sequences of the sequence number 1 correspond to the 367 th to 480 th sequences of the sequence number 9, and the 19 th to 480 th sequences of the sequence number 5 correspond to the 19 th to 480 th sequences of the sequence number 9. The 19 th to 132 th bit sequences of sequence No. 3 correspond to the 367 th to 480 th bit sequences of sequence No. 11, and the 19 th to 480 th bit sequences of sequence No. 7 correspond to the 19 th to 480 th bit sequences of sequence No. 11. Further, the sequences at positions 1 to 18 in each of the sequence numbers 1, 3, 5, 7, 9 and 11 are all the same sequence.

As described later, these thrombomodulin of the present invention can be obtained from transformed cells prepared by transfecting DNA encoding these peptides (specifically, the base sequences such as SEQ ID NO.2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, or SEQ ID NO. 12) into host cells with a vector.

Further, these peptides may have the above amino acid sequence, and may have a sugar chain added thereto or not, and this point is not particularly limited. In addition, in gene manipulation, the kind, position or degree of addition of the sugar chain varies depending on the kind of host cell used, and it can be used arbitrarily. The binding position and type of the sugar chain are known from the disclosure of Japanese patent application laid-open No. 11-341990, and the same sugar chain may be added to the same position in the thrombomodulin of the present invention. The ratio of the fucosyl biantennary and fucosyl triantennary N-linked sugar chains bound to the thrombomodulin of the present embodiment is, for example, (100:0) to (60:40), preferably (95:5) to (60:40), and more preferably (90:10) to (70: 30). The ratios of these sugar chains can be measured by a two-dimensional sugar chain map described in Biochemical experiments experiment 23, glycoprotein parent-derived oligosaccharide interlocking study (Biochemical experiments 23, glycoprotein sugar chain study), academic Press center (1990), or the like. Further, when the sugar composition of the thrombomodulin of the present embodiment is examined, neutral sugars, amino sugars, and sialic acid are detected, and the proportions thereof are, independently, 1% to 30% by weight, preferably 2% to 20% by weight, and more preferably 5% to 10% by weight, based on the protein content. These sugar contents can be measured by the methods described in Experimental experiments on New chemical construction experiment No. seat 3 saccharine I-sugar タンパク (up) (New Biochemical experiment lecture No. 3 saccharine I-glycoprotein (up))), Tokyo chessin (1990) and the methods described in neutral sugar (phenol-sulfuric acid method, aminosugar (Morgan-Elsen method), sialic acid (periodate acid) -resorcinol method).

As described later, the thrombomodulin is obtained by gene manipulation, but when it is obtained by gene manipulation, the nucleotide sequence encoding the amino acid sequence at positions 1 to 18 of SEQ ID NO.9, the nucleotide sequence encoding the amino acid sequence at positions 1 to 16 of SEQ ID NO.9, or other known signal sequences (for example, the signal sequence of human tissue plasminogen activator (International publication No. 88/9811)) can be used as signal sequences that can be used for expression.

When the DNA sequence encoding thrombomodulin is introduced into a host cell, a method of introducing the DNA sequence encoding thrombomodulin into a vector (particularly preferably an expression vector which can be expressed in animal cells) is preferable. The expression vector refers to a DNA molecule comprising a promoter sequence, a sequence that imparts a ribosome binding site to mRNA, a DNA sequence encoding a protein to be expressed, a splicing signal, a terminator sequence for terminating transcription, a replication initiation sequence, and the like, and examples of preferable animal cell expression vectors include MulliganRC et al [ proc natl acadsiiusa 1981; 78: 2072-; 101:387-402, and pBP69T (69-6) reported by academic Press. In addition, there are other ways to introduce expression vectors capable of expression in microorganisms.

Examples of host cells that can be used for producing these peptides include animal cells. Examples of the animal cells include Chinese Hamster Ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCCCL-81) cells, BHK cells, MDCK cells derived from dog kidney, hamster AV-12-664 cells, and examples of the human cells include HeLa cells, WI38 cells, human 293 cells, and PER. C6 cells. CHO cells are generally highly preferred, and among CHO cells, dihydrofolate reductase (DHFR) deficient CHO cells are further preferred.

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

The thrombomodulin in the present embodiment may be produced by a conventionally known method or according to a conventionally known method, and for example, the method of the above-mentioned Shanben et al (Japanese patent application laid-open No. Sho 64-6219) or Japanese patent application laid-open No. Hei 5-213998 can be referred to. That is, the human thrombomodulin gene may be further modified as necessary after it is prepared, for example, as a DNA encoding the amino acid sequence of SEQ ID NO.9 by gene manipulation techniques. As such a modification, for example, in order to prepare a DNA encoding the amino acid sequence of SEQ ID NO. 11 (specifically, the base sequence of SEQ ID NO. 12), the codon encoding the 473 rd amino acid of SEQ ID NO.9 (specifically, the 1418 th base of SEQ ID NO. 10) is modified according to ZollerMJ et al [ Methodsinenzymology 1983; 468-500, academic Press. For example, a DNA obtained by converting the base T at position 1418 of SEQ ID NO.10 into the base C can be formed using a synthetic DNA for mutation having the base sequence represented by SEQ ID NO. 13.

The DNA thus prepared may be introduced into, for example, Chinese Hamster Ovary (CHO) cells to prepare transformed cells, which may be appropriately selected, and purified thrombomodulin may be prepared from a culture solution obtained by culturing the cells by a known method. As described above, it is preferable to transfect the DNA encoding the amino acid sequence of SEQ ID NO.9 (SEQ ID NO. 10) into the above host cell.

The method for producing thrombomodulin in the present embodiment is not limited to the above-mentioned method, and for example, it may be extracted and purified from urine, blood, other body fluids, etc., or from a tissue producing thrombomodulin, a tissue culture solution thereof, etc., or may be subjected to a cleavage treatment with a protease, if necessary.

When the transformed cells are cultured, a medium used in ordinary cell culture can be used, and it is preferable to culture the transformed cells in advance in various media and select the optimal medium. For example, a medium in which a known medium such as an MEM medium, a DMEM medium, or a 199 medium is used as a minimal medium and further modified or supplemented with various media can be used. Examples of the culture method include a serum culture in which the culture is performed in a medium to which serum is added, and a serum-free culture in which the culture is performed in a medium to which serum is not added. The culture method is not particularly limited, and serum-free culture is preferable.

In the serum culture, when serum is added to the medium, bovine serum is preferable. The bovine serum includes fetal bovine serum, newborn calf serum, adult bovine serum, and the like, and any bovine serum suitable for cell culture can be used. In the serum-free culture, a commercially available medium can be used as the serum-free medium. Serum-free media suitable for various cells are commercially available, for example, CD-CHO, CHO-S-SFMII and CHO-III-PFM are commercially available from Invitrogen for CHO cells, ISCHO and ISCHO-CD media are commercially available from Irvine scientific, and the like. These media may be used as they are, or they may be used with modifications or supplements. Further, as a serum-free medium, a DMEM medium supplemented with insulin, transferrin and selenious acid at 5mg/L, respectively, can be exemplified. As described above, the medium is not particularly limited as long as it can produce the thrombomodulin of the present embodiment. The culture method is not particularly limited, and any culture method such as batch culture, repeated batch culture, fed-batch culture, perfusion culture, and the like may be used.

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

Further, when soluble thrombomodulin is produced using a DNA having the base sequence of SEQ ID NO.10, a diversity of C-terminal amino acids may be found, and a peptide having 1 amino acid residue in a smaller amount may be produced. That is, the C-terminal amino acid may be modified as in the case where the amino acid at position 515 forms the C-terminus and the amino acid at position 515 is amidated. In addition, there are also cases where a peptide having 2 amino acid residues less is produced. That is, the amino acid at position 514 may form the C-terminus. Therefore, it is possible to prepare a peptide or a mixture thereof rich in diversity of the N-terminal amino acid and the C-terminal amino acid. Preferably, the amino acid at position 515 or the amino acid at position 516 forms the C-terminus, and more preferably, the amino acid at position 516 forms the C-terminus. In addition, there are other ways in which it is preferable that the amino acid at position 514 form the C-terminus. The above modifications, diversifications, and the like are also the same for the DNA having the base sequence of SEQ ID NO. 12.

The thrombomodulin obtained by the above method may be a mixture of peptides having diversity at the N-terminus and the C-terminus. Specifically, there can be mentioned a mixture of peptides having the sequence at the 19 th to 516 th positions, 19 th to 515 th positions, 19 th to 514 th positions, 17 th to 516 th positions, 17 th to 515 th positions or 17 th to 514 th positions in SEQ ID NO. 9.

Then, the method for isolating and purifying thrombomodulin from the culture supernatant or culture obtained as described above can be performed according to the conventional methods [ horiba tailing, U.S. Pat. No. 5,300,500 (basic experiments on protein-enzyme), 1981 ]. For example, it is also preferable to use a chromatographic carrier having a functional group having a charge opposite to that of thrombomodulin immobilized thereon, and ion exchange chromatography or adsorption chromatography utilizing an interaction between thrombomodulins. In addition, affinity chromatography utilizing specific affinity with thrombomodulin is also a preferred example. As a preferred example of the adsorbent, thrombin, which is a ligand of thrombomodulin, or an antibody against thrombomodulin, is used. As such an antibody, an antibody of thrombomodulin having an appropriate property or recognizing an appropriate epitope can be used, and examples thereof include those described in, for example, Japanese patent publication No. 5-42920, Japanese patent application laid-open No. 64-45398, Japanese patent application laid-open No. 6-205692, and the like. Further, gel filtration chromatography or ultrafiltration using the molecular weight of thrombomodulin can be mentioned. Further, there are a chromatographic carrier having a hydrophobic group immobilized thereon and a hydrophobic chromatogram utilizing hydrophobic bonding with a hydrophobic site possessed by thrombomodulin. Furthermore, hydroxyapatite can be used as a carrier for adsorption chromatography, and examples thereof include those described in Japanese patent application laid-open No. 9-110900. These methods may be appropriately combined. The degree of purification may be selected depending on the intended use, and is preferably such that a single band is obtained as a result of electrophoresis (preferably SDS-PAGE), or a single peak is formed as a result of gel filtration HPLC or reverse phase HPLC of the separated purified product. Of course, when two or more thrombomodulin are used, it is preferable that substantially only a band of thrombomodulin is formed, and it is not required that a single band is formed.

Specifically, the purification method of the present invention is exemplified by a method of purifying thrombomodulin using its activity as an index, and examples thereof include the following methods: the culture supernatant or the culture was roughly purified by Q-Sepharose FastFlow on an ion exchange column to recover a fraction having thrombomodulin activity, followed by main purification on a DIP-thrombin-agarose (dissopropylthrombinagarose) column on an affinity column to recover a fraction having strong thrombomodulin activity, the recovered fraction was concentrated, and a thrombomodulin active fraction was obtained in a pure form by gel filtration [ GomiKetal, Blood 1990; 75:1396-1399]. Examples of the thrombomodulin activity as an index include a promoting activity of thrombin-based activation of protein C. In addition, a preferred purification method is exemplified below.

Selecting proper ion exchange resin with good adsorption condition with thrombomodulin, and carrying out ion exchange chromatography purification. A particularly preferred example is a method using Q-Sepharose FastFlow equilibrated with 0.02mol/LTris hydrochloric acid buffer (pH7.4) containing 0.18 mol/LNaCl. After washing appropriately, the crude thrombomodulin can be obtained by elution with, for example, 0.02mol/LTris hydrochloric acid buffer solution (pH7.4) containing 0.3 mol/LNaCl.

Subsequently, for example, a substance having specific affinity for thrombomodulin may be immobilized on a resin and subjected to affinity chromatography purification. Preferable examples include a DIP-thrombin-agarose column and an anti-thrombomodulin monoclonal antibody column. The DIP-thrombin-agarose column is equilibrated in advance with, for example, 20mmol/L Tris hydrochloric acid buffer solution (pH7.4) containing 100mmol/L NaCl and 0.5mmol/L calcium chloride, and the above-mentioned crude purified product is filled, washed appropriately, and eluted with, for example, 20mmol/L Tris hydrochloric acid buffer solution (pH7.4) containing 1.0mol/L NaCl and 0.5mmol/L calcium chloride, whereby thrombomodulin of the purified product can be obtained. In addition, in the anti-thrombomodulin monoclonal antibody column, the following methods can be exemplified: a0.1 mol/L NaHCO3 buffer solution (pH8.3) containing 0.5mol/L NaCl in which an anti-thrombomodulin monoclonal antibody is dissolved is brought into contact with Sepharose 4FF (GEHealthcare Bio-Sciences) which has been activated beforehand by CNBr, and the column packed with the resin in which Sepharose 4FF is coupled to the anti-thrombomodulin monoclonal antibody is equilibrated beforehand with, for example, 20mmol/L phosphate buffer solution (pH7.3) containing 0.3mol/L NaCl, washed appropriately and then eluted with, for example, 100mmol/L glycine hydrochloride buffer solution (pH3.0) containing 0.3mol/L NaCl. The eluate is neutralized with an appropriate buffer solution to obtain a purified product.

The purified product thus obtained was adjusted to pH3.5, and then filled in a cation exchanger (preferably a strong cation exchanger SP-Sepharose FF (GEHealthcare Bio-Sciences)) equilibrated with 100mmol/L glycine hydrochloride buffer (pH3.5) containing 0.3mol/L NaCl, and washed with the same buffer to obtain a non-adsorbed fraction. The obtained fraction is neutralized with an appropriate buffer solution to obtain a purified product of high purity. They are preferably concentrated by ultrafiltration.

Further, it is preferable to perform buffer exchange by gel filtration. For example, a high purity purified product concentrated by ultrafiltration can be packed in a Sephacryl S-300 column or S-200 column equilibrated with 20mmol/L phosphate buffer solution (pH7.3) containing 50mmol/L NaCl, and the high purity purified product can be recovered by performing separation with 20mmol/L phosphate buffer solution (pH7.3) containing 50mmol/L NaCl, confirming the promoting activity of thrombin-based protein C activation, and recovering the active fraction to obtain a buffer-exchanged high purity purified product. The thus obtained high-purity purified product is preferably filtered with an appropriate virus-removing membrane, for example, Planova15N (asahi chemical corporation) for safety, and then concentrated by ultrafiltration to a desired concentration. Preferably, filtration is finally carried out through sterile filtration membranes.

According to the present invention, there is provided a drug for treatment and/or amelioration of sepsis, which contains thrombomodulin as an active ingredient, and which is to be administered to a severe sepsis patient having 1 or more kinds of organ dysfunction, wherein the value of the International Normalized Ratio (INR) of a plasma sample of the patient is a value greater than 1.4.

That is, the drug for use in the treatment and/or amelioration of sepsis in the present embodiment is a drug for use in the treatment and/or amelioration of sepsis in a severe sepsis patient who has 1 or more kinds of organ dysfunction and whose plasma sample has a value of INR of more than 1.4.

Furthermore, also provided is an agent for reducing the likelihood of death in a human patient with severe sepsis, the human patient with severe sepsis having 1 or more organ dysfunctions, the value of INR of a plasma sample of the patient being a value greater than 1.4.

As one of preferable effects of treatment and/or improvement of sepsis, for example, "prevention of death of a patient due to sepsis" can be mentioned. In addition, a preferable effect of "preventing deterioration of the general state of the patient due to sepsis" can be also given.

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

The sepsis in the present embodiment is Systemic Inflammatory Response Syndrome (SIRS) caused by infection. That is, in addition to the presence of infection, one may cite the SIRS project ((1) body temperature >38 ℃ or <36 ℃; (2) heart rate > 90/min; 3) respiratory rate > 20/min or PaCO2<32 torr; (4) a white blood cell count of >12,000/. mu.L or < 4000/. mu.L or immature white blood cells > 10%), and sepsis can be basically diagnosed based on the disease state.

There are several methods as a diagnostic method of sepsis. This is summarized in levym et al, crit.care.med., 31: 1250-. Examples of the method include a method of making the diagnosis by a doctor or a method of using a test value. As an example of the latter, the following method is included: at 1) body temperature >38 ℃ or <36 ℃; 2) heart rate > 90/min; 3) respiratory rate > 20/min or artificial respiration is necessary; 4) (ii) leukocyte count >12000/μ L or <4000/μ L, or blast cell > 10%; within these 4, SIRS was diagnosed when 2 was satisfied, and SIRS proved to be or suspected to be causative of microorganisms was diagnosed as sepsis [ larosa. Methods for approximating this are described in Mebersoftefuran college of chestPhysicans/SocietoyofCritical CareImedicineContonsConserence CritiCareMed, 20, 864-874 (1992).

Examples of the state of sepsis (sepsis) include bacteremia, sepsis (septicema), Systemic Inflammatory Response Syndrome (SIRS), sepsis (SIRS which is proved to be or suspected to be caused by microorganisms), severe sepsis, septic shock, refractory septic shock, and multiple organ dysfunction (hereinafter sometimes referred to as MODS) (harrison's internal science institute 15 th edition 124 item P828-833medical sciences international). The above conditions may be exemplified by the symptoms effective as the therapeutic agent and/or the ameliorating agent of the present invention.

The sepsis is not particularly limited as long as it satisfies the above diagnostic criteria, and sepsis associated with blood coagulation disorder (septicemia) is preferred. The blood coagulation disorder is not particularly limited as long as the INR of the plasma sample of the patient is greater than 1.2, preferably greater than 1.3, and more preferably greater than 1.4.

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

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

The Systemic Inflammatory Response Syndrome (SIRS) may be, for example, a state in which DIC is prepared as described above.

Examples of severe sepsis include sepsis accompanied by one or more of metabolic acidosis, organ hypoperfusion, acute encephalopathy, oliguria, hypoxemia, or organ dysfunction such as disseminated intravascular coagulation, or hypotension. In sepsis, a state in which organ dysfunction, organ hypoperfusion, or hypotension is present is called severe sepsis (severesepsilon). Organ hypoperfusion or perfusion abnormalities include lactic acidosis, oliguria, confusion, and the like. In severe sepsis, hypotension continues even when a sufficient infusion load is applied, and is called septic shock (septichcock).

The severe sepsis in the present embodiment is more specifically as follows.

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

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

As the Multiple Organ Dysfunction (MODS), there is a multiple organ dysfunction in which 1 or more organs are in functional failure and medical intervention is required to maintain homeostasis.

INR in the present embodiment is an examination index defining blood coagulation abnormality. INR refers to prothrombin time (hereinafter sometimes abbreviated as PT) normalized to the difference between manufacturing lots of a thromboplastin preparation, and is defined as follows.

INR value ═ clotting time (seconds) of the test sample/control sample ^ (ISI value)

In the formula, the blood coagulation time (sec) of the sample to be measured indicates PT of the plasma sample to be measured.

In addition, ISI represents the international susceptibility index.

As described above, the severe sepsis in the present embodiment can be exemplified by sepsis accompanied by one or two or more of metabolic acidosis, acute encephalopathy, oliguria, hypoxemia, disseminated intravascular coagulation and other organ dysfunction and hypotension symptoms. Severe refers to a serious condition at risk of life. Particularly, severe sepsis includes sepsis with 1 or more kinds of organ dysfunction. The organ dysfunction is not particularly limited, and may be any organ dysfunction that causes dysfunction due to sepsis, and is preferably organ dysfunction necessary for life maintenance. The 1 or more organ dysfunctions include 1 or more organ dysfunctions selected from the group consisting of circulatory organ disorders, respiratory organ disorders, renal dysfunction and liver dysfunctions, preferably 1 or more organ dysfunctions selected from the group consisting of respiratory organ disorders, circulatory organ disorders and renal dysfunctions, and more preferably 1 or more organ dysfunctions selected from the group consisting of respiratory organ disorders and circulatory organ dysfunctions. The number of organ dysfunctions is not particularly limited as long as it is1 or more, and two or more thereof may be preferred. In particular, it is preferable to have both of respiratory organ disorders and circulatory organ disorders.

The circulatory disorder is not particularly limited as long as it is generally known as a circulatory disorder, and examples thereof include a decrease in blood pressure and shock.

The respiratory disorder is not particularly limited as long as it is generally known as a respiratory disorder, and examples thereof include hypoxemia, acute lung injury, and dyspnea.

The renal dysfunction is not particularly limited as long as it is generally known as a renal dysfunction, and examples thereof include renal dysfunction, oliguria, and renal failure.

The liver function disorder is not particularly limited as long as it is generally known as a liver function disorder, and examples thereof include liver function disorder, jaundice, and liver failure.

These organ dysfunctions are generally known as described in the publications known before the application, "the blood loss disease peripheral interpretation promotion 12392, therapeutic (sepsis interpretation and therapeutic strategy)" (journal of medicine, p38, by yotakuwa 2006), or "survingsepsepsissepampaign: international hormone for administration of regenerative and septic shock 2008" (crittamed. 2008jan; 36 (327): 296-.

Since it is assumed that patients with severe sepsis are likely to be caused by factors other than sepsis, such as drug-induced organ dysfunction, it is preferable that patients with organ dysfunction including only the liver or the kidney are excluded. In addition, as a result of organ dysfunction, Thrombocytopenia (Thrombocytopenia) is known to occur. The number of platelets in a patient to whom the drug of the present embodiment is administered is not particularly limited as long as it is less than 30 ten thousand/. mu.L, and is preferably less than 20 ten thousand/. mu.L, and more preferably less than 15 ten thousand/. mu.L.

The value of INR of the plasma sample of the sepsis patient in the present embodiment is not particularly limited as long as it is greater than 1.4, and in the case where INR is greater than 1.4, thrombomodulin is more effective for sepsis patients having 1 or more kinds of organ dysfunction. The upper limit of INR is, for example, 2.0 or less, preferably 1.9 or less, more preferably 1.8 or less, further preferably 1.7 or less, and particularly preferably 1.6 or less. It is also preferably 1.5 or less in some cases. In addition, it is sometimes preferable not to include INR ═ 1.7.

Note that "INR greater than 1.4" may be sometimes referred to as "INR > 1.4".

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

Clinical symptoms of DIC vary depending on the type of underlying disease, and it is preferable to diagnose DIC by scoring DIC with several test values as described below and scoring at a certain or higher score, in addition to observing bleeding symptoms and organ symptoms. Examples of the test values include the number of platelets in blood, the concentration of plasmin-decomposed fibrin and fibrinogen decomposition products (hereinafter sometimes abbreviated as "FDP"), the concentration of D-dimer, the fibrinogen concentration, and the prothrombin time. Furthermore, diagnosis of DIC (advanced intravascular coagulation (DIC)) by lowering platelets, increasing the concentration of D-dimer or FDP, etc. without scoring DIC may be performed by using に Seki する report on investigation using diagnostic standard of Disseminated Intravascular Coagulation (DIC) for diagnosis of blood coagulation disorders of specific disorders in major life, in 11 years of research report, 1999:65-72, export study of について (study on early diagnosis of DIC for specific disorders in major life, in 11 years of research report, 1999:73-77, 1999, report on blood coagulation disorders of specific disorders in major life, 1999: アンケート in clinical study of DIC diagnosis, , 1999, 40:362-364).

In addition, in this embodiment, a sepsis patient whose INR value of a plasma sample of the patient is more than 1.2, preferably more than 1.3, and more preferably more than 1.4 may be referred to as a generalized DIC patient, and the drug for treatment and/or improvement of sepsis in this embodiment may be used as a drug for treatment and/or improvement of DIC in some cases.

The drug in this embodiment may be used for DIC in some cases. Sepsis is also localized to SIRS caused by a critical clinical invasion of infectivity, and is closely related to DIC which is a disease causing infectious diseases. DIC is often complicated in sepsis, and the drug of the present embodiment may be used for patients with such DIC complicated sepsis. That is, the drug of the present embodiment may be used for patients having or suspected of having either or both of DIC or sepsis.

INR in the present embodiment can be measured, for example, as follows. That is, tissue thromboplastin and Ca were added to plasma (sample to be measured) obtained by adding sodium citrate²⁺The time (PT) until coagulation (fibrin deposition) was measured, and the time was determined in seconds, and evaluated in terms of the relative ratio (activity ratio) to the control sample. The activity ratio is determined by "clotting time (seconds) of a sample to be tested/clotting time (seconds) of a control sample", and the difference in test value is caused by the test apparatus due to the difference in sensitivity of the tissue thromboplastin used. The INR value is conceived to eliminate such a difference, and by evaluating the PT with the INR value corrected by an international sensitivity index (hereinafter, sometimes abbreviated as ISI), the inter-equipment difference can be eliminated to obtain a result of the criterion. ISI represents how different from international standards. ISI is determined by the respective tissue thromboplastin reagent, which is attached to the reagent. Examples of the thromboplastin reagent include, but are not limited to, ThromborelS (registered trademark: manufactured by Sysmex) and Thromboplastin C + (registered trademark: manufactured by Sysmex). Human placenta thromboplastin (ISI value close to 1.0) was used in thrombobolis (registered trademark), and rabbit brain thromboplastin (ISI value of about 1.8) was used in thromboplastin c + (registered trademark).

ISI is given to the tissue thromboplastin reagent, and the INR value is calculated by the calculation formula of formula 1.

The control sample is not particularly limited as long as it is a commercially available normal human mixed plasma, and for example, a commercially available normal human mixed sodium citrate plasma commercially available from Kojin-bioco.

For the treatment of sepsis, the following basic treatments are generally performed based on the publicly known literature (survivingsepsis campaigns: international guide for management of sepsis and sepsis show: critcamed. 200836296-327, critcamed. 200332(3)1250-56), and thrombomodulin may be used in combination with other agents, but the combination is not limited to these agents.

In a case where hypotension continues even after the Central Venous Pressure (CVP) rises to a target value, dopamine may be administered to a patient with septic shock in order to increase the mean blood pressure to at least 60 mmHg. Also, in the case of dopamine in excess of 20 μ g/kg/min, other vasoconstrictors (typically norepinephrine) are sometimes administered.

For the treatment of sepsis-causing pathogenic bacteria, antibiotics are generally used. Selection of antibiotics requires a well-founded inference based on the suspected cause, clinical condition, knowledge of microorganisms, knowledge of a map of susceptibility common to a particular hospital building, and previous culture examination results.

The thorough normalization of blood glucose values in septic patients improves the clinical outcome of critically ill patients.

When an antibiotic substance is used, a sample such as blood, body fluid, or a wound can be examined, and a drug effective against pathogenic bacteria can be selected. For example, in septic shock or the like of unknown cause, gentamicin or tobramycin may be administered together with a third-generation cephalosporin. Further, vancomycin was added when resistant staphylococci or enterococci were suspected.

The dosage is generally adjusted by continuous intravenous injection of insulin so that the blood glucose level is maintained at 80 to 110mg/dL (4.4 to 6.1 mmol/L).

Corticosteroid therapy has shown efficacy in the treatment of sepsis and is therefore sometimes administered in supplemental doses.

Recombinant activated protein C (rhAPC; Drotrecogin α) may be administered to patients at high risk of death (APACHEII score ≧ 25, multiple organ failure due to sepsis, septic shock, and ARDS due to sepsis) without contraindication (bleeding, etc.).

Although the target patient is limited, concentrated red blood cells may be used for transfusion with Hb7.0g/dL to 9.0g/dL as a target.

Erythropoietin (erythropoetin (epo)) is sometimes administered to sepsis patients when erythropoiesis is impaired due to renal failure.

In severe sepsis, DVT may be administered prophylactically by administering low-dose heparin or low-molecular heparin.

The medicament of the present invention contains a carrier. As the carrier usable in the present invention, a water-soluble carrier is preferable, and usually, an isotonic agent, a buffer, a thickener, a surfactant, a preservative, a painless agent, a pH adjuster, and the like which are allowable as additives of medicines are preferable. For example, it can be prepared by adding sucrose, glycerin, and other pH regulators of inorganic salts as additives. Further, amino acids, salts, sugars, surfactants, albumin, gelatin and the like may be added as required as disclosed in Japanese patent application laid-open Nos. 1-6219 and 6-321805. The method of adding these is not particularly limited, and the following methods may be mentioned: in the case of freeze-drying, for example, a method in which a solution containing at least one selected from an immunosuppressant and a therapeutic agent for hematopoietic malignancy and a solution containing thrombomodulin are mixed and then the mixed additive is added, as is generally performed; or a method comprising mixing an additive in advance with at least 1 selected from an immunosuppressant dissolved in water, distilled water for injection or an appropriate buffer solution, and a therapeutic agent for hematopoietic malignancy, adding and mixing a solution containing thrombomodulin, preparing a solution by this method, and freeze-drying the solution. When the drug of the present invention is a drug obtained by combining the respective drug components, the respective drugs are preferably produced by adding a carrier by an appropriate production method. The drug of the present invention may be provided in the form of an injection solution, or may be provided in the form of a lyophilized preparation dissolved at the time of use.

In the preparation step, an ampoule or a Vial (visual) is filled with, for example, 0.5 to 10mL of a solution containing 0.1 to 10mg of thrombomodulin, water for injection, and an additive, and the mixture is prepared as an aqueous solution preparation for injection. Further, a method of preparing a lyophilized preparation by freezing and drying under reduced pressure can be exemplified.

The drug of the present invention is preferably administered by a non-oral administration method, for example, intravenous administration, intramuscular administration, subcutaneous administration, or the like. In addition, oral administration, intrarectal administration, intranasal administration, sublingual administration, and the like may be performed. When the drug of the present invention is a drug in which the respective drug components are combined, the respective drug components are preferably administered by an appropriate administration method.

When intravenous administration is carried out, a method of administering a desired amount at a time (intravenous bolus) or intravenous administration by drip may be mentioned.

From the viewpoint of short administration time, a method of administering a desired amount at a time (intravenous bolus) is preferable. In particular, patients with sepsis are in many cases urgent and administration in a short time is sometimes preferable. In the case of single administration, the time required for administration by a syringe generally varies, and the time required for administration varies depending on the amount of liquid to be administered, and may be, for example, 5 minutes or less, preferably 3 minutes or less, more preferably 2 minutes or less, further preferably 1 minute or less, and particularly preferably 30 seconds or less. The lower limit is not particularly limited, but is preferably 1 second or more, more preferably 5 seconds or more, and further preferably 10 seconds or more. The dose is not particularly limited, and may be any of the above-mentioned preferred doses. In addition, intravenous administration by instillation is preferable in terms of ease of keeping the blood level of thrombomodulin constant.

The 1-day dose of the drug of the present invention varies depending on the age, body weight, degree of disease, administration route, etc. of a patient, and usually the upper limit is preferably 20mg/kg or less, more preferably 10mg/kg or less, further preferably 5mg/kg or less, particularly preferably 2mg/kg or less, most preferably 1mg/kg or less, and the lower limit is preferably 0.001mg/kg or more, more preferably 0.005mg/kg or more, further preferably 0.01mg/kg or more, particularly preferably 0.02mg/kg or more, most preferably 0.05mg/kg or more, in terms of the amount of thrombomodulin.

The above-mentioned preferable dose is not particularly limited in the case of intravenous bolus injection, but the upper limit of the dose per 1 day is preferably 1mg/kg or less, more preferably 0.5mg/kg or less, further preferably 0.1mg/kg or less, particularly preferably 0.08mg/kg or less, and most preferably 0.06mg/kg or less, and the lower limit thereof is preferably 0.005mg/kg or more, more preferably 0.01mg/kg or more, further preferably 0.02mg/kg or more, and particularly preferably 0.04mg/kg or more.

When administered to a patient weighing more than 100kg, the blood volume is not proportional to the body weight, and it may be preferable to administer a fixed dose of 6mg from the viewpoint of a relative decrease in blood volume relative to the body weight.

In intravenous drip administration, the above-mentioned preferred dose is not particularly limited, but the upper limit of the dose per 1 day is preferably 1mg/kg or less, more preferably 0.5mg/kg or less, further preferably 0.1mg/kg or less, particularly preferably 0.08mg/kg or less, most preferably 0.06mg/kg or less, and the lower limit thereof is preferably 0.005mg/kg or more, more preferably 0.01mg/kg or more, further preferably 0.02mg/kg or more, particularly preferably 0.04mg/kg or more.

Administered 1 time every 1 day or several times as needed. The administration interval may be 1 time for 2to 14 days, preferably 1 time for 2to 7 days, and more preferably 1 time for 3 to 5 days.

[ description of sequence Listing ]

Sequence number 1: amino acid sequence coded by gene for producing TME456

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

Sequence number 3: amino acid sequence coded by gene for producing TME456M

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

Sequence number 5: amino acid sequence encoded by gene for producing TMD12

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

Sequence number 7: amino acid sequence encoded by gene for producing TMD12M

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

Sequence number 9: amino acid sequence encoded by gene for producing TMD123

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

Sequence number 11: amino acid sequence encoded by gene for producing TMD123M

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

Sequence number 13: synthetic DNA for mutagenesis used for site-directed mutagenesis

Examples

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

The thrombomodulin of the present invention used in the test examples was prepared according to the method of the above-mentioned Shanben et al (the method described in Japanese patent application laid-open No. Sho 64-6219). An example of the production thereof is shown below. The thrombomodulin obtained in the present production example was confirmed for safety by a single and repeated intravenous administration test using rats and monkeys, a mouse reproduction test, a local irritation test, a safety pharmacological test, a virus inactivation test, and the like.

Production example 1

< obtaining of thrombomodulin >

A thrombomodulin was obtained by transfecting DNA encoding the amino acid sequence of SEQ ID NO.9 (specifically, the base sequence of SEQ ID NO. 10) into Chinese Hamster Ovary (CHO) cells and obtaining a high-purity purified product from the culture broth of the transformed cells by the purification method of the conventional method described above, wherein the active fraction was recovered in 20mmol/L phosphate buffer (pH7.3) containing 50mmol/L of NaCl. Further, the solution was concentrated by ultrafiltration to obtain 11.0mg/mL of a thrombomodulin (in this specification, it may be abbreviated as TMD 123).

< Polysorbate solution preparation >

0.39g of polysorbate 80 was weighed into a glass beaker, and dissolved by adding 30mL of water for injection.

< preparation and filling of drug solution >

Into a 5L stainless steel container, 2239mL of the above-obtained TMD123 solution (equivalent to 24.63g based on the amount of soluble thrombomodulin protein, to which 5% was added in excess) was added. Further, the polysorbate solution obtained above was added thereto, and 27.9g of sodium chloride was added thereto. 600mL of water for injection was added and stirred. A1 mol/L hydrochloric acid solution was added to adjust the pH to 6.0. Further, water for injection was added to a total amount of 3940g, and the mixture was uniformly mixed and stirred. The resulting liquid medicine was filtered and sterilized by a filter having a pore size of 0.22 μm (MCGL 10S manufactured by Millipore). The filtrates were filled in ampoules with 1.1g each to give a TMD123 preparation.

Production example 2

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

Production example 3

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

Production example 4

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

Production example 5

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

Production example 6

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

Production example 7 preparation of placebo preparation

< Polysorbate solution preparation >

0.4g of polysorbate 80 was weighed into a glass beaker, and dissolved by adding 30mL of water for injection.

< preparation and filling of drug solution >

2000mL of water for injection was added to a 5L stainless steel container. Further, the polysorbate solution obtained above was added. Further, water for injection was added to a total amount of 4000g, and the mixture was uniformly mixed and stirred. The resulting liquid medicine was filtered and sterilized by a filter having a pore size of 0.22 μm (MCGL 10S manufactured by Millipore). The filtrates were filled into ampoules at 1.1g, respectively, to give placebo preparations.

[ example 1]

< test method >

TMD-123 manufactured according to manufacturing example 1 was used as thrombomodulin, and tests of randomized double-blind Placebo (Placebo) subjects were performed with sepsis and patients suffering from DIC as subjects. The total number of subjects was 750 patients, and 741 patients who had been administered test drugs (370 TMD-123 administration groups and 371 placebo administration groups) were tested. TMD-123 was administered 1 time 1 day at 0.06mg/kg for 6 days as a continuous bolus intravenous injection. As the placebo, a placebo prepared according to manufacturing example 7 was used.

In order to suppress the side effect manifestation due to overdosing, patients with a body weight of more than 100kg were given 6-day continuous iv boluses with 1 time per 1 day at a fixed dose of 6 mg.

The INR value in plasma of the patient before administration of the test drug was measured according to the method described in formula 1 above.

In addition, patients with organ dysfunction of only liver or kidney were removed from the subject patients. This is because, in a patient with organ dysfunction of only liver or kidney, it is assumed that organ dysfunction may be caused by factors other than sepsis, such as drug-induced organ dysfunction.

The conversion period (pellet ) at day 28 after the start of administration was confirmed, and the Mortality (Mortality) of each patient group was calculated.

In addition, the mortality Difference between TMD-123 and placebo was calculated as Difference (Difference).

< test results >

Among patients with sepsis without organ dysfunction, the Difference between the mortality rate when TMD-123 was given and the mortality rate when placebo was given was the greatest (6.1%) among patients with INR >1.5 in plasma before the administration of the test drug and the mortality rate when INR >1.6 was the next (4.5%). The mortality difference in INR >1.4 patients was around 1.7% and did not show as high a mortality difference absolutely or relatively (table 1).

That is, as is clear from Table 1, when the maximum of the lower limit value of INR is 1.5 to 1.6 and the lower limit value of INR is not more than 1.5 to 1.6, the difference in mortality is extremely reduced.

In contrast, in patients with severe sepsis having 1 or more organ dysfunctions selected from the group consisting of circulatory organ disorders, respiratory organ disorders, renal dysfunction and liver dysfunction, the mortality rate difference was the greatest (9.7%) in patients with INR >1.4 (instead of INR >1.5), and showed a high mortality rate difference absolutely or relatively compared to other INR lower limit values (table 2). Further, as a whole, in patients with severe sepsis having 1 or more kinds of organ dysfunction selected from the group consisting of circulatory organ disorders, respiratory organ disorders, renal dysfunction, and liver dysfunction, the mortality rate difference between TMD-123 and placebo was large compared to sepsis patients without organ dysfunction. (the former: 5.4%, the latter: -1.1%)

In addition, in patients with severe sepsis having 1 or more organ dysfunctions selected from the group consisting of circulatory organ disorders, respiratory organ disorders, renal dysfunction, and liver dysfunction, the mortality rate difference was 16.0% in patients with 1.4< INR ≦ 1.6, which absolutely showed significant values (table 3). As is clear from Table 3, the mortality difference was 10 to 12% for all the other INR upper limit values, and a remarkably high mortality difference was relatively exhibited.

On the other hand, in patients without organ dysfunction, a relatively prominent tendency was observed only in some parts, and the mortality rate difference obtained was at most 7.1% (patient group with 1.4< INR ≦ 1.7) (table 4).

[ TABLE 1]

[ TABLE 2]

[ TABLE 3]

[ TABLE 4]

Industrial applicability

The thrombomodulin-containing drug of the present invention is useful as a drug capable of effectively treating and/or improving sepsis in a severe sepsis patient (in which INR of a plasma sample of a patient is a value of more than 1.4).

Claims

1. Use of thrombomodulin for the preparation of a medicament for the treatment and/or amelioration of sepsis, which comprises thrombomodulin as an active ingredient, for administration to a severe sepsis patient who has more than 1 organ dysfunction and whose plasma sample has a value of the international normalized ratio INR of a value greater than 1.4.

2. The use as claimed in claim 1, wherein the medicament is for administration to critically ill septic patients having more than 1 organ dysfunction, the value of the international normalized ratio INR of the patient's plasma samples being a value greater than 1.4 and equal to or less than 1.6.

3. The use according to claim 1 or 2, wherein the severe sepsis patient is a severe sepsis patient excluding a sepsis patient having only organ dysfunction of liver or kidney.

4. The use of claim 1, wherein the medicament is for administration to a critically ill septic patient having more than 1 organ dysfunction selected from the group consisting of liver, kidney, respiratory organ and circulatory organ.

5. The use of claim 1 or 4, wherein the medicament is for administration to a critically ill septic patient having more than 1 organ dysfunction selected from the group consisting of respiratory organs and circulatory organs.

6. The use of claim 1 or 4, wherein the thrombomodulin is soluble thrombomodulin.

7. The use according to claim 1 or 4, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a DNA encoding the amino acid sequence set forth in SEQ ID NO.9 or SEQ ID NO. 11 into a host cell.

8. The use according to claim 1 or 4, wherein the thrombomodulin is a peptide of an amino acid sequence of (i-1) below, which is a peptide having a thrombomodulin activity,

(i-1) the amino acid sequence at positions 19 to 516 in the amino acid sequence of any one of SEQ ID NO.9 or SEQ ID NO. 11.

9. The use of claim 1 or 4, wherein the thrombomodulin is administered in an amount of 0.005mg/kg to 1mg/kg by intravenous bolus injection within 5 minutes.

10. The use according to claim 1 or 4, wherein the sepsis is severe sepsis accompanied by blood coagulation disorder.

11. The use of claim 4, wherein the circulatory dysfunction is shock.

12. The use of claim 1 or 4, wherein the organ dysfunction is caused by sepsis.

13. The use according to claim 1 or 4, wherein the medicament is for administration to patients with severe sepsis, excluding patients with a value of 1.7 for the international normalized ratio INR.

14. The use of claim 1 or 4, wherein the thrombomodulin is for intravenous administration to a patient.

15. Use of thrombomodulin for the preparation of a medicament for reducing the mortality of septic patients, which comprises thrombomodulin as an active ingredient, for administration to severe sepsis patients, who have more than 1 organ dysfunction, the value of the international normalized ratio INR of the patient's plasma sample being a value greater than 1.4.

16. The use of claim 15, wherein the medicament is for administration to critically ill septic patients having more than 1 organ dysfunction, the value of the international normalized ratio INR of the patient's plasma samples being a value greater than 1.4 and equal to or less than 1.6.

17. The use according to claim 15 or 16, wherein the severe sepsis patient is a severe sepsis patient excluding a sepsis patient having only organ dysfunction of liver or kidney.

18. The use of claim 15, wherein the medicament is for administration to a critically ill septic patient having more than 1 organ dysfunction selected from the group consisting of liver, kidney, respiratory organ and circulatory organ.

19. The use of claim 15 or 18, wherein the medicament is for administration to a critically ill septic patient having more than 1 organ dysfunction selected from the group consisting of respiratory organs and circulatory organs.

20. The use of claim 15 or 18, wherein the thrombomodulin is soluble thrombomodulin.

21. The use according to claim 15 or 18, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a DNA encoding the amino acid sequence set forth in SEQ ID NO.9 or SEQ ID NO. 11 into a host cell.

22. The use according to claim 15 or 18, wherein the thrombomodulin is a peptide of the amino acid sequence of (i-1) below, which is a peptide having a thrombomodulin activity,

23. The use of claim 15 or 18, wherein the thrombomodulin is administered in an intravenous bolus injection in an amount of 0.005mg/kg to 1mg/kg within 5 minutes.

24. Use according to claim 15 or 18, wherein sepsis is severe sepsis associated with coagulation disorders.

25. The use of claim 18, wherein the circulatory organ dysfunction is shock.

26. The use of claim 15 or 18, wherein the organ dysfunction is caused by sepsis.

27. The use of claim 15 or 18, wherein the medicament is for administration to patients with severe sepsis, excluding patients with a value of 1.7 for the international normalized ratio INR.

28. The use of claim 15 or 18, wherein the thrombomodulin is for intravenous administration to a patient.