WO2008038777A1 - Agent thérapeutique pour le syndrome de coagulation intravasculaire disséminée - Google Patents

Agent thérapeutique pour le syndrome de coagulation intravasculaire disséminée Download PDF

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WO2008038777A1
WO2008038777A1 PCT/JP2007/068998 JP2007068998W WO2008038777A1 WO 2008038777 A1 WO2008038777 A1 WO 2008038777A1 JP 2007068998 W JP2007068998 W JP 2007068998W WO 2008038777 A1 WO2008038777 A1 WO 2008038777A1
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thrombomodulin
plasma
seq
drug
concentration
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PCT/JP2007/068998
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English (en)
Japanese (ja)
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Kazuhisa Tsuruta
Yoshikazu Aoki
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Asahi Kasei Pharma Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/366—Thrombomodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

Definitions

  • the present invention relates to a therapeutic and / or ameliorating agent for disseminated intravascular blood coagulation syndrome or sepsis comprising thrombomodulin as an active ingredient, which has a higher therapeutic effect.
  • DIC Disseminated intravascular coagulation
  • a large amount of vascular coagulation-promoting substances are caused by tissue disorders in various diseases.
  • the blood coagulation system is extremely enhanced and small blood clots are generated in the blood vessels throughout the body, clogging the small blood vessels, thereby consuming insufficient platelets and coagulation factors to control bleeding. As a result, it is a disease that causes abnormal coagulation.
  • blood clotting factors accumulate in the microvessels of the kidney / lung and cause ischemia, often causing circulatory disturbance and shock.
  • Sepsis is known as a state in which an infection mainly caused by bacteria progresses and exhibits severe general findings. Sepsis is more commonly positioned as a systemic inflammatory response syndrome caused by a serious clinical invasion that is infectious. Dic caused by sepsis and infection is closely related, and DIC often accompanies sepsis.
  • thrombomodulin is known as a substance that specifically binds to thrombin and inhibits the blood clotting activity of thrombin and at the same time significantly enhances the protein C activation ability of thrombin, and has a strong blood clotting inhibitory action. It is known to have. It is known to prolong the coagulation time by thrombin and to suppress platelet aggregation by thrombin. Protein C plays an important role in the blood coagulation and fibrinolysis system. This protein is activated by the action of thrombin and becomes activated protein C.
  • Non-Patent Document 1 This activated protein C inactivates the active factor V and VIII of the blood coagulation factor in vivo, and is involved in the production of plasminogen activator with thrombolytic activity.
  • Non-Patent Document 1 thrombomodulin is said to be useful as an anticoagulant or thrombolytic agent by promoting the activation of protein C by this thrombin, and it is reported that it is effective for the treatment and prevention of diseases associated with hypercoagulation.
  • Non-patent Document 2 There are also reports on animal experiments.
  • thrombomodulin has been discovered and acquired as a glycoprotein expressed on vascular endothelial cells of various animal species including humans, and subsequently successfully cloned. That is, the gene of human thrombomodulin precursor including a signal peptide is cloned from a human lung cDNA library using a genetic engineering technique, and the entire gene sequence of thrombomodulin is analyzed to obtain a signal peptide (usually 18 amino acid residues). An amino acid sequence of 575 residues including a group is exemplified (Patent Document 1).
  • the mature thrombomodulin from which the signal peptide has been cleaved has an N-terminal region from the N-terminal side of the mature peptide (1-226 position: position indication when the signal peptide is considered to be 18 amino acid residues, the same applies hereinafter), 6 regions of EGF-like structure (227-462), O-type glycosylation region (463-498), transmembrane region (499-521), and cytoplasmic region (522-557) It consists of 5 regions and has the same activity as the full-length thrombomodulin (ie, the minimum activity unit). Among the 6 EGF-like regions, the region is mainly from the N-terminal side. It is known that this is the 4th, 5th and 6th EGF-like structure!
  • Soluble thrombomodulin exists. Soluble thrombomodulin may be prepared so as not to contain at least a part or all of the transmembrane region, for example, three regions of an N-terminal region, six EGF-like regions, and an O-type glycosylation region.
  • Soluble thrombomodulin consisting of only amino acids (ie consisting of amino acid sequences 19 to 516 of SEQ ID NO: 9) can be obtained by application of recombinant technology, and its recombination Body soluble thrombomodulin has been confirmed to have the activity of natural thrombomodulin! (Patent Document 1).
  • Other examples of soluble thrombomodulin! / And some reports Patent Documents 2 to 9).
  • Another example is a soluble thrombomodulin derived from human urine as a natural type (Patent Documents 10 and 11).
  • thrombomodulin has been reported to be effective in the treatment of DIC (Non-patent Document 5).
  • thrombomodulin is used for other purposes such as acute coronary syndrome (ACS), thrombosis, peripheral vascular occlusion, obstructive arteriosclerosis, vasculitis, functional disorders secondary to cardiac surgery, and organ transplant complications.
  • Angina pectoris transient cerebral ischemic attack, pregnancy toxemia, diabetes, liver VOD (Liver veno-occlusive disease), deep vein thrombosis (DVT) venous thrombosis) and the like, and adult respiratory distress syndrome (ARDS);) 3 ⁇ 4 It is expected to be used for the treatment and prevention of disease.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 64-6219
  • Patent Document 2 JP-A-2-255699
  • Patent Document 3 Japanese Patent Laid-Open No. 3-133380
  • Patent Document 4 Japanese Patent Laid-Open No. 3-259084
  • Patent Document 5 Japanese Patent Laid-Open No. 4 210700
  • Patent Document 6 Japanese Patent Laid-Open No. 5-213998
  • Patent Document 7 WO92 / 00325
  • Patent Document 8 WO92 / 03149
  • Patent Document 9 W093 / 15755
  • Patent Document 10 Japanese Patent Laid-Open No. 3-86900
  • Patent Document 11 Japanese Patent Laid-Open No. 3-218399
  • Non-Patent Document 1 Koji Suzuki, History of Medicine, Vol. 125, 901 (1983)
  • Non-Patent Document 2 K. Gomi et al. Blood 75. 1396-1399 (1990)
  • Non-Patent Document 3 M. Zushi et al., J. Biol. Chem., 246, 10351-10353 (1989)
  • Non-Patent Document 4 D. Z. Wen et al., Biochemistry, 26, 4350—4357 (1987)
  • Non-Patent Document 5 S. M. Bates et al., Br. J. Pharmacol., 144, 1017-1028 (200
  • An object of the present invention is to provide a therapeutic and / or ameliorating agent for DIC or sepsis having a higher therapeutic effect, or a method for treating and / or improving the same.
  • anticoagulant therapy with an anticoagulant or supplementation with concentrated platelets' fresh frozen plasma is generally performed. Since the main form of DIC is excessive activation of the blood coagulation system, anticoagulant therapy to suppress it is widely performed in parallel with the treatment for the underlying disease! Heparin and / or AT preparations are often used to treat and / or improve DIC! In addition, antibiotics and / or activated protein C (only in countries approved as pharmaceuticals) are often used to treat and / or improve sepsis. In sepsis associated with DIC, a drug selected from the above drugs or a combination of these drugs is often used.
  • the inventors of the present invention have found that when thrombomodulin is administered to these patients, the plasma thrombomodulin concentration is maintained at 200 ng / ml or 1.3 units / ml or more, thereby showing high efficacy. Furthermore, the present inventors have found that when thrombomodulin is administered to these patients, the effectiveness of maintaining the plasma prothrombinase activity inhibition rate is maintained at 85% or higher, and the present invention is completed. It was.
  • the present invention provides a therapeutic and / or ameliorating agent for DIC or sepsis having a higher therapeutic effect, or a method for treating and / or improving the same. Deliver power S.
  • the present invention has been completed by a test using humans and careful analysis of the results, and cannot be envisaged by those skilled in the art.
  • the present invention includes the following.
  • a drug containing thrombomodulin as an active ingredient which is administered to a patient with disseminated intravascular blood coagulation syndrome or a sepsis patient to treat and / or improve disseminated intravascular blood coagulation syndrome or sepsis
  • a drug comprising thrombomodulin as an active ingredient which is administered to a patient with disseminated intravascular blood coagulation syndrome to treat and / or improve disseminated intravascular blood coagulation syndrome, Plasma thrombomodulin concentration is at least 200 ng / ml or more, or at least 1.3 units / ml or more, or plasma prothrombinase activity inhibition rate is low Both drugs for being maintained above 85%;
  • a drug comprising thrombomodulin as an active ingredient, wherein the drug is administered to a patient with disseminated intravascular blood coagulation syndrome to treat and / or improve the disseminated intravascular blood coagulation syndrome.
  • a drug containing thrombomodulin as an active ingredient which is administered to a patient with disseminated intravascular blood coagulation syndrome to treat and / or improve disseminated intravascular blood coagulation syndrome.
  • a drug comprising thrombomodulin as an active ingredient, wherein the drug is administered to a patient with disseminated intravascular blood coagulation syndrome to treat and / or improve disseminated intravascular blood coagulation syndrome.
  • the thrombomodulin is an amino acid represented by SEQ ID NO: 1, 3, 5, 7, 9 or SEQ ID NO: 11. [1] to [2] above, which is a peptide obtained from a transformed cell prepared by transfecting a DNA encoding an acid sequence into a host cell;
  • [3-2] A peptide obtained by a transformed cell prepared by transfecting a DNA encoding the amino acid sequence of the thrombomodulin S, SEQ ID NO: 1 or SEQ ID NO: 3 into a host cell, The drug according to any one of [1] to [2];
  • [3-3] A peptide obtained by a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of the thrombomodulin S, SEQ ID NO: 5 or SEQ ID NO: 7, The drug according to any one of [1] to [2];
  • [3-4] A peptide obtained by a transformed cell prepared by transfecting a DNA encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11 into a host cell, [1] The drug according to any one of [2];
  • thrombomodulin S a peptide having a sequence of positions 19 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3, or a peptide having a homologous mutation sequence of the above sequence and having a thrombomodulin activity [ 1] to [2], or a drug according to any one of [3-2];
  • the thrombomodulin linker according to any one of the above [1] to [2] or [3-2], which is a peptide having a sequence at positions 19 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3. Drugs;
  • thrombomodulin S a peptide having a sequence at positions 19 to 480 in each of SEQ ID NO: 5 or SEQ ID NO: 7, a peptide having a homologous mutant sequence of the above sequence and having a thrombomodulin activity, or their [1] to [2], or [3-3]! /, which is any one of a mixture;
  • thrombomodulin S a peptide having a sequence of positions 19 to 480 in each of SEQ ID NO: 5 or SEQ ID NO: 7, or a peptide having a homologous mutation sequence of the above sequence and having a thrombomodulin activity [1] to [2] or the drug according to any one of [3-3];
  • the thrombomodulator a peptide having a sequence of positions 19 to 516 in each of SEQ ID NO: 9 or SEQ ID NO: 11, a peptide having a sequence of positions 19 to 515 in each, and the amino acid sequence of the peptide
  • a peptide having thrombomodulin activity having an amino acid sequence in which one or more amino acids are substituted, deleted or added, a peptide having a homologous variant of the above sequence and having thrombomodulin activity, or a mixture thereof
  • the drug according to any one of [1] to [2] or [3-4], which is: [6-2] The 19th to 516th positions in each of the thrombomodulator, SEQ ID NO: 9 or SEQ ID NO: 11, respectively.
  • a peptide having a sequence, a peptide having a sequence of positions 19 to 515, and one or more amino acids in the amino acid sequence of the peptide are substituted
  • a peptide having an amino acid sequence deleted, added, or having thrombomodulin activity, or the above sequence The agent according to any one of [1] to [2] or [3-4], which is a peptide having a homologous mutation sequence of thrombomodulin activity;
  • thrombomodulin having 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in each of SEQ ID NO: 9 or 11
  • [6-5] Having a thrombomodulator, 19-516, 19-515, 17-516, or 17-515 in each of SEQ ID NO: 9 or 11
  • the drug according to any one of [1] to [2] or [3-4], which is a peptide to be synthesized, or a mixture thereof;
  • the thrombomodulin a peptide comprising the sequence of positions 19 to 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515 in IJ number 9 [1 ] ⁇ [2]
  • the thrombomodulin linker ⁇ ⁇ ⁇ Peptides consisting of sequences 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in IJ No. 11, or a mixture thereof [6] of [1] to [2] or [3-4] which is a compound, or a drug according to any one of the above; [6-11]
  • the thrombomodulin linker The drug according to any one of the above [1] to [2] or [3-4], which is a peptide comprising the sequence at position 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515 ;
  • [7] The drug according to any one of [1] to [611] above, wherein the drug is a drug for maintaining a plasma thrombomodulin concentration of at least 200 ng / ml for 24 hours or more.
  • Drug [7-2] The drug according to any one of [1] to [7] above, wherein the drug is a drug for maintaining plasma thrombomodulin concentration of at least 200 ng / ml for 72 hours or longer.
  • the drug is a drug for maintaining plasma thrombomodulin concentration of at least 1.3 units / ml for 144 hours or longer. Or the drug according to any one of [92];
  • [0023] [11] The above-mentioned [1] to [6 11], which is a drug for maintaining the plasma prothrombinase activity inhibition rate at least 85% or more for 24 hours or more.
  • [0025] A method of administering thrombomodulin to a disseminated intravascular blood coagulation syndrome patient or a sepsis patient, wherein the plasma thrombomodulin concentration is at least 200 ng / ml or more.
  • the plasma thrombomodulin concentration is at least 200 ng / ml or more.
  • [3 ′] a peptide obtained from a transformed cell prepared by transfecting a host cell with DNA encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11
  • the administration method is a method of administration by maintaining the plasma thrombomodulin concentration at least 200 ng / ml for 72 hours or more. Investment method as described in;
  • [6 ′] The method according to any one of [1 ′] to [5 ′] above, wherein the administration method is an administration method by maintaining a plasma thrombomodulin concentration of at least 200 ng / ml for 144 hours or more. Administration method;
  • the plasma thrombomodulin concentration is at least 1.3 units / ml or more
  • the administration method has a plasma prothrombinase activity inhibition rate of at least 85% or more.
  • the administration method has a plasma prothrombinase activity inhibition rate of at least 85% or more.
  • the administration method according to any one of [1 ′] to [4 ′] or [9 ′], which is an administration method by maintaining at least 144 hours at a time;
  • the symptoms of DIC patients or septic patients can be effectively treated and / or ameliorated, and according to the method of the present invention, DI C or sepsis is effective. Treatment and / or improvement.
  • DIC As a treatment and / or improvement of DIC, for example, “Preventing death of a patient due to DIC” is one of the preferable effects, and “Preventing deterioration of the patient's general condition by DIC” is also a preferable effect. As mentioned.
  • DIC vascular coagulation-promoting substances flow out in large quantities due to tissue damage in various diseases, the function of the coagulation system is extremely enhanced, and small thrombi develop in the blood vessels of the whole body (microthrombus formation) It is a disease or syndrome that causes abnormal hemostasis as a result of clogging of small blood vessels and the consumption and lack of platelets and coagulation factors necessary to control bleeding. Specifically, intravascular fibrin formation may cause bleeding symptoms due to consumable coagulation and fibrinolysis and organ failure symptoms due to microthrombosis. DIC is sometimes called disseminated intravascular coagulation syndrome or generalized intravascular coagulation syndrome.
  • DIC is an extension of systemic inflammatory response j ⁇ , Ski group (Sytemic Inflammatory Response Syndrome SI RS) caused by severe clinical invasion regardless of infectivity or non-infectivity. It may be caused by.
  • SIRS is a pathological condition caused by increased production of inflammatory site force-in associated with severe tissue injury or severe infection, and when this reaction is excessive, neutrophil activation and vascular endothelial cells are induced. Activation of the cell causes DIC. That is, it is a DIC preparation stage (hereinafter may be abbreviated as preDIC), and the treatment and / or amelioration agent of the present invention can be used effectively in preDIC, and the treatment and / or amelioration agent of the present invention can be used. Includes treatment and / or improvement of preDIC.
  • Diagnosis of DIC includes DIC scores based on the following test values in addition to observation of bleeding symptoms and organ symptoms. Diagnosis of DIC is preferable when a certain score is reached. Test values include, for example, blood platelet count, fibrin degraded by plasmin, fibrinogen degradation product (hereinafter abbreviated as FDP) concentration, D-dimer concentration, fibrinogen concentration, or prothrombin time. It is done. In addition, it can be diagnosed as preDIC without DIC score, such as decreased platelets, D-dimer, or increased FDP concentration (Masao Nakagawa.
  • DIC Disseminated Intravascular Coagulation
  • Diagnostic criteria for attaching DIC scores include, for example, Overt DIC diagnostic criteria (Taylor FB et al., Thromb. Haemost. 2001: 86: 1327-1330), acute DIC diagnostic criteria (Gando S. et al., Clin Appl Thromb Hemost. 2005: 11 (1): 71-76), or revision of the DIC diagnostic criteria of the Ministry of Health and Welfare (Nobuo Aoki, Atsushi Hasegawa: “Auxiliary test results and findings for diagnosis” in the DIC diagnostic criteria. Specific example of the Ministry of Health and Welfare specific disease blood coagulation disorder investigation research group 1992 research report 1988: p37-41).
  • the DIC diagnostic method is not particularly limited as long as it is the above-described diagnostic method, but a diagnostic method with a DIC score is preferred. In addition, there is another mode in which preDIC diagnosis is preferred when treatment from the viewpoint of therapeutic effect, medical costs, or patient QOL requires early treatment for DIC. [0039] As a diagnostic method for assigning a DIC score, in addition to the above-described diagnostic methods, a diagnostic method for assigning a similar DIC score is also possible.
  • the application of the drug of the present invention is not particularly limited to the type of DIC, but is more preferably applied to DIC derived from hematopoietic malignant tumor or DIC derived from infectious disease, and applied to DIC derived from infectious disease Is more preferable. Another preferred embodiment is application to DIC derived from a hematopoietic malignant tumor.
  • the drug of the present invention can also be used for sepsis. Sepsis is also positioned as SIRS caused by infectious and serious clinical invasion, and is closely related to DIC that causes infection. DIC often accompanies sepsis, and the drug of the present invention can also be used for septic patients with DIC. That is, in the present invention, it can be used for patients suffering from DIC or sepsis, or both, or patients suspected of suffering.
  • Sepsis is caused by treatments for diseases such as infections, malignant tumors, cirrhosis, renal failure, diabetes, abnormal labor, and injuries such as indwelling catheters, infusion devices, dialysis, and tracheostomy.
  • diseases such as infections, malignant tumors, cirrhosis, renal failure, diabetes, abnormal labor, and injuries
  • injuries such as indwelling catheters, infusion devices, dialysis, and tracheostomy.
  • systemic shock is triggered by septic shock, that is, rapid drop in blood pressure, peripheral circulatory failure, and damage to vital organs such as the lungs, kidneys, liver, heart, gastrointestinal tract, and even the central nervous system. Death from disability.
  • DIC CAD and pulmonary capillary injury associated with neutrophil activation and migration accumulation in the lung parenchyma, resulting in pulmonary interstitial edema, bleeding and acute respiratory failure.
  • Distress syndrome (ARDS) is induced and the prognosis is very poor.
  • the status of septic patients includes, for example, bacteremia, septicemia (s-significant ticemia), systemic inflammatory response syndrome (SIRS), sepsis (SIRS in which microorganisms have been proven or suspected), serious disease Sepsis, septic shock, refractory septic shock, or multiple organ dysfunction
  • MODS Harmonic Internal Science, 15th edition, 124th paragraph, P828-833 Medicano Science Inc. International.
  • MODS Hard Internal Science, 15th edition, 124th paragraph, P828-833 Medicano Science Inc. International.
  • Each of the above conditions is exemplified as a symptom in which the therapeutic and / or ameliorating agent of the present invention is effective.
  • the bacteremia is exemplified by a state in which the presence of bacteria in the blood, which is proven to be positive in blood culture, is confirmed.
  • septicemia examples include conditions in which the presence of microorganisms or other toxins in the blood is confirmed.
  • SIRS Systemic inflammatory response syndrome
  • Examples of severe sepsis include sepsis with one or more symptoms of organ failure or hypotension such as metabolic acidosis, acute encephalopathy, oliguria, hypoxemia or disseminated intravascular coagulation.
  • septic shock examples include those with hypotension (blood pressure 90 mmHg or less or 40 mm Hg or less than normal blood pressure) that do not respond to resuscitation with fluid replacement and are associated with organ failure.
  • refractory septic shock examples include those in which septic shock persists for more than 1 hour and does not respond to fluid replacement vasopressors.
  • MODS multi-organ dysfunction
  • Staphylococci, enterococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, enteropactor, etc. are mainly observed as causative bacteria of sepsis. These bacterial infections show high fever, chills, tachycardia, and strong systemic symptoms, often leading to confirmed infection in arterial venous blood, cerebrospinal fluid, and bone marrow fluid. In addition to infectious bacteria that have invaded the body, sepsis-causing bacteria include E.
  • LPS lipopolysaccharide
  • the causative bacteria are detected, their sensitivity to antibiotics is measured, and then the most appropriate antibiotic is administered to the causative bacteria, and diuresis, fluid replacement, electrolyte correction, It is necessary to focus on host defenses such as improvement of hypoproteinemia, nutritional supplementation, and administration of ⁇ -globulin [Katu M., Encyclopedia of Medical Science, 37: 263-265 (1984). ].
  • the evaluation method is not particularly limited! /, But various effectiveness by doctors (for example, improvement of bleeding symptoms) Degree, organ symptom improvement degree), various test values (for example, coagulation test values), those obtained by scoring those test values, or a combination of the above.
  • various blood coagulation test values FDP is performed to grasp the enhanced state of blood coagulation system and fibrinolytic system in the living body, but in particular, DIC and various thrombotic diseases are diagnosed, pathologic condition, and therapeutic effect. It is useful as an index for determination. In DIC patients, FDP generally decreases with improvement of the high symptoms.
  • the thrombomodulin in the present invention has an action of (1) selectively binding to thrombin and (2) promoting the activation of protein C by thrombin. (3 It is preferable that the effect of (1) prolonging the coagulation time by thrombin and / or (4) the effect of inhibiting platelet aggregation by thrombin is usually observed. These actions of thrombomodulin are sometimes called thrombomodulin activity.
  • the thrombomodulin activity has the actions of (1) and (2) above, and (1) to (1) above.
  • the action of accelerating the activation of protein C by thrombin is, for example, the activation of protein C by a test method clearly described in various known literatures such as JP-A-64-6219. It is possible to easily confirm the active amount and the presence or absence of the action of promoting the action. Similarly, it can be easily confirmed whether it has an action of prolonging the coagulation time by thrombin and / or an action of suppressing platelet aggregation by thrombin.
  • the thrombomodulin in the present invention is not particularly limited as long as it has thrombomodulin activity, but is preferably a soluble thrombomodulin.
  • Preferred examples of the solubility of soluble thrombomodulin include water (eg, in the absence of a surfactant such as Triton X-100 and polidocanol, usually near neutrality) in water such as distilled water for injection. mg / ml or more, or 10 mg / ml or more, preferably 15 mg / ml or more, or 17 mg / ml or more, more preferably 20 mg / ml or more, 25 mg / ml or more, or 30 mg / ml or more.
  • soluble thrombomodulin could be dissolved, it is clear when dissolved, for example, when observed with the naked eye at a brightness of about 1000 lux, just under a white light source. It is understood that the absence of an insoluble material to the extent that it is clearly recognized is a straightforward indicator. Moreover, it can also filter and can confirm the presence or absence of a residue.
  • the thrombomodulin in the present invention is known as the central site of thrombomodulin activity in human thrombomodulin! /, And includes the amino acid sequence at positions 19 to 132 of SEQ ID NO: 1. That is, it is not particularly limited as long as it includes the amino acid sequence at positions 19 to 132 of SEQ ID NO: 1.
  • the amino acid sequence at positions 19 to 132 of SEQ ID NO: 1 may be naturally or artificially mutated as long as it has an action of promoting the activation of protein C by thrombin, ie, thrombomodulin activity.
  • Number 1 th One or more amino acids in the amino acid sequence at positions 19 to 132 may be substituted, deleted, or added.
  • the degree of mutation allowed is not particularly limited as long as it has thrombomodulin activity.For example, 50% or more homology is exemplified as an amino acid sequence, and 70% or more homology is preferred. 90% or higher homology is more preferable 95% or higher homology is particularly preferable 98% or higher homology is most preferable. Such amino acid sequences are called homologous mutation sequences. As described later, these mutations can be easily obtained by using ordinary gene manipulation techniques.
  • the sequence of SEQ ID NO: 3 is a force in which Val, which is an amino acid at position 125 in the sequence of SEQ ID NO: 1, is mutated to Ala.
  • Val which is an amino acid at position 125 in the sequence of SEQ ID NO: 1
  • amino acids at positions 19 to 132 of SEQ ID NO: 3 It is also preferable to include arrays! /.
  • the thrombomodulin in the present invention includes SEQ ID NO: 1 or SEQ ID NO:
  • Preferred examples include peptides comprising the 19th to 132nd positions or the 17th to 13th positions in each of SEQ ID NO: 1 or SEQ ID NO: 3; and a peptide having a thrombomodulin activity comprising at least a homologous variant of the above sequence.
  • Peptides consisting of the 19th to 132nd positions of SEQ ID NO: 1 or SEQ ID NO: 3 are more preferred.
  • a peptide consisting of a homologous mutant sequence at positions 19 to 132 or positions 17 to 132 in SEQ ID NO: 1 or SEQ ID NO: 3 and having at least thrombomodulin activity is more preferable.
  • the amino acid sequence at positions 19 to 480 of SEQ ID NO: 5 which preferably includes the amino acid sequence at positions 19 to 480 of SEQ ID NO: 5, It is not particularly limited as long as it is included.
  • the amino acid sequence at positions 19 to 480 of SEQ ID NO: 5 may be a homologous mutant sequence as long as it has an action of promoting the activation of protein C by thrombin, ie, thrombomodulin activity.
  • the sequence of SEQ ID NO: 7 is a force in which Val, which is the amino acid at position 473 of the sequence of SEQ ID NO: 5, is mutated to Ala.
  • the thrombomodulin in the present invention is the amino acids at positions 19 to 480 of SEQ ID NO: 7. It is also preferable to include arrays! /.
  • the thrombomodulin in the present invention has at least the ligated IJ at positions 19 to 480 of SEQ ID NO: 5 or 7 or a homologous mutant sequence thereof, and has at least thrombomodulin activity. Including the peptide sequence! /, Not particularly limited!
  • a peptide comprising the sequence from positions 19 to 480 or positions 17 to 480 in SEQ ID NO: 5 or SEQ ID NO: 7, respectively, or the above Peptides comprising a homologous sequence of the sequence and having at least thrombomodulin activity can be mentioned as a preferred example, and peptides consisting of the 19th to 480th positions of SEQ ID NO: 5 or SEQ ID NO: 7 are more preferred.
  • a peptide consisting of a homologous mutant sequence at positions 19 to 480 or positions 17 to 480 in SEQ ID NO: 5 or 7 and having at least thrombomodulin activity is more preferable.
  • the amino acid sequence at positions 19 to 515 of SEQ ID NO: 9 preferably includes the amino acid sequence at positions 19 to 515 of SEQ ID NO: 9. It is not particularly limited as long as it is included.
  • the amino acid sequence at positions 19 to 515 of SEQ ID NO: 9 may be a homologous mutant sequence as long as it has an action of promoting the activation of protein C by thrombin, ie, thrombomodulin activity.
  • the sequence of SEQ ID NO: 11 is a force in which Val, which is the amino acid at position 473 of the sequence of SEQ ID NO: 9, is mutated to Ala.
  • Val which is the amino acid at position 473 of the sequence of SEQ ID NO: 9
  • thrombomodulin the sequence from positions 19 to 515 of SEQ ID NO: 11 is used. It is also preferable to include the amino acid sequence! /.
  • a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9 is particularly preferable. A mixture of these is also a preferred example.
  • positions 19 to 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515, IJ force, Ranaru Petit there is also another embodiment in which the door is particularly preferred. Mixtures of these are also preferred examples.
  • a peptide comprising these homologous mutant sequences and having at least thrombomodulin activity is also preferred.
  • the peptide having a homologous mutation sequence is as described above.
  • One or more of the amino acid sequence of the target peptide that is, one or more amino acids, more preferably several (for example, 1 to 20) And preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3 amino acids) which may be substituted, deleted or added.
  • the degree of mutation allowed is not particularly limited as long as it has thrombomodulin activity.For example, 50% or more homology is exemplified as an amino acid sequence, and 70% or more homology is preferred. 90% or higher homology is more preferable, 95% or higher homology is particularly preferable, and 98% or higher homology is most preferable.
  • the thrombomodulin in the present invention includes a peptide consisting of the sequence of SEQ ID NO: 14 (462 amino acid residues) and a sequence of SEQ ID NO: 8 (272 amino acid residues) in JP-A-64-6219.
  • Peptides or peptides consisting of the sequence of SEQ ID NO: 6 (236 amino acid residues) are also preferred examples.
  • the thrombomodulin in the present invention is not particularly limited as long as it is a peptide having at least the 19th to 132nd amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and among them, SEQ ID NO: 5 or SEQ ID NO: 7 is preferably a peptide having at least the 19th to 515th amino acid sequences of SEQ ID NO: 9 or SEQ ID NO: 11 Is more preferable.
  • the peptide having at least the amino acid sequence of positions 19 to 515 of SEQ ID NO: 9 or SEQ ID NO: 11 includes positions 19 to 516 and positions 19 to 515 of SEQ ID NO: 9 or SEQ ID NO: 11, respectively.
  • a more preferred example is a peptide consisting of the sequence at positions 17-516 or 17-515. Further, in each of SEQ ID NO: 9 or SEQ ID NO: 11, the peptide consisting of positions 19-516, 19-515, 17-516, or 17-515, IJ force, A mixture of SEQ ID NO: 9 or SEQ ID NO: 11 is also a more preferable example.
  • the position 17 in each of SEQ ID NO: 9 or SEQ ID NO: 11 (30:70)-(50:50) are exemplified as the mixing ratio of the peptide starting from the 19th position and the peptide starting from the 19th position, and (35:65)-(45:55) are preferred! /, As an example Can be mentioned.
  • Examples of the mixing ratio of the peptide ending at position 515 and the peptide ending at position 516 in SEQ ID NO: 9 or 11 are (70:30) to (90:10), (75 : 25) to (85: 15) are preferred! /, For example.
  • the mixing ratio of these peptides can be determined by an ordinary method.
  • the 19th to 132nd sequence of SEQ ID NO: 1 corresponds to the 367th to 480th sequence of SEQ ID NO: 9
  • the 19th to 480th sequence of SEQ ID NO: 5 is the sequence of SEQ ID NO: 9. It corresponds to the 19th to 480th array.
  • the 19th to 132nd sequences of SEQ ID NO: 3 correspond to the 367th to 480th positions of IJ No. 11 to the 367th to 480th IJ Nos.
  • the 480th rank IJ is equivalent to the 19th to 480th sequence of IJ No. 11.
  • sequences at positions 1 to 18 in each of SEQ ID Nos: 1, 3, 5, 7, 9, and 11 are all the same sequence.
  • the thrombomodulin in the present invention is a DNA encoding a peptide such as SEQ ID NO: 1, 3, 5, 7, 9, or 11 (specifically, SEQ ID NO: 2, SEQ ID NO: 4, respectively) It can be obtained from a transformed cell prepared by transfecting a host cell with a vector such as SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12).
  • these peptides only have to have the amino acid sequence described above, and this point is not particularly limited, whether or not a sugar chain is attached.
  • the type of sugar chain, the addition position and the degree of addition differ depending on the type of host cell to be used, and any of them can be used.
  • the bonding position and type of sugar chain the facts described in JP-A-11-341990 are known, and the same sugar chain may be added at the same position for thrombomodulin in the present invention.
  • the signal sequence that can be used for expression is the above-mentioned sequence number 9 of SEQ ID NO: 9.
  • the DNA sequence encoding thrombomodulin is preferably introduced by being incorporated into a vector, particularly preferably an expression vector that can be expressed in an animal cell.
  • a vector particularly preferably an expression vector that can be expressed in an animal cell.
  • molecular and preferred animal cell expression vectors include RC Mulligan et al. [Proc. Natl. Acad. Sci. US A. 78. 2072 (1981)], pSV2—X, PM Howley et al. [Method in Emzymology, 101, 387, Academic Press (1983)].
  • Host cells that can be used in the production of these peptides include Chinese nomstar ovary (CHO) cells, COS-1 cells, J 03-7 cells, and £ 0 (Haccho C CCL- 81 ) Cells, BHK cells, Inu kidney-derived MDCK cells, Nomstar AV-12-664 cells, etc., and human-derived cells include HeLa cells, WI38 cells, and human 293 cells.
  • CHO cells where CHO cells are very common and preferred, DHFR-CHO cells are more preferred.
  • microorganisms such as E. coli are often used in the genetic manipulation process and peptide production process, and it is preferable to use a host vector system suitable for each.
  • An appropriate vector system can also be selected for cells.
  • the gene of thrombomodulin used in the gene recombination technology has been cloned, and a production example using the gene recombination technology of thrombomodulin is disclosed, and further, a purification method for obtaining the purified product is also disclosed. [Japanese Unexamined Patent Publication Nos. 64-6219, 2-255699, 5-213998, 5-310787, 7-155176, J. Biol Chem., 264: 10351—10353 (1989)].
  • the thrombomodulin used in the present invention can be obtained by using the method described in the above report. Or it can manufacture by applying to the method as described in them.
  • JP-A-64-6219 discloses Escherichia coli K-12 strain DH5 (ATCC Deposit No. 67283) containing plasmid PSV2TMJ2 containing DNA encoding full-length thrombomodulin.
  • a strain Esscherichia coli DH5 / pSV 2TM J2
  • FERM BP-5570 re-deposited with this strain at Life Research Institute (currently the National Institute of Advanced Industrial Science and Technology (AIST)
  • the thrombomodulin of the present invention can be prepared by a known gene manipulation technique.
  • the thrombomodulin used in the present invention may be prepared by a conventionally known method or a method similar thereto.
  • a human-derived thrombomodulin gene can be made into a DNA encoding the amino acid sequence of SEQ ID NO: 9, for example, by genetic manipulation techniques, and further modified as necessary.
  • this modification for example, in order to obtain a DNA encoding the amino acid sequence of SEQ ID NO: 11 (specifically, consisting of the base sequence of SEQ ID NO: 12), position 473 of the amino acid sequence of SEQ ID NO: 9 is used.
  • the codon that encodes the amino acid (especially the base at position 1418) is described in Method Enzymology [Methods in Enzymology, 100: 468 (1983), Academic Press]. According to the method, site-specific mutation is performed. For example, base T at position 1418 of SEQ ID NO: 10 can be converted to DNA converted to base C using a synthetic DNA for mutation having the base sequence shown in SEQ ID NO: 13.
  • the DNA thus prepared is incorporated into, for example, Chinese nomstar ovary (CHO) cells to form transformed cells, which are appropriately selected from the culture solution obtained by culturing the cells by a known method.
  • Purified thrombomodulin can be produced.
  • the method for producing thrombomodulin used in the present invention is not limited to the above-mentioned method. For example, it can be extracted and purified from urine, blood, other body fluids, etc. These tissue culture fluids can also be extracted and purified, and if necessary further cleaved with proteolytic enzymes. Is also possible.
  • the amino acid at position 17, 18, 19, or 22 in SEQ ID NO: 9 may be N-terminal.
  • the N-terminal amino acid may be modified so that glutamic acid at position 22 is converted to pyroglutamic acid.
  • the amino acid at the 17th or 19th position is preferably N-terminal, and the amino acid at the 19th position is preferably the N-terminal.
  • it is preferred that the amino acid at position 17 is the N-terminus, and there is another embodiment.
  • thrombomodulin when thrombomodulin is produced using DNA having the base sequence of SEQ ID NO: 10, diversity of C-terminal amino acids may be observed, and a peptide having a short amino acid residue may be produced. . That is, the C-terminal amino acid may be modified such that the amino acid at position 515 becomes the C-terminal and the position 515 is amidated. Therefore, N-terminal amino acids and C-terminal amino acids have a variety of peptides, or mixtures thereof.
  • the amino acid at position 515 is preferably the C-terminus. In another embodiment, the amino acid at position 516 is preferably C-terminal.
  • the above modification and diversity are the same for the DNA having the nucleotide sequence of SEQ ID NO: 12.
  • Thrombomodulin obtained by the above method may be a mixture of peptides in which diversity is observed at the N-terminus and C-terminus. Specifically, a mixture of peptides consisting of the sequences of positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9 can be mentioned.
  • the method for isolating thrombomodulin from the culture supernatant or culture obtained as described above can be performed according to a known method [edited by Takeshi Horio, basic experimental method for protein / enzyme]. it can.
  • adsorption chromatography using ion exchange chromatography utilizing an interaction between a thrombomodulin and a chromatography matrix carrier in which a functional group having a charge opposite to that of thrombomodulin is immobilized.
  • affinity chromatography utilizing specific affinity with thrombomodulin.
  • thrombin which is a ligand of thrombomodulin, is used.
  • an antibody of rombomodulin is given.
  • an antibody of thrombomodulin that recognizes an appropriate property or an appropriate epitope can be used.
  • JP-B-5-42920, JP-A-64-45398, JP-A-6-20569 The example described in No. 2 gazette etc. is mentioned.
  • gel filtration chromatography and ultrafiltration using the molecular weight size of thrombomodulin can be mentioned.
  • hydrophobic chromatography using a hydrophobic bond between a chromatographic carrier on which a hydrophobic group is immobilized and a hydrophobic site of thrombomodulin can be mentioned.
  • hydroxyapatite as a carrier for adsorption chromatography, and examples thereof include those described in JP-A-9-110900. These methods can be combined appropriately.
  • the degree of purification depends on the force that can be selected according to the purpose of use, for example, the electrophoretic movement, preferably the force that gives SDS-PAGE results as a single band, or the gel filtration HPLC or reverse phase HPLC results of the isolated purified product. It is desirable to purify until a single peak is reached.
  • the band is substantially composed of only thrombomodulin, and it is not required to be a single band.
  • a specific example of the purification method in the present invention is a method of purification using thrombomodulin activity as an index.
  • the culture supernatant or culture is roughly purified with Q-Sepharose Fast Flow of an ion exchange column.
  • the fraction having thrombomodulin activity was collected, and the main fraction was purified with a DIP-thrombin agarose column on the affinity column to collect the fraction with strong thrombomodulin activity, and the collected fraction was concentrated.
  • a purification method [Gomi K. et al., Blood, 75: 1396-1399 (1990)] to obtain a pure product of a thrombomodulin active fraction by gel filtration.
  • the target thrombomodulin activity include the activity of promoting protein C activation by thrombin.
  • Other preferred purification methods are as follows.
  • a particularly preferred example is a method using Q-Sepharose Fast Flow equilibrated with 0.02 M Tris-HCl buffer (pH 7.4) containing 0.18 M NaCl. After washing as appropriate, for example with 0.02 M Tris-HCl buffer (pH 7.4) containing 0.3 M NaCl.
  • the crude product thrombomodulin can be obtained by elution.
  • a substance having specific affinity for thrombomodulin can be immobilized on a resin and purified by affinity chromatography.
  • Preferable examples include a DIP-thrombin monogarose column and an anti-thrombomodulin monoclonal antibody column.
  • the DIP-thrombin agarose column is pre-equilibrated with, for example, 20 mM Tris-HCl buffer (pH 7.4) containing lOOmM NaCl and 0.5 mM calcium chloride, charged with the above crude product, and appropriately washed.
  • 20 mM Tris-HCl buffer (pH 7.4) containing 1. OM NaCl and 0.5 mM calcium chloride.
  • a cation exchanger preferably a strong cation, equilibrated with lOOmM dalysin hydrochloride buffer (pH 3.5) containing 0.3 ⁇ NaCl.
  • lOOmM dalysin hydrochloride buffer pH 3.5
  • the obtained fraction can be neutralized with an appropriate buffer and obtained as a highly purified product. These are preferably concentrated by ultrafiltration.
  • buffer exchange by gel filtration.
  • a Sephacryl S-300 column or S-200 column equilibrated with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl was charged with a highly purified product concentrated by ultrafiltration, and 50 mM Developed with 20 mM phosphate buffer (pH 7.3) containing NaCl, confirmed the activity of promoting the activation of protein C by thrombin, collected the active fraction, and replaced the buffer.
  • Purified products can be obtained.
  • the purified high-purity product thus obtained is preferably filtered using an appropriate virus-removing membrane such as Branova 15N (Asahi Kasei Medical Co., Ltd.) to enhance safety. Can concentrate to concentration S.
  • the thrombomodulin thus obtained can be made into a lyophilized preparation by an ordinary method. That is, a method of freezing a solution containing thrombomodulin and an additive that is added if necessary and drying by sublimating water under reduced pressure is exemplified.
  • the plasma thrombomodulin concentration can also be expressed by thrombomodulin weight or thrombomodulin activity.
  • the plasma is preferably a vein-derived plasma, which may be an artery-derived plasma or a vein-derived plasma. In addition, it may be preferable to use plasma derived from arteries.
  • the method for measuring the thrombomodulin concentration in plasma by thrombomodulin weight display or thrombomodulin activity display (hereinafter sometimes referred to as thrombomodulin concentration by weight display or thrombomodulin concentration by activity display) is not particularly limited. For example, it can be measured by enzyme immunoassay, fluorescent immunoassay or radioimmunoassay. Among them, the method for measuring plasma thrombomodulin concentration by enzyme immunoassay is preferred.
  • a blood sample is collected in a heparin-containing blood collection tube, and rapidly centrifuged to collect plasma. Subsequently, the plasma is appropriately diluted and added to the well of the enzyme immunoassay plate on which the anti-thrombomodulin antibody is immobilized.
  • the anti-thrombomodulin antibody used is preferably an antibody that recognizes the active site of thrombomodulin! /. After sufficient time, the well is washed with a suitable buffer. Continue! Add anti-thrombomodulin antibody solution labeled with appropriate enzyme to the well.
  • the antibody used is an antibody different from the antibody used for immobilization, and an antibody that recognizes the active site of thrombomodulin is preferred.
  • the enzyme used for labeling is not particularly limited, and for example, horse radish peroxidase can be used. After sufficient time, the well is washed with a suitable buffer. Subsequently, a substrate solution for the labeling enzyme is added to the well. Group for labeling enzyme used in substrate solution The quality is not particularly limited! /, But o-phenylenediamine can be used, for example, when horseradish peroxidase is used as the labeling enzyme. Stop the enzyme-substrate reaction by a suitable method after a certain period of time. The method for stopping the enzyme / substrate reaction is not particularly limited. For example, the reaction between horse radish peroxidase and o-phenylenediamine can be stopped by adding a sulfuric acid solution.
  • a substrate reaction product corresponding to the amount of thrombomodulin in the sample is generated.
  • the amount of the substrate reaction product can be measured by colorimetric determination or the like according to the properties.
  • the amount of thrombomodulin in a sample can be determined using a separately prepared calibration curve.
  • a calibration curve can be created by using a standard product.
  • the amount of thrombomodulin in plasma can be determined by multiplying the amount of thrombomodulin in the sample thus determined by the dilution factor.
  • the amount of thrombomodulin in the sample is obtained as the protein weight concentration (eg, ng / ml).
  • the method for measuring the protein weight of the standard product is not particularly limited, and for example, a colorimetric method or a Raleigh method can be used.
  • the protein weight concentration obtained is calculated as the weight of thrombomodulin used as a standard product.
  • the standard product of thrombomodulin is not particularly limited, but the ability to appropriately use a standard product of thrombomodulin of the same molecular type as that of thrombomodulin that is normally administered is exemplified by TMD123 described in Production Example 1 below as a preferred example.
  • the amount of thrombomodulin in the sample is expressed as the thrombomodulin concentration (for example, units / ml) indicated by the activity.
  • the thrombomodulin concentration for example, units / ml
  • the activity can get.
  • a standard product use a standard product described in Niimi S. et al., Biologics, 30: 69-76 (2002), or a self-standard product whose content unit is measured using the standard product and the method described in the literature. Is preferred.
  • the specific activity of the thrombomodulin (thrombomodulin activity per protein weight, eg, unit / mg protein) is obtained and used as a conversion factor for plasma. It is possible to convert thrombomodulin concentration by medium weight display into plasma thrombomodulin activity. For example, the activity table can be obtained by multiplying the specific activity by the thrombomodulin concentration by weight. The indicated thrombomodulin concentration can be determined. Conversely, the thrombomodulin concentration by activity display can be converted to the thrombomodulin concentration by weight using the specific activity as a conversion factor.
  • the specific activity measurement method of the thrombomodulin is not particularly limited!
  • the thrombomodulin activity in a solution containing the purified thrombomodulin is determined by the method described in the above-mentioned literature, and the thrombomodulin protein in the solution is determined.
  • the weight is obtained by the single-Lee method, and the former is divided by the latter.
  • thrombomodulin concentration in plasma it is possible to directly determine the thrombomodulin concentration in plasma by the above-described various immunoassays.
  • the thrombomodulin activity can be measured by the method to determine the thrombomodulin concentration in the plasma.
  • the thrombomodulin isolation method to be used is not particularly limited. For example, a chromatographic method using a carrier bound with an anti-thrombomodulin antibody can be used.
  • Prothrombinase is an enzyme complex composed of activated factor X, activated factor V, phospholipid, and Ca 2+ ions, and generates thrombin from prothrombin.
  • the generated thrombin causes the generation and growth of thrombus by generating fibril monomers and activated factor XIII, and aggregating platelets.
  • activated factor V and activated factor VIII are generated to further activate the blood coagulation system.
  • thrombomodulin specifically binds to thrombin and inhibits the thrombin clotting activity, and at the same time significantly enhances the protein C activation ability of thrombin.
  • Activated protein C produced by the thrombin thrombomodulin complex inhibits the blood coagulation system by inhibiting the amount of thrombin produced by inhibiting prothrombinase. That is, plasma prothrombinase activity is inhibited through the action of thrombomodulin to promote protein C activation of thrombin. Therefore, the rate at which plasma prothrombinase activity is inhibited (inhibition rate) is an important pharmacokinetic parameter for investigating the blood coagulation inhibitory action of thrombomodulin expressed in vivo.
  • the method for measuring plasma prothrombinase activity is not particularly limited! /, But is determined by the method described in, for example, Mohri M. et al., Thoromb. Haemost., 82: 1687-1693 (1999).
  • the inhibition rate of prothrombinase activity in plasma is the same as that of prothrombin in standard plasma.
  • the rate of inhibition of prothrombinase activity in plasma (%) ((8 ⁇ B) / A) X100 or (( ⁇ ′— ⁇ ) / ⁇ ′) X100 is preferred.
  • the standard plasma to be used is not particularly limited. For example, blood is collected from a plurality of healthy individuals in a blood collection tube containing sodium citrate, rapidly centrifuged, and the collected plasma is pooled. Can be created.
  • Plasma thrombomodulin concentration and plasma prothrombinase activity inhibition rate can be converted to each other as follows. That is, the prothrombinase activity of the plasma sample added to the standard plasma so that it contains various concentrations of the purified thrombomodulin and the thrombomodulin is not added! / Standard plasma is measured by, for example, the method described in the above-mentioned literature. And determine the inhibition rate of prothrombinase activity in the plasma sample. By plotting the relationship between the plasma thrombomodulin concentration and the plasma prothrombinase activity inhibition rate thus obtained, the plasma thrombomodulin concentration and the plasma prothrombinase activity inhibition rate can be converted into each other.
  • the dosage of the drug of the present invention is such that plasma thrombomodulin concentration is at least 20 Ong / ml or at least 1 ⁇ 3 units / ml or more by administration to DIC patients or septic patients, or plasma proto There is no particular limitation as long as it is administered so that the inhibition rate of lombinase activity is maintained at least 85% or more for the preferable time described below. Specifically, it is sufficient to adjust the dose of the drug of the present invention so as to satisfy the above numerical value while measuring the plasma thrombomodulin concentration or the plasma prothrombinase activity inhibition rate.
  • the method may vary depending on the patient and the patient being administered, so it cannot be generally stated, but first, the following administration should be carried out, and the plasma thrombomodulin concentration or plasma prothrombinase activity inhibition rate should be finely adjusted. .
  • 5mg / kg or less is preferred, lmg / kg or less is more preferred, and lmg / kg or less is more preferred.
  • 8 mg / kg or less is a particularly preferable example, and 0.005 mg / kg or more is preferable 0.
  • Olmg / kg or more is more preferable 0.02 mg / kg or more is more preferable 0.05 mg / kg Above is particularly favorable A good example.
  • the plasma thrombomodulin concentration is at least 200 ng / ml or at least 1.
  • the lower limit is preferably 24 hours or more, more preferably 72 hours or more, and more preferably 144 hours or more
  • the preferred upper limit is not particularly limited as long as symptoms such as side effects do not occur.
  • the plasma thrombomodulin concentration is not particularly limited as long as it is 200 ng / ml or more, but preferably 3 OOng / ml or more, more preferably 500 ng / ml or more.
  • the upper limit of plasma thrombomodulin concentration is not particularly limited as long as it does not cause toxicity, but it is preferably 7000 ng / ml or less, more preferably 5000 ng / ml or less, force S. In some cases, it may be preferably 3000 ng / ml or less, or 1000 ng / ml or less, or 500 ng / ml or less.
  • the plasma thrombomodulin concentration is not particularly limited as long as it is 1.3 units / ml or more, but 1.6 units / ml or more is preferable, and 2 units / ml or more is more preferable.
  • the upper limit of plasma thrombomodulin concentration is not particularly limited as long as it does not cause toxicity, but 45 units / ml or less is preferred, 30 units / ml or less is more preferred, and 20 units / ml or less is further preferred.
  • the plasma prothrombinase activity inhibition rate is not particularly limited as long as it is 85% or more, but 90% or more is preferable, and 95% or more is more preferable.
  • the upper limit of the inhibition rate of plasma prothrombinase activity is preferably 100% or less.
  • the dosing interval may be daily, but is preferably once every 2 to 14 days, more preferably once every 3 to 10 days, more preferably 4 tons, or once every 7 days. It is a storehouse.
  • the intravenous administration is preferably once a day, but is not limited thereto.
  • thrombomodulin when thrombomodulin is an active ingredient, Can be administered by a commonly used administration method, that is, a parenteral administration method such as intravenous administration, intramuscular administration, or subcutaneous administration.
  • a parenteral administration method such as intravenous administration, intramuscular administration, or subcutaneous administration.
  • intramuscular administration or subcutaneous administration is preferable in that the administration interval can be increased because the blood concentration is maintained for a long time.
  • intravenous administration is preferred.
  • oral administration, rectal administration, intranasal administration and sublingual administration are also possible.
  • a method of administering a desired amount at a time is preferable in that the administration time is short.
  • force that usually has a range of time required for administration with a syringe Force that depends on the amount of liquid to be administered is usually 2 minutes or less.
  • the following is more preferable, and more preferably 30 seconds or less.
  • the lower limit is not particularly limited, but 1 second or more is preferable, 5 seconds or more is more preferable, and 10 seconds or more is more preferable.
  • the dose is preferably V as described above, and the dose is not particularly limited.
  • Intravenous administration is not particularly limited as long as it is preferable in terms of the ease with which the blood concentration of thrombomodulin can be kept relatively constant, but the upper limit of the daily dosage is lmg / kg or less is preferred 0.5 mg / kg or less is more preferred 0. lmg / kg or less is more preferred 0.8 mg / kg or less is particularly preferred 0.6 mg / kg or less is most preferred
  • the lower limit is preferably 0.005 mg / kg or more, more preferably 0.01 mg / kg or more, more preferably 0.02 mg / kg or more, and particularly preferably 0.04 mg / kg or more.
  • the upper limit is preferably 4 hours or less, more preferably 3 hours or less, more preferably 2 hours or less, particularly preferably 1 hour or less 30 minutes
  • the most preferable lower limit is 10 minutes or more, 15 minutes or more is more preferable, and 20 minutes or more is more preferable.
  • a lyophilized preparation When a lyophilized preparation is used, it can be administered to a patient after use by dissolving in water, for example, distilled water (or water for injection), physiological saline or the like.
  • an effective amount of thrombomodulin may be prepared by mixing with a carrier that can be used as a drug. That is, an effective amount of thrombomodulin for treating the above diseases is mixed with a known appropriate amount of carrier and effectively administered to a patient.
  • a suitable formulation can be prepared.
  • thickeners such as sucrose, glycerin, methylcellulose, carboxymethylcellulose, pH adjusters for various inorganic salts, etc. can be prepared as calorie-free IJ.
  • SEQ ID NO: 2 Base sequence encoding the amino acid sequence of SEQ ID NO: 1
  • SEQ ID NO: 3 Amino acid sequence encoded by the gene used for the production of TME456M
  • SEQ ID NO: 4 Base sequence encoding the amino acid sequence of SEQ ID NO: 3
  • SEQ ID NO: 5 amino acid sequence encoded by the gene used for the production of TMD12
  • SEQ ID NO: 6 base sequence encoding the amino acid sequence of SEQ ID NO: 5
  • SEQ ID NO: 7 amino acid sequence encoded by the gene used for the production of TMD12M
  • SEQ ID NO: 8 base sequence encoding the amino acid sequence of SEQ ID NO: 7
  • SEQ ID NO: 9 amino acid sequence encoded by the gene used for the production of TMD123
  • SEQ ID NO: 10 base sequence encoding the amino acid sequence of SEQ ID NO: 9
  • SEQ ID NO: 11 Amino acid sequence encoded by the gene used for production of TMD123M
  • SEQ ID NO: 12 Base sequence encoding the amino acid sequence of SEQ ID NO: 11
  • SEQ ID NO: 13 Synthetic DNA for mutation used for site-specific mutation
  • the thrombomodulin in the present invention used in the test examples was produced according to the method of Yamamoto et al. (The method described in JP-A-664-6219). The production example is shown below.
  • arginine hydrochloride (Ajinomoto Co., Inc.) was weighed, and 5 L of water for injection was added and dissolved. 1M sodium hydroxide solution was added to adjust the pH to 7.3.
  • the total amount of the above additive solution was placed in a 20 L stainless steel container, and 2398 ml of the TMD 123 solution obtained above (corresponding to 26.88 g as the protein mass of soluble thrombomodulin, but charged in excess of 12%) was added and stirred. Further, water for injection was added to make the total volume 12 L, and the mixture was stirred uniformly.
  • This chemical solution was sterilized by filtration through a filter having a pore size of 0.22 ⁇ 111 (Millipore MCGL1 OS). 1 ml of the filtrate was filled into the vial, and the rubber stopper was half stoppered.
  • freeze-drying was performed in the following order: freeze-drying ⁇ filling with nitrogen ⁇ full plugging of rubber stoppers ⁇ tightening of caps to obtain a preparation containing TMD 123 containing 2 mg of soluble thrombomodulin and 40 mg of arginine hydrochloride in one container. It was.
  • a DNA encoding the amino acid sequence of SEQ ID NO: 11 (specifically, the nucleotide sequence of SEQ ID NO: 12) is transfected into a chayote nomstar ovary (CHO) cell, and the culture solution of this transformed cell Obtain a thrombomodulin (hereinafter sometimes abbreviated as TMD123M) solution purified by the above-described conventional purification method, and obtain a lyophilized preparation of TMD123M by the same method as described above.
  • TMD123M thrombomodulin
  • a DNA encoding the amino acid sequence of SEQ ID NO: 1 (specifically, consisting of the base sequence of SEQ ID NO: 2) is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • a chayuznoumster ovary (CHO) cell is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • TME456 conventional purification method
  • a DNA encoding the amino acid sequence of SEQ ID NO: 3 (specifically, consisting of the base sequence of SEQ ID NO: 4) is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • a chayuznoumster ovary (CHO) cell a chayuznoumster ovary (CHO) cell
  • TME456M thrombomodulin purified by the above-mentioned conventional purification method
  • TME456M freeze-dried preparation of TME45 6M by the same method as described above.
  • a DNA encoding the amino acid sequence of SEQ ID NO: 5 (specifically, consisting of the base sequence of SEQ ID NO: 6) is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • a chayuznoumster ovary (CHO) cell is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • thrombomodulin hereinafter abbreviated as TMD12
  • a DNA encoding the amino acid sequence of SEQ ID NO: 7 (specifically, consisting of the base sequence of SEQ ID NO: 8) is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • a chayuznoumster ovary (CHO) cell is transfected into a chayuznoumster ovary (CHO) cell, and the culture solution of this transformed cell is obtained.
  • TMD12M thrombomodulin purified by the conventional purification method described above (hereinafter sometimes abbreviated as TMD12M), and obtain a lyophilized preparation of TMD12 M in the same manner as described above.
  • the treatment and / or amelioration agent of the present invention prepared by the method shown below is administered to DIC patients diagnosed with DIC based on the DIC diagnostic criteria of the Ministry of Health and Welfare, and the 7th day of administration (the first day of administration is the first day) (Corresponding to 144 hours after the start of administration)
  • the 7th day of administration (the first day of administration is the first day) (Corresponding to 144 hours after the start of administration)
  • leukemia and related diseases, aplastic anemia, and bone marrow megakaryocyte depletion such as after administration of antitumor agents are prominent and severe thrombocytopenia is observed. .
  • test solution prepared in Preparation Example 1 was adjusted to 0.012 ml / kg (0.006 mg / kg), 0.04 ml / kg (0.02 mg / kg). Collected at a rate of 0.12ml / kg (equivalent to 0.06mg / kg) and added to physiological saline to make 100ml. The physiological saline solution was added once a day. Intravenous infusion over 30 minutes was repeated for 6 days.
  • a 3 ml blood sample of the subject patient was collected in a blood collection tube containing heparin, mixed by inversion and centrifuged, and plasma was collected and stored frozen. Subsequently, the plasma was diluted with a phosphate buffer containing about 1M sodium chloride and 0.05% Tween 80, and finally a sample solution containing 0.9M sodium chloride was prepared.
  • a standard product containing TMD123 (manufactured by Asahi Kasei Pharma) was diluted in the same manner to prepare a standard solution containing 0 to 10 ng / ml of TMD123.
  • anti-TMD 123 antibody (R4D 1 manufactured by Asahi Kasei Pharma) labeled with horseradish peroxidase was used per well. 50 1 / ml was added and left at room temperature for 2 hours. After washing well with phosphate buffer containing 0 ⁇ 05% Tween 80, o-phenidylamine solution (1 Omg o-diendiamine and 35% hydrogen peroxide 10 1 in 20 ml phosphate 'In citrate buffer 100 1 was added per well. After leaving outside light at room temperature for 20 to 30 minutes, 4.5 M sulfuric acid was added at 50 1 per well.
  • the absorbance at 492 nm was measured, and a calibration curve was prepared from the absorbance values of the wells added with the standard solution.
  • the absorbance value of the well to which the sample solution was added was applied to a calibration curve, and the thrombomodulin concentration in the sample solution was multiplied by the dilution factor to obtain the plasma thrombomodulin concentration.
  • the degree of improvement in coagulation test values and the degree of general improvement were used. That is, using the various coagulation test values before administration and on the 7th day (144 hours) and the clinical symptom improvement (bleeding symptom improvement, organ symptom improvement) determined by the doctor on the 7th day.
  • the degree of coagulation test value improvement and general improvement were calculated as follows.
  • each clot value is scored using Table 1.
  • Table 1 the changes in the total score of the four items of FDP, platelet count, fibrinogen, and prothrombin time ratio in the non-leukemic group are shown in Table 2, and in the leukemia group, the three items of FDP, fibrinogen, and prothrombin time ratio.
  • the change in the total score of the scores was applied to Table 3. Changes in the score total score for D-dimer, TAT (thrombin. AT complex), and PIC (plasmin (plasmin inhibitor complex)) were applied to Tables 2 and 3.
  • the degree of general improvement is the degree of improvement in clinical symptoms (bleeding symptom improvement, organ symptom improvement) determined on the 7th day after administration by the doctor, and the degree of coagulation test value improvement obtained above.
  • the evaluation was made according to the following criteria. That is, in the non-leukemia group, A to E determined from Table 2 were applied to Table 4 and a to e were selected, and this was applied to Table 5 to evaluate the general improvement degree. In the leukemia group, A to E determined from Table 3 were applied to Table 6 to evaluate the overall improvement. In this way, the overall improvement on the 7th day (144 hours) of each patient was classified into “significant improvement”, “moderate improvement”, “mild improvement”, “invariant”, and “deterioration”. . In this study example, the ratio of patients with “moderate improvement” or higher was the overall improvement rate.
  • the test solution is administered according to the administration method described in ⁇ Dose and administration method> above, and the plasma thrombomodulin concentration, the degree of coagulation test value, and the general improvement level are examined on the seventh day (44 hours after administration).
  • the test solution was administered according to the administration method described in ⁇ Dose and administration method> above, and the plasma thrombomodulin concentration, the degree of coagulation test value, and the general improvement level are examined on the seventh day (44 hours after administration).
  • plasma thrombomodulin concentrations were determined for each patient population within 8 to 34 patients within a certain range (3-fold width) on both sides, and the improvement rate of plasma thrombomodulin concentration was determined. Plotted.
  • the rate of improvement of the coagulation test value and the rate of general improvement showed almost similar patterns.
  • the plasma thrombomodulin concentration (plasma thrombomodulin weight concentration) is 200 ng / ml or higher, the overall improvement rate is 60% or more and the coagulation test value improvement rate is 50% or more.
  • the effective plasma thrombomodulin concentration (plasma thrombomodulin weight concentration) was considered.
  • TMD123 thrombomodulin
  • the specific activity of 6 lots of TMD123 solution produced according to Production Example 1 was measured.
  • the thrombomodulin activity of each TMD123 solution was measured by the method described in Niimi S. et al., Biologicals, 30: 69-76 (2002). At that time, the standard described in this document was used as a standard for measuring thrombomodulin activity.
  • the thrombomodulin protein weight concentration of each TMD123 solution was determined using the Raleigh method. Specifically, an appropriate amount of each TMD123 solution was accurately weighed, water was added accurately, and a solution containing 50 to 150; ⁇ as a protein in 1 ml was prepared as a sample solution.
  • a standard albumin solution was taken, water was added accurately, and four kinds of liquids containing 0 to 150 g in lml were prepared and used as standard solutions.
  • the sample solution and the standard solution were each placed in a test tube at a rate of 1. Oml, and 2.5ml of alkaline copper solution was added to each test tube, shaken, and allowed to stand at room temperature for 10 minutes or longer.
  • Folin reagent diluted 8 times 2. Oml was added and shaken and allowed to stand at 37 ° C for more than 30 minutes. Absorbance at a wavelength of 750 nm was measured using water as a control.
  • the calibration curve force with the standard solution was also used to determine the protein content (mg / ml) in 1 ml of the sample solution, and by multiplying the dilution factor, the thrombomodulin protein weight concentration (mg / ml) of each TMD 123 solution was obtained.
  • the thrombomodulin activity (unit / ml) of each TMD123 solution thus obtained was divided by the thrombomodulin protein weight concentration (mg / ml) to determine the specific activity (unit / mg protein).
  • the average specific activity of 6 lots of TMD123 solution was 6400 units / mg protein. From this result, the minimum effective plasma thrombomodulin weight concentration of 200 ng / ml is The modulin activity was found to correspond to 1 ⁇ 3 units / ml.
  • Plasma samples containing various concentrations (0.002—1 g / ml) of TMD123 were prepared by adding a high-concentration TMD123 solution prepared according to Production Example 1 to normal human plasma.
  • This plasma sample and human normal plasma (standard plasma) without addition of TMD123 were measured, and prothrombinase activity was measured by the method described in Mohri M. et al., Thoromb. Haemost., 82: 1687-1693 (1999). did.
  • the inhibition rate of the prothrombinase activity of the plasma sample with respect to the prothrombinase activity obtained using standard plasma was expressed as the inhibition rate of prothrombinase activity, and the relationship with the TMD 123 concentration was plotted.
  • TMD123 inhibited prothrombinase activity in a concentration-dependent manner. From this result, it was found that the lowest effective plasma thrombomodulin weight concentration of 200 ng / ml corresponds to a prothrombinase activity inhibition rate in plasma of 85%.
  • the therapeutic and / or ameliorating agent of the present invention prepared by the method shown below is administered to DIC patients whose symptoms are induced by hematopoietic malignancies or infections diagnosed with DIC based on the DIC diagnostic criteria of the Ministry of Health and Welfare. Then, the change in the FDP value of the patient at the 24th to 144th hour before administration was examined.
  • test solution prepared in Preparation Example 2 was collected at a rate of 0.12 ml / kg (equivalent to 0.06 mg / kg) and added to physiological saline to make 100 ml.
  • physiological saline solution 1 Intravenous infusion was performed once a day for 30 minutes. This was repeated for 6 days.
  • Plasma of specimens collected at various blood sampling points before and after administration of the treatment and / or ameliorating agent of the present invention to healthy individuals and patients diagnosed with DIC based on the DIC diagnostic criteria of the Ministry of Health and Welfare Medium thrombomodulin concentration was measured.
  • the population pharmacokinetics (PPK) solution was calculated from these various plasma thrombomodulin concentrations. Analysis was performed.
  • test solutions were prepared by adding physiological saline to various freeze-dried preparations of TMD123 obtained according to Production Example 1.
  • the test solution prepared in Preparation Example 3 was intravenously administered to 20 healthy individuals.
  • the liquid volume was 10 ml / person, and the dose was 0.03 mg / person, 0.1 mg / person, or 0.3 mg / person, and was administered as a single intravenous drip for 2 hours (4 per dose).
  • Man Four healthy subjects were repeatedly infused once a day for 2 hours at a dose of 10 ml / person and a dose of 0.2 mg / person for 3 days.
  • four healthy subjects were intravenously infused once for 1 minute at a dose of 1.5 ml and a dose of 0.3 mg / person.
  • a total of 348 plasma thrombomodulin concentrations were measured in the above-mentioned studies conducted on healthy individuals.
  • Test Example 1 conducted in DIC patients, a total of 305 plasma thrombomodulin concentrations were measured from 116 DIC patients. PPK analysis was performed on the above 653 measured plasma thrombomodulin concentrations. As one of the analyses, the changes in plasma thrombomodulin concentrations in the elderly (65 years and older, 56 cases) and non-elderly people (under 65 years, 80 cases) with respect to the age at which pharmacokinetics were observed were analyzed. Simulated based on the model formula.
  • Fig. 4 shows a graph simulating changes in plasma thrombomodulin concentrations in elderly and non-elderly when thrombomodulin (TMD123) was administered to DIC patients at a dose of 0.06 mg / kg.
  • TMD123 thrombomodulin
  • the plasma thrombomodulin concentration exceeded the lowest effective plasma thrombomodulin concentration (200 ng / ml) in the elderly and non-elderly people from the start of administration until 144 hours.
  • the plasma thrombomodulin concentration at a dose of 0.02 mg / kg is simulated to change at one-third of the concentration in Figure 4.
  • plasma thrombomodulin concentration is the lowest effective plasma thrombomodulin concentration (both in the elderly and non-elderly) from the beginning of administration until 144 hours, except for a short time just before 24 hours after administration. 200 ng / ml).
  • Test Example 1 9 cases were diagnosed as sepsis by a doctor at the same time as DIC (3 in the 0.006 mg / kg group, 3 in the 0.02 mg / kg group, 0.06 mg / kg). 3 subjects in the administration group), before administration, immediately after administration on day 1 (+0 hours), immediately after administration on day 6 (120 hours), on administration day 7 (24 hours after administration on day 6) The plasma thrombomodulin concentration at 144 hours) was measured. In addition, the degree of coagulation test value improvement and general improvement on the seventh day (144 hours) of administration was examined, and the relationship between these and the dose was examined.
  • Table 8 shows the relationship between plasma thrombomodulin concentration and the degree of improvement. Both results showed that thrombomodulin was also effective in sepsis depending on plasma thrombomodulin concentration. Efficacy has been demonstrated by administering thrombomodulin so that the plasma thrombomodulin concentration exceeds the lowest effective plasma thrombomodulin concentration (200 ng / ml), especially when the plasma thrombomodulin concentration is 550 ng / ml or more (0 ⁇ 06 mg / kg In the dose group), 2 out of 3 cases showed a marked improvement in both degrees of improvement.
  • the symptoms of DIC patients or septic patients can be effectively treated and / or improved, and according to the method of the present invention, DIC or sepsis can be effectively treated. And / or can be improved.
  • FIG. 2 is a graph showing the relationship between the time from the start of administration and plasma thrombomodulin (TMD123) concentration at each dose (0.006, 0.02, 0.06 mgZkg) in Test Example 1.
  • FIG. 3 is a graph showing the relationship between thrombomodulin (TMD123) concentration and prothrombinase activity inhibition rate in human normal plasma in Test Example 2.
  • FIG. 4 is a graph simulating changes in plasma thrombomodulin concentration in elderly and non-elderly patients when thrombomodulin (TMD123) was administered to a DIC patient at a dose of 0.06 mg / kg in Test Example 4. .

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Abstract

L'invention concerne un agent thérapeutique et/ou améliorant ou un procédé thérapeutique et/ou améliorant présentant un effet thérapeutique important. L'invention concerne notamment un agent thérapeutique et/ou améliorant comprenant la thrombomoduline en tant qu'ingrédient actif. L'agent est administré à un patient présentant le syndrome de coagulation intravasculaire disséminée ou une sepsie, de manière à ce que le niveau de thrombomoduline dans le plasma atteigne une valeur supérieure ou égale à 200 ng/ml ou supérieure ou égale à 1,3 unités/ml, ou que le taux d'inhibition de l'activité prothrombinase dans le plasma soit maintenu à un niveau supérieur ou égal à 85 % chez le patient.
PCT/JP2007/068998 2006-09-29 2007-09-28 Agent thérapeutique pour le syndrome de coagulation intravasculaire disséminée WO2008038777A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013073545A1 (fr) * 2011-11-15 2013-05-23 旭化成ファーマ株式会社 Médicament pour le traitement et/ou l'amélioration de la sepsie
US8772239B2 (en) 2011-11-15 2014-07-08 Asahi Kasei Pharma Corporation Medicament for therapeutic treatment and/or improvement of sepsis
KR20140095559A (ko) * 2011-11-15 2014-08-01 아사히 가세이 파마 가부시키가이샤 패혈증의 치료 및/또는 개선을 위한 의약
JPWO2013073545A1 (ja) * 2011-11-15 2015-04-02 旭化成ファーマ株式会社 敗血症の治療及び／又は改善のための医薬
US9034823B2 (en) 2011-11-15 2015-05-19 Asahi Kasei Pharma Corporation Medicament for therapeutic treatment and/or improvement of sepsis
RU2580756C2 (ru) * 2011-11-15 2016-04-10 Асахи Касеи Фарма Корпорейшн Лекарственное средство для терапевтического лечения и/или улучшения состояния при сепсисе
AU2012337838B2 (en) * 2011-11-15 2016-04-21 Asahi Kasei Pharma Corporation Medicament for therapeutic treatment and/or improvement of sepsis
KR101672108B1 (ko) * 2011-11-15 2016-11-02 아사히 가세이 파마 가부시키가이샤 패혈증의 치료 및/또는 개선을 위한 의약
US9555080B2 (en) 2011-11-15 2017-01-31 Asahi Kasei Pharma Corporation Medicament for therapeutic treatment and/or improvement of sepsis
US9592275B2 (en) 2011-11-15 2017-03-14 Asahi Kasei Pharma Corporation Medicament for therapeutic treatment and/or improvement of sepsis

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