JP5924611B2 - Medicament for prevention and / or treatment of engraftment syndrome associated with hematopoietic cell transplantation - Google Patents

Description

  The present invention relates to a medicament for use in hematopoietic cell transplantation, comprising thrombomodulin as a main active ingredient, and for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation.

  There are three types of cells in blood, white blood cells, red blood cells, and platelets, and these are produced by the differentiation and proliferation of hematopoietic stem cells present in the bone marrow. In hematopoietic malignant tumors, abnormalities occur in specific cells differentiated from hematopoietic stem cells, and blood cells that do not have normal functions are produced in large quantities. As a result, the number of normal blood cells decreases, and it is a disease that eventually results in death due to complications of symptoms such as anemia and bleeding and infectious diseases. Hematopoietic malignancies mainly consist of leukemia, malignant lymphoma, and multiple myeloma. Leukemia affects 260,000 people worldwide and 210,000 people die annually. Malignant lymphoma and multiple myeloma affect 410,000 people worldwide and die 240,000 people worldwide. Also, in the United States, leukemia, malignant lymphoma, and multiple myeloma account for 2.9%, 5.3%, and 1% of tumor patients, respectively.

  Hematopoietic cell transplantation is a treatment for restoring normal hematopoietic function by transplanting hematopoietic cells of a healthy person to a patient with a hematopoietic malignant tumor. In recent years, in addition to hematopoietic malignant tumors, it has also been used as a relief therapy for myelosuppression, which is the greatest side effect of high-dose chemotherapy and radiation therapy for innate immune deficiencies, inborn errors of metabolism, and solid tumors. Hematopoietic cell transplantation is roughly classified into autologous transplantation using own bone marrow or peripheral blood stem cells, and allogeneic transplantation using related or unrelated hematopoietic cells. As the hematopoietic cells used for hematopoietic cell transplantation, peripheral blood of the donor or umbilical cord blood collected from the bone marrow of the donor and administered with granulocyte colony stimulating factor (G-CSF) is used.

Hematopoietic cell transplantation consists of 1) pre-transplantation treatment, 2) hematopoietic cell infusion (transplantation), and 3) prevention and treatment of complications. Pre-transplantation treatment involves the administration of large doses of chemotherapeutic agents such as cyclophosphamide and total body irradiation (Total BI) 7-7 days prior to transplantation to kill bone marrow cells of patients undergoing hematopoietic cell transplantation . Next, hematopoietic cells obtained from the donor are infused. The number of cells required for transplantation is approximately 3 × 10 ⁸ nucleated cells per kg body weight for bone marrow transplantation, 2 × 10 ⁶ or more CD34 positive cells per kg body weight for peripheral blood stem cells, and 1 kg body weight for cord blood transplantation 2 × 10 ⁷ or more is desirable.

  Engraftment syndrome (ES) is a condition that presents symptoms such as fever, erythema-like skin rash, and non-cardiogenic pulmonary edema at the time of neutrophil engraftment after hematopoietic stem cell transplantation. Engraftment syndrome broadly encompasses these series of symptoms. A similar pathology is sometimes referred to as capillary leak syndrome (CLS), focusing on the symptoms associated with extensive increased vascular permeability. It has been reported that when engraftment syndrome develops, the hospitalization period is prolonged, the morphine administration period is prolonged, and the mortality rate is increased.

  On the other hand, thrombomodulin is known as a substance that specifically binds to thrombin and inhibits the blood coagulation activity of thrombin and at the same time significantly promotes the protein C activation ability of thrombin, and has a strong blood coagulation inhibitory action. Are known. It is known to prolong the coagulation time by thrombin and to suppress platelet aggregation by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis system, and is activated by the action of thrombin to become activated protein C. This activated protein C is known to be involved in the production of plasminogen activator having a thrombolytic action, inactivating activated factor V and activated factor VIII of blood coagulation factor in vivo. (Non-Patent Document 1). Therefore, thrombomodulin is said to be useful as an anticoagulant or thrombolytic agent by accelerating the activation of protein C by this thrombin, and is an animal experiment that is effective in the treatment and prevention of diseases associated with hypercoagulation. There is also a report about (Non-patent Document 2).

  Conventionally, thrombomodulin has been discovered and acquired as a glycoprotein expressed on vascular endothelial cells of various animal species including humans, and has been successfully cloned. That is, the gene of a human thrombomodulin precursor containing 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 having 18 amino acid residues). The amino acid sequence of 575 residues including (exemplified) has been clarified (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 (position 1-226: position indication when the signal peptide is considered to be 18 amino acid residues, the same applies hereinafter), 5 of an EGF-like structure region (227-462th), an O-type glycosylation region (463-498th), a transmembrane region (499-521th), and an intracytoplasmic region (522-557th) It consists of two areas. The portion having the same activity as the full-length thrombomodulin (ie, the minimum active unit) is a portion mainly composed of the 4th, 5th and 6th EGF-like structures from the N-terminal side in the region having 6 EGF-like structures. Is known (Non-patent Document 3).

  The full-length thrombomodulin is difficult to dissolve unless in the presence of a surfactant, and as a formulation, the addition of a surfactant is essential, whereas the soluble thrombomodulin that can be dissolved neatly in the absence of a surfactant Exists. Soluble thrombomodulin may be prepared so as not to contain at least a part or all of the transmembrane region, for example, an N-terminal region, a region having six EGF-like structures, 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 the recombinant soluble thrombomodulin has the activity of natural thrombomodulin. (Patent Document 1). There are some other reports as examples of soluble thrombomodulin (Patent Documents 2 to 9). Or as a natural type, soluble thrombomodulin derived from human urine is exemplified (Patent Documents 10 and 11).

  Incidentally, in genes, polymorphic mutations have been found in humans as observed in many cases due to natural mutations or mutations at the time of acquisition, and human thrombomodulin consisting of the amino acid sequence of 575 residues described above. The amino acid at position 473 of the precursor is currently identified as Val and Ala. In the base sequence encoding this amino acid, it corresponds to a mutation between T and C at position 1418, respectively (Non-patent Document 4). However, there is no difference in activity and physical properties, and both can be considered substantially the same.

  Conventional thrombomodulin uses include, for example, myocardial infarction, thrombosis (eg, acute or chronic cerebral thrombosis, arterial or venous acute or chronic peripheral thrombosis), embolism (eg, acute or chronic phase) Cerebral embolism, arterial or venous acute or chronic peripheral embolism, etc.), peripheral vascular occlusion (eg, Buerger's disease, Raynaud's disease, etc.), obstructive arteriosclerosis, functional disorder secondary to cardiac surgery, transplanted organ Used in the treatment and prevention of diseases such as complications of the blood vessels, intravascular blood coagulation syndrome (DIC), angina pectoris, transient ischemic attack, pregnancy toxemia, deep vein thrombosis (DVT) It is expected that. Examples of applications for diseases other than diseases associated with hypercoagulation such as thrombosis and DIC include liver damage (Patent Document 12), absorbable bone disease (Patent Document 13), and wound healing (Patent Document 14). Furthermore, wound healing (Patent Document 15) and protection of brain tissue (Patent Document 16) are disclosed as applications in which thrombomodulin is used in combination with other active ingredients.

  VOD has been described as a use of thrombomodulin for complications associated with hematopoietic cell transplantation (Patent Document 17). In this patent document, the increase in GPT and bilirubin was suppressed by administering 1 mg / kg or 3 mg / kg of thrombomodulin to a drug-induced VOD model in which monocrotaline was administered to beagle dogs with reference to Non-patent document 5. Is described.

JP-A 64-6219 JP-A-5-213998 JP-A-2-255699 Japanese Patent Laid-Open No. 3-133380 JP-A-3-259084 JP-A-4-210700 WO92 / 00325 Publication WO92 / 03149 WO93 / 15755 JP-A-3-86900 JP-A-3-218399 JP-A-8-3065 Japanese Patent Laid-Open No. 8-301783 Japanese Patent Laid-Open No. 9-20877 US Pat. No. 5,976,523 US Pat. No. 5,827,832 US Pat. No. 5,916,874

Koji Suzuki, History of Medicine 1983; 125: 901 Gomi K et al, Blood 1990; 75: 1396-1399 Zushi M et al, J Biol Chem 1989; 246: 10351-10353 Wen DZ et al, Biochemistry 1987; 26: 4350-4357 Epstein RB et al, Transplant 1992; 54 (1): 12-16

  An object of the present invention is to provide a medicament for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation, or a method thereof.

  In recent years, hematopoietic cell transplantation has improved the therapeutic results due to new chemotherapeutic agents and combinations of drugs, but the fatality rate in the case of complications is still high, and complications due to improved hematopoietic cell transplantation methods There is a need for further improvement in transplantation results through effective prevention or treatment. As a result of intensive research aimed at finding a medicine that satisfies and satisfies the prevention and / or treatment of engraftment syndrome associated with hematopoietic cell transplantation, the present inventors have surprisingly been able to obtain hematopoiesis by administering thrombomodulin. It was found that clinical symptoms of engraftment syndrome associated with cell transplantation are improved. References disclosing thrombomodulin include Am. J. et al. Physiol. Lung Cell. Mol. Physiol. 273: L889-L894, 1997 and Japanese Patent Application Laid-Open No. 2008-189574, but these do not describe any engraftment syndrome associated with hematopoietic cell transplantation.

  That is, the present invention relates to a medicament for containing a thrombomodulin as an active ingredient and for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation. The medicament of the present invention can be improved upon hematopoietic ability after hematopoietic cell transplantation by administering it at the time of hematopoietic cell transplantation, which is expected as a radical treatment method for hematopoietic malignant tumors. In addition, the bone marrow image and peripheral blood cell count of patients with hematopoietic malignant tumors accompanying hematopoietic cell transplantation can be normalized, and hematopoietic malignant tumors can be treated and / or improved more effectively.

Specific examples of the present invention include the following.
[1] A medicament comprising thrombomodulin as an active ingredient for preventing and / or treating engraftment syndrome associated with hematopoietic cell transplantation.
[2] The medicament according to [1], wherein the thrombomodulin is a soluble thrombomodulin.
[3] The above [1] or [3], 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 shown in SEQ ID NO: 9 or 11 2].
[3-2] The thrombomodulin is obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence described in 1, 3, 5, 7, 9, or SEQ ID NO: 11. The medicine according to [1] or [2] above, which is a peptide.
[3-3] The thrombomodulin is a peptide obtained by a transformed cell prepared by transfecting a host cell with DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, [1] Or the medicine according to [2].
[3-4] The above-mentioned [1], wherein the thrombomodulin is a peptide obtained by a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. Or the medicine according to [2].

[4] The thrombomodulin is a peptide comprising any one of the following amino acid sequences (i-1) or (i-2), and the peptide is a peptide having thrombomodulin activity: High purity soluble thrombomodulin according to
(I-1) the amino acid sequence of positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11; or (i-2) one or more of the amino acid sequences of (i-1) above An amino acid sequence in which the amino acids are substituted, deleted, or added.
[4-2] The above-mentioned [1] or [4], wherein the thrombomodulin is a peptide comprising any of the following amino acid sequences (i-1) or (i-2), and the peptide is a peptide having thrombomodulin activity: 2];
(I-1) the amino acid sequence at positions 19 to 132 in the amino acid sequence of either SEQ ID NO: 1 or SEQ ID NO: 3, or (i-2) one or more of the amino acid sequences of (i-1) above An amino acid sequence in which the amino acids are substituted, deleted, or added.

[4-3] The above-mentioned [1] or [4], wherein the thrombomodulin is a peptide comprising any of the following amino acid sequences (i-1) or (i-2), and the peptide is a peptide having thrombomodulin activity: 2];
(I-1) the amino acid sequence of positions 19 to 480 in the amino acid sequence of SEQ ID NO: 5 or 7; or (i-2) one or more of the amino acid sequences of (i-1) above An amino acid sequence in which the amino acids are substituted, deleted, or added.
[4-4] The medicament according to [1] or [2], wherein the thrombomodulin is a peptide having a sequence of positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

[4-5] The above [1] or [2], wherein the thrombomodulin is a peptide of any of the following (i-1) to (i-3), and the peptide is a peptide having thrombomodulin activity: Medicines;
(I-1) a peptide comprising the sequence of positions 19 to 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515 in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. ,
(I-2) One of the sequences of positions 19 to 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515 in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. Or a peptide comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, or added, or (i-3) a group consisting of the peptide described in (i-1) and the peptide described in (i-2). A mixture of two or more peptides selected from.

[4-6] The thrombomodulin is located at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. The medicament according to [1] or [2] above, which is a peptide having a sequence or a mixture thereof.
[4-7] The thrombomodulin is located at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. The medicament according to [1] or [2] above, which is a peptide consisting of a sequence or a mixture thereof.

[4-8] Any of the above [4], [4-2], [4-3], or [4-5], wherein the thrombomodulin activity is an activity having the following properties (1) to (4): The medicine according to any one of the above.
(1) an action of selectively binding to thrombin,
(2) the action of promoting the activation of protein C by thrombin,
(3) the action of prolonging the coagulation time by thrombin, and (4) the action of inhibiting platelet aggregation by thrombin;
[5] The medicament according to any one of [1] to [4-8], wherein the engraftment syndrome is a pathological condition involving at least one selected from the group consisting of rash, pulmonary edema, and diarrhea.
In addition, when the term number quoted as a range such as [1] to [4-8] is shown in the range, and a term having a branch number such as [4-2] is arranged in the range, It means that a term having a branch number such as [4-2] is also cited. The same applies to the following.

[5-2] The engraftment syndrome is a pathological condition accompanied by at least two selected from the group consisting of fever exceeding 38 ° C., rash, pulmonary edema, weight gain, decreased albumin level, dyspnea, and hypoxia The medicine according to any one of [1] to [4-8].
[5-3] The medicament according to any one of [1] to [4-8], wherein the engraftment syndrome is a pathological condition of pulmonary edema.
[5-4] The medicament according to any one of [1] to [4-8], wherein the engraftment syndrome is a pathological condition of pulmonary edema and weight gain.

[6] A medicine comprising a combination of the medicine according to any one of [1] to [5-4] above and at least one selected from the group consisting of an immunosuppressant and a hematopoietic malignant tumor therapeutic agent, The medicament for preventing and / or treating engraftment syndrome associated with hematopoietic cell transplantation.
[6-2] A medicine comprising a combination of the medicine according to any one of [1] to [5-4] above and an immunosuppressive agent for preventing and / or preventing engraftment syndrome associated with hematopoietic cell transplantation. The medicament for treatment.
[6-3] A drug comprising a combination of the drug according to any one of [1] to [5-4] above, an immunosuppressant and a hematopoietic malignant tumor therapeutic agent, The medicament for preventing and / or treating wearing syndrome.
[6-4] The medicine according to any one of [1] to [5-4], an immunosuppressant, a hematopoietic malignant tumor therapeutic agent, and a preventive and / or therapeutic agent for complications associated with hematopoietic cell transplantation A pharmaceutical comprising a combination of at least one selected from the group consisting of the above, for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation.

[7] The medicament according to any one of [1] to [5-4] for administration together with at least one selected from the group consisting of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent.
[7-2] The medicament according to any one of [1] to [5-4] for administration together with an immunosuppressant.
[7-3] The medicament according to any one of [1] to [5-4] for administration together with an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent.
[7-4] The above [1] to be administered together with at least one selected from the group consisting of an immunosuppressive agent, a hematopoietic malignant tumor therapeutic agent, and a preventive and / or therapeutic agent for complications associated with hematopoietic cell transplantation [5-4] The medicine according to any one of [5-4].
[7-5] any one of [1] to [5-4] for administration together with an immunosuppressive agent, a hematopoietic malignant tumor therapeutic agent, and a prophylactic and / or therapeutic agent for complications associated with hematopoietic cell transplantation The pharmaceutical described.

[8] A pharmaceutical comprising thrombomodulin as an active ingredient for improving the hematopoietic ability accompanying hematopoietic cell transplantation in a hematopoietic malignant tumor.
[9] Use of thrombomodulin for the manufacture of a medicament for preventing and / or treating engraftment syndrome associated with hematopoietic cell transplantation.
[9-2] Use of thrombomodulin for the manufacture of a medicament for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation, which is described in any one of [1] to [7-5] above The use having features.
[10] A method for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation, comprising a step of administering thrombomodulin to a mammal.
[10-2] A method for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation, comprising a step of administering thrombomodulin to a mammal, comprising the above-mentioned [1] to [7-5] Said method which has the characteristics in any one of these.

  With the medicament containing thrombomodulin of the present invention, engraftment syndrome associated with hematopoietic cell transplantation can be prevented and / or treated.

The time-dependent change of the various test values after hematopoietic cell transplantation is implemented in MDS patients is shown. The day of hematopoietic cell transplantation was displayed as Day0. The period of administration of ATIII (1,500 units), granulocyte colony-stimulating factor (G-CSF, 450 μg), and TMD123 (rTM, 380 units / kg [0.06 mg / kg]) is indicated by a bold black line on the figure frame. Displayed. In addition, the dates of platelet transfusion (PL, 10 units) and red blood cell transfusion (RBC, 2 units) were indicated by arrows. The body weight increase indicating the progress of edema was observed until the 5th day of rTM administration, but the body weight decreased after the 6th day of rTM administration. In the above figure, ■ Body weight (kg) indicates body weight. O FDP (μg / ml) indicates a fibrinogen and fibrin degradate product (Fibrinogen and Fibrin Degradate Product). ΔPT (sec) indicates the prothrombin time. ◆ D-dimer (μg / mL) indicates D-dimer. ◇ SFMC (μg / mL) indicates a soluble fibrin monomer complex. In the figure below, ○ BT (° C.) indicates body temperature. ◆ Plt (× 104 / μl) indicates the platelet count. ΔAT (%) indicates antithrombin. (3) WBC (/ μL) indicates the white blood cell count.

Hereinafter, the present invention will be specifically described.
The thrombomodulin in the present invention is known to have the action of (1) selectively binding to thrombin and (2) promoting the activation of protein C by thrombin. Moreover, it is preferable that (3) the action of prolonging the coagulation time by thrombin and / or (4) the action of suppressing the platelet aggregation by thrombin is usually observed. The action of these thrombomodulins is sometimes called thrombomodulin activity. The thrombomodulin activity preferably has the effects (1) and (2) above, and further has all the effects (1) to (4) above.

  The binding action of thrombomodulin with thrombin is described in, for example, Thrombosis and Haemostasis 1993 70 (3): 418-422 and The Journal of Biological Chemistry 1989 Vol. 264, no. 9 pp. It can be confirmed by the test methods described in various known documents including 4872-4876. The action of accelerating the activation of protein C by thrombin is, for example, the action of accelerating the activation of protein C by a test method clearly described in various known literatures including JP-A-64-6219. The amount of activity and its presence can be easily confirmed. In addition, the action of prolonging the coagulation time by thrombin and / or the action of suppressing platelet aggregation by thrombin can be easily confirmed in the same manner.

  The thrombomodulin in the present invention is not particularly limited as long as it has thrombomodulin activity, but is preferably a soluble thrombomodulin. A preferable example of the solubility of soluble thrombomodulin is 1 mg / mL in water, for example, distilled water for injection (in the absence of a surfactant such as Triton X-100 or polidocanol, usually near neutrality). 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. Preferably, 60 mg / mL or more is mentioned, and depending on the case, 80 mg / mL or more, or 100 mg / mL or more is mentioned, respectively. In determining whether or not soluble thrombomodulin could be dissolved, it was clear and clearly recognized when dissolved, for example, directly under a white light source at a brightness of about 1000 lux. It is understood that the absence of such an insoluble material is a simple indicator. Moreover, it can also filter and can confirm the presence or absence of a residue.

  The thrombomodulin in the present invention preferably includes the amino acid sequence at positions 19 to 132 of SEQ ID NO: 1, which is known as the central site of thrombomodulin activity in human thrombomodulin. It is not particularly limited as long as it includes the amino acid sequence at position 132. 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. In the amino acid sequence at positions 19 to 132, one or more amino acids 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, 70% or more homology is preferable, and 80% or more homology is more. Preferably, a homology of 90% or more is further preferred, a homology of 95% or more is particularly preferred, and a homology of 98% or more is most preferred. An amino acid sequence in which one or more amino acids in the amino acid sequence are substituted, deleted, or added in this way is called a homologous mutant sequence. As described later, these mutations can be easily obtained by using ordinary gene manipulation techniques.

  The sequence of SEQ ID NO: 3 is obtained by mutating Val, which is the amino acid at position 125 of SEQ ID NO: 1, to Ala, and includes the amino acid sequences of positions 19 to 132 of SEQ ID NO: 3 as thrombomodulin in the present invention. It is also preferable.

  As described above, the thrombomodulin in the present invention includes at least a sequence at positions 19 to 132 of SEQ ID NO: 1 or SEQ ID NO: 3, or a homologous mutant sequence thereof, and includes at least a peptide sequence having thrombomodulin activity. Although not particularly limited, preferred examples include peptides consisting of the 19th to 132nd positions or the 17th to 132nd positions in SEQ ID NO: 1 or SEQ ID NO: 3, respectively, or peptides having at least thrombomodulin activity consisting of homologous mutant sequences of the above sequences. Peptides consisting of sequences 19 to 132 of SEQ ID NO: 1 or SEQ ID NO: 3 are more preferred. There is also another embodiment in which 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.

  Further, as another embodiment of the thrombomodulin in the present invention, it preferably includes the amino acid sequence at positions 19 to 480 of SEQ ID NO: 5, and includes the amino acid sequence at positions 19 to 480 of SEQ ID NO: 5. There is no particular limitation. 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 the one in which Val, which is the amino acid at position 473 of SEQ ID NO: 5, is mutated to Ala. The thrombomodulin in the present invention includes the amino acid sequence of positions 19 to 480 of SEQ ID NO: 7. It is also preferable.

  Thus, the thrombomodulin in the present invention includes at least a sequence at positions 19 to 480 of SEQ ID NO: 5 or 7 or a homologous mutant sequence thereof, and includes a peptide sequence having at least thrombomodulin activity. Preferable examples include, but not limited to, a peptide consisting of the 19th to 480th positions or the 17th to 480th positions in SEQ ID NO: 5 or 7 respectively, or a peptide consisting of homologous mutants of the above sequences and having at least thrombomodulin activity. Peptides consisting of the 19th to 480th positions of SEQ ID NO: 5 or SEQ ID NO: 7 are more preferred. In addition, there is another embodiment in which a peptide having a thrombomodulin activity consisting of homologous mutant sequences at positions 19 to 480 or positions 17 to 480 in SEQ ID NO: 5 or SEQ ID NO: 7 is preferred.

In addition, as another embodiment of the thrombomodulin in the present invention, it preferably includes the amino acid sequence at positions 19 to 515 of SEQ ID NO: 9, and includes the amino acid sequence at positions 19 to 515 of SEQ ID NO: 9. There is no particular limitation. 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 obtained by mutating Val, which is the amino acid at position 473 of SEQ ID NO: 9, to Ala, and includes the amino acid sequences of positions 19 to 515 of SEQ ID NO: 11 as thrombomodulin in the present invention. It is also preferable.

  As described above, the thrombomodulin in the present invention includes at least a sequence at positions 19 to 515 of SEQ ID NO: 9 or SEQ ID NO: 11, or a homologous mutant sequence thereof, and includes a peptide sequence having at least thrombomodulin activity. Although not particularly limited, a peptide comprising a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in each of SEQ ID NO: 9 or SEQ ID NO: 11, or homology of the above sequences A peptide comprising a mutated sequence and having at least thrombomodulin activity is a more preferred example, and comprises a sequence of positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9. Peptides are particularly preferred. Mixtures of these are also preferred examples. In addition, there is another embodiment in which a peptide comprising a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 11 is particularly preferred. Mixtures of these are also preferred examples. Furthermore, a peptide comprising these homologous mutant sequences and having at least thrombomodulin activity is also a preferred example.

  The peptide having a homologous mutation sequence is as described above, but one or more, that is, one or more amino acids, more preferably several (for example, 1 to 20, preferably 1) in the amino acid sequence of the peptide of interest. 1 to 10, more preferably 1 to 5, and particularly preferably 1 to 3 amino acids). 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, 70% or more homology is preferable, and 80% or more homology is more. Preferably, a homology of 90% or more is further preferred, a homology of 95% or more is particularly preferred, and a homology of 98% or more is most preferred.

  Furthermore, the thrombomodulin in the present invention includes a peptide consisting of SEQ ID NO: 14 (462 amino acid residues), a peptide consisting of SEQ ID NO: 8 (272 amino acid residues), or SEQ ID NO: 6 (residue of 236 amino acids) in JP-A-64-6219. Peptides comprising the group) 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 amino acid sequence at positions 19 to 132 of SEQ ID NO: 1 or SEQ ID NO: 3. Among them, the 19th to SEQ ID NO: 5 or SEQ ID NO: 7 A peptide having at least an amino acid sequence at position 480 is preferable, and a peptide having at least an amino acid sequence at positions 19 to 515 of SEQ ID NO: 9 or 11 is more preferable. Examples of the peptide having at least the 19th to 515th amino acid sequence of SEQ ID NO: 9 or 11 include the 19th to 516th positions, the 19th to 515th positions, and the 19th position in SEQ ID NO: 9 or 11 respectively. Peptides consisting of the sequence at positions -514, 17-516, 17-515, or 17-514 are more preferred examples. In addition, the sequences of positions 19 to 516, positions 19 to 515, positions 19 to 514, positions 17 to 516, positions 17 to 515, or positions 17 to 514 in SEQ ID NO: 9 or SEQ ID NO: 11, respectively. A more preferred example is a mixture of the peptides consisting of SEQ ID NO: 9 and SEQ ID NO: 11 respectively.

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

Examples of the mixing ratio of peptides ending at positions 514, 515, and 516 in SEQ ID NO: 9 or 11 are (0: 0: 100) to (0:90:10). In some cases, (0:70:30) to (10: 90: 0), (10: 0: 90) to (20:10:70) are exemplified.
The mixing ratio of these peptides can be determined by an ordinary method.

  The sequence of positions 19 to 132 of SEQ ID NO: 1 corresponds to the sequence of positions 367 to 480 of SEQ ID NO: 9, and the sequence of positions 19 to 480 of SEQ ID NO: 5 is the sequence of positions 19 to It corresponds to the 480th sequence. In addition, the 19th to 132nd sequence of SEQ ID NO: 3 corresponds to the 367th to 480th sequence of SEQ ID NO: 11, and the 19th to 480th sequence of SEQ ID NO: 7 is the 19th to 19th sequence of SEQ ID NO: 11. It corresponds to the 480th sequence. Furthermore, the sequences at positions 1 to 18 in each of SEQ ID NOs: 1, 3, 5, 7, 9, and 11 are all the same sequence.

  As described later, these thrombomodulins in the present invention are DNAs encoding these peptides (specifically, nucleotides 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). Sequence) can be obtained from a transformed cell prepared by transfecting a host cell with a vector.

  Further, these peptides only have to have the amino acid sequence described above, and may or may not have a sugar chain, and this point is not particularly limited. In the genetic manipulation, 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. Regarding the binding position and type of sugar chains, the facts described in JP-A No. 11-341990 are known, and the same sugar chain may be added at the same position for thrombomodulin in the present invention. As will be described later, it is not specified to be obtained by genetic manipulation, but when obtained by genetic manipulation, as a signal sequence that can be used for expression, amino acids 1 to 18 of SEQ ID NO: 9 are used. A nucleotide sequence encoding the sequence, a nucleotide sequence encoding the amino acid sequence at positions 1 to 16 of SEQ ID NO: 9, and other known signal sequences such as a signal sequence of human tissue plasminogen activator can be used (International Publication 88). / 9811 publication).

  When introducing a DNA sequence encoding thrombomodulin into a host cell, a DNA sequence encoding thrombomodulin is preferably incorporated into a vector, particularly preferably an expression vector that can be expressed in animal cells. An expression vector is a DNA molecule comprising a promoter sequence, a sequence that gives a ribosome binding site to mRNA, a DNA sequence that encodes a protein to be expressed, a splicing signal, a termination terminator sequence for transcription termination, a replication origin sequence, etc. Examples of animal cell expression vectors include pSV2-X reported by Mulligan RC et al. [Proc Natl Acad Sci USA 1981; 78: 2072-2076] and Howley PM et al [Methods in Emzymology 1983; 101: 387-402. PBP69T (69-6) reported by Academic Press].

  Examples of host cells that can be used for producing these peptides include Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, and canine kidney-derived MDCK. Cells, hamster AV-12-664 cells, etc., and human-derived cells include HeLa cells, WI38 cells, and human 293 cells. CHO cells are very common and preferred, and among CHO cells, DHFR-CHO cells are more preferred.

  In addition, microorganisms such as Escherichia coli are often used in the process of gene manipulation and peptide production, and it is preferable to use a host-vector system suitable for each, and an appropriate vector system is selected for the above host cells. can do. The gene of thrombomodulin used in the gene recombination technique has been cloned, and an example of production using the gene recombination technique of thrombomodulin is disclosed, and further, a purification method for obtaining the purified product is also known. [Japanese Patent Laid-Open Nos. 64-6219, 2-255699, 5-213998, 5-310787, 7-155176, J Biol Chem 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 by following the method described therein. For example, JP-A-64-6219 discloses Escherichia coli K-12 strain DH5 (ATCC Deposit No. 67283) containing plasmid pSV2TMJ2 containing DNA encoding full-length thrombomodulin. Moreover, the strain (Escherichia coli DH5 / pSV2TM J2) (FERM BP-5570) of Life Research Institute (the National Institute of Advanced Industrial Science and Technology (AIST)) can be used as this strain. Using the DNA encoding this full-length thrombomodulin as a raw material, 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. For example, refer to the method of Yamamoto et al. [Japanese Patent Laid-Open No. 64-6219] or Japanese Patent Laid-Open No. 5-213998. Can be. That is, 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. As this modification, for example, a codon encoding the amino acid at position 473 of SEQ ID NO: 9 to form a DNA encoding the amino acid sequence of SEQ ID NO: 11 (specifically, consisting of the base sequence of SEQ ID NO: 12) In particular, site-directed mutagenesis is performed according to the method of Zoller MJ et al. [Methods in Enzymology 1983; 100: 468-500, Academic Press]. For example, the 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 hamster ovary (CHO) cells to be appropriately selected as transformed cells, and thrombomodulin purified by a known method from a culture solution obtained by culturing the cells. Can be manufactured. As described above, it is preferable to transfect the host cell with DNA encoding the amino acid sequence of SEQ ID NO: 9 (SEQ ID NO: 10). The method for producing thrombomodulin used in the present invention is not limited to the above-mentioned method, and for example, it can be extracted and purified from urine, blood, other body fluids, etc., and the thrombomodulin producing tissue or these It can be extracted and purified from tissue culture medium or the like, and if necessary, further cleaved with a proteolytic enzyme.
When the thrombomodulin in the present invention is produced by the above cell culture method, diversity may be recognized in the N-terminal amino acid due to post-translational modification of the protein. For example, the amino acid at position 17, 18, 19, or 22 in SEQ ID NO: 9 may be the N-terminus. In addition, for example, the N-terminal amino acid may be modified so that glutamic acid at position 22 is converted to pyroglutamic acid. The amino acid at position 17 or 19 is preferably N-terminal, and the amino acid at position 19 is more preferably N-terminal. There is also another embodiment in which the amino acid at position 17 is preferably N-terminal. The same examples can be given for SEQ ID NO: 11 with respect to the above modifications and diversity.

  Furthermore, when soluble 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. In addition, a peptide having a short two amino acid residues may be produced. That is, the amino acid at position 514 may be the C-terminus. Therefore, a peptide rich in diversity in N-terminal amino acid and C-terminal amino acid, or a mixture thereof may be produced. The amino acid at position 515 or the amino acid at position 516 is preferably C-terminal, and the amino acid at position 516 is more preferably C-terminal. There is also another embodiment in which the amino acid at position 514 is preferably C-terminal. The above modifications and diversity are the same for DNA having the base sequence of SEQ ID NO: 12.

  The 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, the peptide comprising the sequence of positions 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 17 to 514 in SEQ ID NO: 9. A mixture is mentioned.

  Subsequently, the method for isolating and purifying thrombomodulin from the culture supernatant or the culture obtained as described above can be performed in accordance with a known method [edited by Takeichi Horio, basic experimental method of protein / enzyme, 1981]. For example, it is also preferred to use ion exchange chromatography or adsorption chromatography using an interaction between a chromatographic carrier having a functional group having a charge opposite to that of thrombomodulin immobilized thereon and thrombomodulin. A preferred example is affinity chromatography using specific affinity with thrombomodulin. Preferable examples of the adsorbent include an example using an antibody of thrombin or thrombomodulin which is a ligand of thrombomodulin. As this antibody, an antibody of thrombomodulin that recognizes an appropriate property or an appropriate epitope can be used. For example, JP-B-5-42920, JP-A-64-45398, JP-A-6-206992 The example described in the gazette gazette etc. is mentioned. Further, gel filtration chromatography and ultrafiltration using the molecular weight size of thrombomodulin can be mentioned. In addition, 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. Moreover, it is also possible to use hydroxyapatite as a carrier for adsorption chromatography, and examples thereof include those described in JP-A-9-110900. These methods can be appropriately combined. The degree of purification can be selected depending on the purpose of use. For example, electrophoresis, preferably SDS-PAGE results can be obtained as a single band, or isolated purified products can be obtained by gel filtration HPLC or reverse phase HPLC. It is desirable to purify until one peak is reached. Of course, in the case of using a plurality of types of thrombomodulin, it is preferable that a band substantially consists of only thrombomodulin, and it does not require 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. For example, the culture supernatant or culture is roughly purified with Q-Sepharose Fast Flow of an ion exchange column to have thrombomodulin activity. Fractions were collected, and then the main fraction was collected with an affinity column DIP-thrombin-agarose (diisopropylphosphorbromine agarose) column to collect a fraction with strong thrombomodulin activity. The collected fraction was concentrated and subjected to gel filtration to purify the thrombomodulin activity fraction. Purification method [Gomi K et al, Blood 1990; 75: 1396-1399] obtained as a product. Examples of the thrombomodulin activity used as an index include activity of promoting protein C activation by thrombin. Other preferred purification methods are as follows.

  Select an appropriate ion exchange resin with thrombomodulin and good adsorption conditions, and perform ion exchange chromatography purification. A particularly preferred example is a method using Q-Sepharose Fast Flow equilibrated with 0.02 mol / L Tris-HCl buffer (pH 7.4) containing 0.18 mol / L NaCl. After appropriate washing, elution is carried out with, for example, 0.02 mol / L Tris-HCl buffer (pH 7.4) containing 0.3 mol / L NaCl to obtain a crude product thrombomodulin.

  Next, for example, a substance having specific affinity for thrombomodulin can be immobilized on a resin and subjected to affinity chromatography purification. Preferred examples include a DIP-thrombin-agarose column and an anti-thrombomodulin monoclonal antibody column. The DIP-thrombin-agarose column is pre-equilibrated with, for example, 20 mmol / L Tris-HCl buffer (pH 7.4) containing 100 mmol / L NaCl and 0.5 mmol / L calcium chloride, and the above crude product is charged. Then, the thrombomodulin is obtained by eluting with 20 mmol / L Tris-HCl buffer (pH 7.4) containing 1.0 mol / L NaCl and 0.5 mmol / L calcium chloride. Can do. In addition, in the anti-thrombomodulin monoclonal antibody column, 0.5 mol / L NaCl-containing 0.1 mol / L NaHCO3 buffer solution in which anti-thrombomodulin monoclonal antibody was dissolved in Sepharose 4FF (GE Healthcare Bioscience) previously activated with CNBr ( A column packed with a resin that was brought into contact with pH 8.3) and coupled with Sepharose 4FF with an anti-thrombomodulin monoclonal antibody was preliminarily treated with, for example, 20 mmol / L phosphate buffer (pH 7.3) containing 0.3 mol / L NaCl. An example is a method of equilibrating and appropriately eluting with a 100 mmol / L glycine hydrochloride buffer (pH 3.0) containing 0.3 mol / L NaCl. The eluate can be neutralized with an appropriate buffer and obtained as a purified product.

  Next, after adjusting the purified product obtained to pH 3.5, a cation exchanger, preferably a strong cation, equilibrated with 100 mmol / L glycine hydrochloride buffer (pH 3.5) containing 0.3 mol / L NaCl. Charge to SP-Sepharose FF (GE Healthcare Bioscience), which is an exchanger, and wash with the same buffer to obtain a non-adsorbed fraction. The obtained fraction can be neutralized with an appropriate buffer and obtained as a highly purified product. These are preferably concentrated by ultrafiltration.

  Furthermore, it is also preferable to perform buffer exchange by gel filtration. For example, a Sephacryl S-300 column or S-200 column equilibrated with 20 mmol / L phosphate buffer (pH 7.3) containing 50 mmol / L NaCl is charged with a highly purified product concentrated by ultrafiltration. And developed with a 20 mmol / L phosphate buffer solution (pH 7.3) containing 50 mmol / L NaCl, and confirmed the activity to promote protein C activation by thrombin to collect the active fraction. Exchanged high-purity products can be obtained. The purified high-purity product thus obtained is preferably filtered using a suitable virus-removing membrane, for example, Planova 15N (Asahi Kasei Medical Co., Ltd.) in order to enhance safety, and then subjected to ultrafiltration for the desired concentration. Can be concentrated. Finally, it is preferable to filter through a sterile filtration membrane.

  In the present invention, hematopoietic cell transplantation includes bone marrow which is the greatest side effect of hematopoietic malignant tumor treatment, congenital immunodeficiency disease, inborn error of metabolism, and high-dose chemotherapy and radiation therapy for solid tumors as described above. Suppression relief therapy is a specific example. The medicament of the present invention can also be used for the above-described hematopoietic cell transplantation.

  As a method for transplanting hematopoietic cells in the present invention, 1) bone marrow cells collected from a donor are infused into a patient, 2) peripheral blood is collected from a donor administered with G-CSF, and only CD34 positive cells are taken out and infused into a patient. Or 3) Thaw frozen umbilical cord blood and infuse it into the patient.

  As donors in the present invention, hematopoietic malignant tumor patients themselves, close relatives of hematopoietic malignant tumor patients whose human leukocyte antigen (HLA) matches or nearly matches, third parties whose HLA matches or nearly matches The method of selection is given as a preferred example.

  One embodiment of the medicament of the present invention can prevent and / or treat engraftment syndrome. Engraftment syndrome is a condition that presents symptoms such as fever, erythema-like skin rash, and non-cardiogenic pulmonary edema during the period until hematopoietic cells engraft after hematopoietic stem cell transplantation. Examples of the period until hematopoietic cell transplantation is engrafted include a period from the day when the engraftment is confirmed to 7 days ago.

  The engraftment syndrome in the present invention is not particularly limited as long as it is a pathological condition involving at least one selected from the group consisting of skin eruption, pulmonary edema (hereinafter sometimes referred to as lung infiltration), and diarrhea (diagnostic criteria of Maiolino et al.). Maiolino A et al, Bone Marlow Transplant 2003; 31: 393-7), preferably a pathological condition of pulmonary edema.

  Further, as one aspect of the engraftment syndrome in the present invention, at least two selected from the group consisting of fever exceeding 38 ° C., rash, pulmonary edema, weight gain, decreased albumin level, dyspnea, and hypoxia are involved. The pathology may be mentioned (Schmid I, Biol Blood Marrow Transplant 2008; 14: 438-44). Among these, pulmonary edema and weight gain are preferable.

  Furthermore, one aspect of the engraftment syndrome in the present invention is a diagnostic condition based on Spitzer's diagnostic criteria (Spitzer TR, Bone Marlow Transplant 2001; 27: 893-8), that is, a major criterion and a minor criterion. The major criteria are 1) non-infectious fever above 38.3 ° C, 2) non-drug erythema-like rash over 25% of the body surface area, 3) wide pulmonary infiltration shadow, hypoxemia Non-cardiogenic pulmonary edema associated with 1) total criteria as follows: 1) Liver dysfunction with 2 mg / dl or more of total bilirubin or more than 2 times normal level of transaminase 2) Renal dysfunction (more than 2 times of serum creatinine) 3) Weight gain of 2.5% or more, 4) Transient encephalopathy of unknown cause. It is diagnosed as engraftment syndrome in the case of a pathological condition that satisfies the above three major criteria, or two major criteria and one or more minor criteria.

  Pulmonary edema refers to a state in which water leaks and accumulates in the parenchyma (bronchi, alveoli), and breathing is impaired by water, resulting in respiratory failure. The lung capillaries originally have small holes for gas exchange, and blood leaks out of the blood vessels and perfuses them to supply oxygen and nutrients to surrounding cells and collect waste products. . In a normal state, the amount of leakage and the amount of return to the blood vessel are balanced, but if the balance is lost for some reason and the amount of leakage exceeds, water will accumulate in the alveoli and bronchi. In pulmonary edema, dyspnea, pink wrinkles, sweating, cyanosis, auscultation of wet rales, etc. are observed, and eventually death occurs due to respiratory failure or heart failure.

  Pulmonary edema is roughly classified into cardiogenic pulmonary edema and non-cardiogenic pulmonary edema based on its cause. In cardiogenic pulmonary edema, due to myocardial infarction or the like, the force of pumping blood from the left ventricle of the heart to the whole body decreases, the internal pressure of the capillary increases, and water leaks out of the blood vessel. Non-cardiogenic pulmonary edema is secondary to severe pneumonia, sepsis, severe trauma, cirrhosis, nephrosis, and drug addiction, and is caused by decreased plasma colloid osmotic pressure (reduced albumin) and increased vascular permeability.

  Examples of pulmonary edema in the present invention include at least one symptom of dyspnea, hypoxemia, pink wrinkles, sweating, and cyanosis. The symptom of pulmonary edema can be confirmed by, for example, chest X-ray photography or auscultation. Improvement of pulmonary edema can be confirmed by, for example, a decrease in lung field (invasion shadow) in which chest X-ray permeability has decreased, an increase in arterial oxygen partial pressure, and a decrease in lung field in which auscultation shows blistering rales. .

Examples of weight gain in the present invention include an increase of 2.5% or more before and after engraftment. Weight gain can be diagnosed by measuring over time using a scale.
Examples of the fever in the present invention include a state in which the body temperature exceeds 38 ° C. As for the body temperature, for example, at least one of rectal temperature, oral temperature, axillary temperature, and eardrum temperature may be measured, and can be measured using a thermometer or thermography corresponding to the measurement of each part.

  Examples of the eruption in the present invention include a non-drug erythema-like eruption over 25% of the body surface area. The diagnosis of skin rash can be made by visual inspection, palpation, and biopsy.

  The decrease in albumin level in the present invention refers to a state in which serum albumin has decreased, and can be diagnosed by measuring the concentration of albumin in serum collected and separated from a patient after hematopoietic cell transplantation. Examples of the decreased albumin value include serum albumin values of less than 3.0 mg / dL, and preferred examples of less than 2.5 mg / dL. The method for measuring serum albumin is not particularly limited, but preferred examples include bromcresol green (BCG) method, bromcresol purple (BCP) method, and BCP improvement method, and BCP improvement method is more preferable.

  The breathing difficulty in the present invention is not particularly limited as long as there is a subjective symptom such as difficulty and effort during breathing. Diagnosis is possible if the patient is aware of dyspnea after hematopoietic cell transplantation.

  Examples of the hypoxia in the present invention include a state where the oxygen partial pressure in arterial blood is 70 mmHg (Torr) or less, and 60 mmHg (Torr) is a preferred example. The arterial blood oxygen partial pressure can be measured by using a blood gas analyzer for blood collected from an artery. Examples of the artery to be collected include, but are not limited to, the brachial artery, femoral artery, and radial artery.

  The cyanosis in the present invention refers to a state in which the skin and mucous membrane are bluish purple as the oxygen concentration in the blood decreases, and can be diagnosed by visual inspection.

  The engraftment of the transplanted hematopoietic cells in the present invention includes that the cells introduced in the hematopoietic cell transplant are engrafted in the bone marrow, and the bone marrow image shows how alive the introduced hematopoietic cells are at a specific time point. One index indicating

  As an index showing improved engraftment of transplanted hematopoietic cells, the number of nucleated cells and the number of megakaryocytes in the bone marrow image can be evaluated. The lower limit of the number of nucleated cells in the bone marrow image is preferably 100,000 / μL or more, and the upper limit is preferably 250,000 / μL or less. The lower limit of the number of megakaryocytes is preferably 50 / μL or more, and the upper limit is preferably 150 / μL or less. The period for recovering the number of nucleated cells and megakaryocytes in the myeloid image to the above range is preferably 6 months after transplantation, more preferably 3 months after transplantation, and further preferably 1 month after transplantation.

  Moreover, it is also preferable to evaluate the period until the peripheral blood neutrophil count reaches 500 / μL or more as an index indicating the improvement of transplanted hematopoietic cell engraftment. In the case of bone marrow transplantation, the upper limit of the reaching period is preferably 28 days or less, and more preferably 21 days or less. Although it does not restrict | limit especially as a minimum, 14 days or more are preferable. In the case of cord blood transplantation, the upper limit of the reaching period is preferably 42 days or less, and more preferably 35 days or less. Although it does not restrict | limit especially as a minimum, 14 days or more are preferable. Setting the engraftment rate of transplanted hematopoietic cells in the above range is also a preferred embodiment for improving the engraftment rate of transplanted hematopoietic cells.

  Specifically, as the survival period of the transplanted hematopoietic cells in the present invention, the lower limit is preferably 1 month or more, more preferably 3 months or more, further preferably 6 months or more, particularly preferably 1 year or more, and 3 years or more. The upper limit is not particularly limited, but 10 years or less is preferable. Setting the survival period of the transplanted hematopoietic cells within the above range is also a preferred embodiment of extending the survival period of the transplanted hematopoietic cells.

Specifically, the upper limit of the number of transplants in the present invention is preferably 3 times or less, more preferably 2 times or less, and most preferably 1 time. Moreover, as a minimum, although it is not limited, 1 time or more is preferable. Most preferably, there is no re-transplantation.
Examples of the peripheral blood cell count in the present invention include the number of red blood cells, white blood cells, and platelets in whole blood. Further, normalizing the peripheral blood cell count is not particularly limited as long as the peripheral blood cell count is increased to a state where it can be determined that the pathology of the hematopoietic organ-created tumor has been improved.

  Normalized blood counts of peripheral blood are exemplified by the number of red blood cells, white blood cells, and platelets in normal whole blood after transplantation. The lower limit of the number of peripheral blood erythrocytes is preferably 3.6 million / μL or more in the case of men, more preferably 3,800,000 / μL or more, and further preferably 4 million / μL or more. The upper limit of the number of peripheral blood erythrocytes is preferably 5.8 million / μL or less, more preferably 5.4 million / μL or less. On the other hand, the lower limit of the peripheral blood red blood cell count for women is preferably 3.3 million / μL or more, more preferably 3.6 million / μL or more. The upper limit of the peripheral blood red blood cell count is preferably 5.2 million / μL or less, more preferably 4.9 million / μL or less.

  The lower limit of the peripheral blood white blood cell count is preferably 4,000 / μL or more. Further, the upper limit of the peripheral blood leukocyte count is preferably 14,000 / μL or less. The lower limit of the peripheral blood platelet count is preferably 80,000 / μL or more, more preferably 120,000 / μL or more, and most preferably 150,000 / μL or more. Further, the upper limit of the peripheral blood platelet count is preferably 500,000 / μL or less, and more preferably 350,000 / μL or less. The period for recovering the peripheral blood cell count to the above range is preferably 6 months after transplantation, more preferably 3 months after transplantation, and even more preferably 1 month after transplantation. Setting the peripheral blood cell count within the above range is also a preferred embodiment of normalizing the peripheral blood cell count.

  The medicament of the present invention can be administered to a patient who receives a hematopoietic cell transplant or a patient who has received a hematopoietic cell transplant. That is, the timing of administration of the medicament of the present invention to a patient may be before receiving a hematopoietic cell transplant or after receiving a hematopoietic cell transplant. When a drug is administered to a patient undergoing hematopoietic cell transplantation, the drug may be administered to the patient prior to receiving hematopoietic cell transplantation, and it is preferable to administer the drug before the start of pretreatment for hematopoietic cell transplantation. In addition, when a drug is administered to a patient who has undergone hematopoietic cell transplantation, the drug may be administered to the patient after receiving hematopoietic cell transplantation, and the drug should be administered by the time when the diagnostic criteria for each complication are satisfied. Is preferred. Furthermore, the medicament of the present invention can be administered to patients undergoing hematopoietic cell transplantation. In this case, it may be administered to the patient simultaneously with the hematopoietic cell transplantation.

  The patient to which the medicament of the present invention is administered is not particularly limited as long as it is a patient who undergoes hematopoietic cell transplantation, a patient who has undergone hematopoietic cell transplantation, or a patient undergoing hematopoietic cell transplantation, but a hematopoietic malignant tumor patient It is preferable that

  The hematopoietic cell transplantation is preferably a hematopoietic cell transplantation performed on a hematopoietic malignant tumor, that is, a hematopoietic cell transplantation in a hematopoietic malignant tumor.

  The hematopoietic malignant tumor patient in the present invention is not particularly limited as long as hematopoietic malignancy is highly impaired, but patients suffering from leukemia, malignant lymphoma, multiple myeloma or related diseases thereof Are given as specific examples, and patients with acute leukemia are preferred examples. Also, chronic leukemia patients may be preferred. An example of an index in which hematopoietic ability is highly impaired includes a state in which abnormal cells are observed in peripheral blood or bone marrow. The presence or absence of abnormal cells can be evaluated, for example, by preparing a smear from peripheral blood or bone marrow fluid obtained by blood sampling or bone marrow puncture and observing with a microscope.

  One aspect of the medicament of the present invention is to improve hematopoietic ability associated with hematopoietic cell transplantation by being administered to a hematopoietic malignant tumor patient receiving hematopoietic cell transplant or a hematopoietic malignant tumor patient receiving hematopoietic cell transplant. it can.

  The degree of improvement in hematopoietic ability can be confirmed by measuring the number of peripheral blood cells. The peripheral blood blood cell count can be measured by an electrical resistance method, an optical measurement method, or a measuring device using the electrical resistance method and the optical measurement method. In addition, measurement by a method using a hemocytometer is also possible.

  One aspect of the medicament of the present invention is to treat and / or improve a hematopoietic malignant tumor by being administered to a hematopoietic malignant tumor patient receiving hematopoietic cell transplant or a hematopoietic malignant tumor patient receiving hematopoietic cell transplant. Can do.

  The degree of treatment and / or improvement of hematopoietic malignancy can be confirmed by 1) measuring the number of abnormal cells in the bone marrow. In addition, in the case of a hematopoietic malignant tumor in which a genetic abnormality has been clarified, it can be confirmed by appropriately combining the Fluorescence in situ hybridization (FISH) method and the Polymerase chain reaction (PCR) method.

  The medicament of the present invention is not particularly limited as long as it contains thrombomodulin as an active ingredient, and the medicament used for hematopoietic cell transplant patients is produced and used as a single or a plurality of preparations as necessary. You can also That is, according to the present invention, a medicament comprising a combination of thrombomodulin and at least one selected from the group consisting of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent, which prevents and / or prevents engraftment syndrome associated with hematopoietic cell transplantation. The medicament for treating, the medicament for improving hematopoietic ability associated with hematopoietic cell transplantation, and the medicament for treating and / or improving a hematopoietic malignant tumor are provided. The pharmaceutical comprising a combination of thrombomodulin and at least one selected from the group consisting of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent is preferably a pharmaceutical comprising a combination of thrombomodulin and an immunosuppressive agent. In addition, there is another embodiment in which a thrombomodulin is preferably a pharmaceutical comprising a combination of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent.

  According to the present invention, there is also provided a medicament comprising a combination of thrombomodulin and at least one selected from the group consisting of an immunosuppressant, a hematopoietic malignant tumor therapeutic agent, and a preventive and / or therapeutic agent for complications associated with hematopoietic cell transplantation. The drug for preventing and / or treating the engraftment syndrome associated with hematopoietic cell transplantation, the drug for improving the hematopoietic ability associated with hematopoietic cell transplantation, and the treatment and / or improving hematopoietic malignant tumor The medicament is provided.

  Examples of the immunosuppressive agent include corticosteroids, alkaloid drugs, calcineurin inhibitors, cyclin-dependent kinase inhibitors, antithymocyte globulin, antilymphocyte globulin, anticytokine antibodies, and anti-CD antibodies. Examples of these agents include prednisolone, methylprednisolone, hydrocortisone, triamcinolone, betamethasone, dexamethasone, cyclosporine, tacrolimus, sirolimus, rapamycin, atgam, Muromonab-CD3, basiliximab, daclizumab, infliximab, etalimestab Tacrolimus, sirolimus, rapamycin and cyclosporine are preferred, but not limited thereto. Furthermore, examples using antimetabolites and alkylating agents described later as immunosuppressants can also be mentioned as preferred examples, and methotrexate, cytarabine, and mycophenolic acid are also preferred but not limited thereto.

  The hematopoietic malignant tumor therapeutic agent is a drug used for the treatment of hematopoietic malignant tumor including a chemotherapeutic agent, and is a DNA or RNA polymerase inhibitor, antimetabolite, anti-malignant tumor enzyme, alkylating agent, Tubular polymerization inhibitors, hormone therapy agents, molecular targeting agents and the like are included. These drugs include doxorubicin, daunorubicin, idarubicin, aclarubicin, etoposide, methotrexate, cytarabine, cladribine, mycophenolic acid, mizoribine, azathioprine, mitomycin C, L-asparaginase, cyclophosphamide, busulfan, melphalan, vincristine, vinblastine, blastine Filgrastim, Lenograstim, Narutograstim, Tretinoin (All-Trans Retinoic Acid: ATRA), Rituximab, Ofatumumab, Alemtuzumab, Gemtuzumab ozogamicin, 131-I Toshitumomab K-1 EC-14 Although -0761 is illustrated, it is not limited to these. These drugs are used alone or in appropriate combination depending on the type of hematopoietic malignant tumor.

  Furthermore, anti-lymphocyte globulin, steroid, mycophenolic acid, C1 esterase inhibitor concentrate, furosemide, torasemide, azosemide, piretanide, hydrochlorothiazide, trichlor as agents for preventing and / or treating complications associated with hematopoietic cell transplantation Examples include methiazide, indapamide, chlorthalidone, spinolactone, canrenoic acid, eplerenone, acetazolamide, dopamine, aminophylline, carperitide and the like, and steroids and mycophenolic acid are preferred, but not limited thereto.

  The pharmaceutical comprising the combination of thrombomodulin and at least one selected from the group consisting of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent according to the present invention is in the state of a combined preparation in which the respective components are combined and mixed. In addition, the respective components may not be mixed and may be in a non-mixed combination that can be separately administered from a plurality of containers.

  As a pretreatment for hematopoietic cell transplantation used in the present invention, in addition to the above-mentioned hematopoietic malignant tumor therapeutic agent, radiotherapy is appropriately combined. As an irradiation method of radiation therapy, a source axis distance (SAD), a source skin distance (SSD), a sweeping beam, a beam transfer method, and a treatment bed transfer method are exemplified. As the dose of TBI and the administration schedule, a single irradiation method or a divided irradiation method is exemplified. 10 to 12 Gy for single irradiation method, 12 Gy for 6 days in divided irradiation method, 12 Gy for 6 days, 15.75 Gy for 7 days, 1.8 Gy for 2 to 3 times a day A method of about 15 Gy, 2 to 3 Gy at a time, and a total of 12 Gy at 1 to 2 times a day is exemplified, but is not limited thereto. As a dose rate of TBI, 5 to 15 cGy / min is exemplified. Further, as a pretreatment without performing radiotherapy called mini-transplantation, fludarabine, cladribine, and antithymocyte globulin may be used in an appropriate combination, but it is not limited thereto.

  The pharmaceutical comprising a combination of thrombomodulin of the present invention and at least one selected from the group consisting of an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent is selected from the group consisting of thrombomodulin, an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent. Can be administered to the patient simultaneously as a complete mixture of at least one active ingredient. In addition, at least one selected from the group consisting of thrombomodulin and an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent in the medicament of the present invention can be administered to the patient simultaneously or sequentially as separate components.

  For example, in the case of a pharmaceutical comprising a combination of thrombomodulin and an immunosuppressive agent, the administration order of thrombomodulin and the immunosuppressive agent is not limited when sequentially administered, but it is preferable to administer the immunosuppressive agent next to thrombomodulin. There is also another preferred embodiment in which thrombomodulin is administered next to the immunosuppressive agent. Thrombomodulin can be administered before or after hematopoietic cell transplantation or during hematopoietic cell transplantation, but is preferably administered before or after hematopoietic cell transplantation, more preferably after hematopoietic cell transplantation. preferable. There is also another embodiment in which it is more preferably administered before hematopoietic cell transplantation. Furthermore, it may be more preferable to administer during hematopoietic cell transplantation. The immunosuppressive agent is preferably administered during or after hematopoietic cell transplantation. The administration order or administration site can be appropriately changed according to the condition of the subject patient.

  By administering the medicament of the present invention together with at least one selected from the group consisting of immunosuppressants and hematopoietic malignant tumor therapeutic agents, engraftment syndrome associated with hematopoietic cell transplantation can be prevented and / or treated. Examples of the immunosuppressive agent include the above-mentioned pharmaceuticals exemplified as immunosuppressive agents, and examples of the hematopoietic malignant tumor therapeutic agent include the above-described pharmaceuticals exemplified as hematopoietic malignant tumor therapeutic agents.

  Specifically, the medicament of the present invention can be administered together with an immunosuppressive agent. In addition, the medicament of the present invention can be administered together with a hematopoietic malignant tumor therapeutic agent. Furthermore, the medicament of the present invention can be administered together with an immunosuppressive agent and a hematopoietic malignant tumor therapeutic agent.

  Further, for example, in the case of a pharmaceutical comprising a combination of thrombomodulin, an immunosuppressive agent, and a hematopoietic malignant tumor therapeutic agent, when sequentially administered, the administration order of the hematopoietic malignant tumor therapeutic agent is the hematopoietic malignant tumor therapeutic agent. Is administered before hematopoietic cell transplantation or after hematopoietic cell transplantation, but it is preferable to administer a therapeutic agent for hematopoietic malignant tumor after administration of thrombomodulin and an immunosuppressive agent. There is also another embodiment in which it is preferable to administer the thrombomodulin and the immunosuppressive agent after administration of the hematopoietic malignant tumor therapeutic agent. The therapeutic agent for hematopoietic malignant tumor can be administered before or after hematopoietic cell transplantation, or during hematopoietic cell transplantation, but is preferably administered before or after hematopoietic cell transplantation. The administration order or administration site can be appropriately changed according to the condition of the subject patient.

  Furthermore, for example, in the case of a pharmaceutical comprising a combination of thrombomodulin, an immunosuppressant, a hematopoietic malignant tumor therapeutic agent, and a prophylactic and / or therapeutic agent for complications associated with hematopoietic cell transplantation, The order of administration of the preventive and / or therapeutic agent for complications associated with cell transplantation is not particularly limited as long as the preventive and / or therapeutic agent for complications associated with hematopoietic cell transplantation is administered before or after hematopoietic cell transplantation. It is preferable to administer a prophylactic and / or therapeutic agent for complications associated with hematopoietic cell transplantation after administration of a thrombomodulin, an immunosuppressive agent, and a hematopoietic malignant tumor therapeutic agent. There is also another embodiment in which it is preferable to administer a thrombomodulin, an immunosuppressive agent, and a hematopoietic malignant tumor therapeutic agent after administration of a prophylactic and / or therapeutic agent for complications associated with hematopoietic cell transplantation. The preventive and / or therapeutic agent for complications associated with hematopoietic cell transplantation can be administered before hematopoietic cell transplantation, after hematopoietic cell transplantation, or during hematopoietic cell transplantation, but is preferably administered after hematopoietic cell transplantation. The administration order or administration site can be appropriately changed according to the condition of the subject patient.

  The medicament of the present invention can contain a carrier. As the carrier that can be used in the present invention, a water-soluble carrier is preferable, and for example, it can be prepared by adding sucrose, glycerin, etc., other inorganic salt pH adjusters and the like as additives. Further, as disclosed in JP-A-1-6219 and JP-A-6-321805, amino acids, salts, carbohydrates, surfactants, albumin, gelatin and the like may be added as necessary. Moreover, it is also preferable to add a preservative, for example, a parabenzoate ester is mentioned as a preferable example, and methyl parabenzoate is mentioned as a particularly preferable example. The amount of the preservative added is typically 0.01 to 1.0% (indicating weight%, the same applies hereinafter), preferably 0.1 to 0.3%. Although these addition methods are not particularly limited, in the case of freeze-drying, as usual, for example, a solution containing at least one selected from an immunosuppressant and a hematopoietic malignant tumor therapeutic agent and a thrombomodulin-containing solution After mixing, or at least one selected from an immunosuppressant or hematopoietic malignant tumor therapeutic agent in which the additive is added and mixed, or the additive is previously dissolved in water, distilled water for injection or an appropriate buffer. After mixing, a method of preparing a solution by a method of adding and mixing a thrombomodulin-containing solution and freeze-drying can be mentioned. When the medicament of the present invention is a medicament comprising a combination of each pharmaceutical ingredient, each medicament is preferably produced by adding a carrier by an appropriate production method. The medicament of the present invention may be provided in the form of an injection solution, or may be provided in a form in which a freeze-dried preparation is dissolved at the time of use.

  In the formulation step, 0.05 to 15 mg, preferably 0.1 to 5 mg of thrombomodulin per ml of water, distilled water for injection or a suitable buffer, and an antiplatelet agent or anticoagulant are added to an ampule or vial. Or a solution containing any one of the thrombolytic agents and the above-mentioned additive, for example, after being filled with 0.5 to 10 mL, frozen and then dried under reduced pressure. Or it can prepare as an aqueous solution injection formulation as it is.

  The medicament of the present invention is desirably administered by a parenteral administration method such as intravenous administration, intramuscular administration, or subcutaneous administration. Oral administration, rectal administration, intranasal administration, sublingual administration and the like are also possible. When the medicament of the present invention is a medicament formed by combining each pharmaceutical ingredient, it is preferable to administer each pharmaceutical ingredient by an appropriate administration method.

In the case of intravenous administration, a method of administering a desired amount at a time or intravenous drip administration can be mentioned.
A method of administering a desired amount at a time is preferable in that the administration time is short. When administered at once, the time required for administration with a syringe usually varies, but the time required for administration depends on the amount of liquid to be administered, but is usually preferably 2 minutes or less, and 1 minute or less. More preferred is 30 seconds or less. The lower limit is not particularly limited, but is preferably 1 second or longer, more preferably 5 seconds or longer, and even more preferably 10 seconds or longer. The dose is not particularly limited as long as it is the above-mentioned preferable dose. Intravenous administration is preferred because it is easy to keep the blood concentration of thrombomodulin constant.

  The daily dose of the medicament in the present invention varies depending on the age, weight, disease level, route of administration, etc. of the patient, but generally, the upper limit of the amount of thrombomodulin is preferably 20 mg / kg or less, preferably 10 mg / kg. kg or less is more preferred, 5 mg / kg or less is more preferred, 2 mg / kg or less is particularly preferred, 1 mg / kg or less is most preferred, and the lower limit is preferably 0.001 mg / kg or more, more preferably 0.005 mg / kg or more. Preferably, 0.01 mg / kg or more is more preferable, 0.02 mg / kg or more is particularly preferable, and 0.05 mg / kg or more is most preferable.

  In the case of intravenous administration, it is not particularly limited as long as it is the above preferable dose, but the upper limit of the daily dose is preferably 1 mg / kg or less, more preferably 0.5 mg / kg or less, and 0.1 mg / kg. kg or less is more preferred, 0.08 mg / kg or less is particularly preferred, 0.06 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, and 02 mg / kg or more is more preferable, and 0.04 mg / kg or more is particularly preferable.

  Administer once a day or several times as needed. The dosing interval can be once every 2 to 14 days, preferably once every 2 to 7 days, more preferably once every 3 to 5 days.

  When the medicament of the present invention containing thrombomodulin thus obtained was administered to a patient with myelodysplastic syndrome (hereinafter sometimes abbreviated as MDS) that developed an engraftment syndrome accompanied by pulmonary edema, the arterial oxygen partial pressure was increased. The disappearance of the infiltrative shadow of the whole lung field of chest X-ray observed in pulmonary edema, the dampness of auscultation, and the suppression and decrease of body weight gain were observed. Complications associated with hematopoietic cell transplantation Excellent preventive and therapeutic effects were observed. That is, from the effect on hematopoietic malignant tumor patients, it was confirmed that the medicament of the present invention is useful as prevention and / or treatment of engraftment syndrome associated with hematopoietic cell transplantation in patients who have undergone hematopoietic cell transplantation. As described above, when the medicament of the present invention is produced, it may be prepared by a known method in which an effective amount of thrombomodulin is mixed with a pharmaceutically acceptable carrier.

  The medicament of the present invention is very useful in that a patient with a hematopoietic malignant tumor that easily bleeds can exhibit a safe and high effect without increasing the risk of bleeding even when hematopoietic cell transplantation is performed. It has been reported that thrombomodulin, which is an active ingredient of the medicament of the present invention, improves bleeding symptoms on the contrary without promoting bleeding (Saito H, et al., J Thromb Haemost 2007; 5: 31-). 41) can also be supported. As a method for evaluating the risk of bleeding, the activation described in coagulation time (for example, Mohri M et al., Thromb Haemost 1999; 82: 1687-93) using a specimen obtained by adding the pharmaceutical agent of the present invention to plasma. The measurement of partial thromboplastin time (APTT) is exemplified. In addition, as an evaluation method of a bleeding state in hematopoietic cell transplantation, use of blood (transfusion) or a plasma preparation as an index is exemplified.

  The medicament of the present invention is also useful in that it can be used without impairing the convenience that it is performed in a short time and has few surgical invasions, which is a feature of hematopoietic cell transplantation. In addition, as described above, since the medicament of the present invention does not increase the risk of bleeding, a test for evaluating that the dose is appropriate is unnecessary. Examples of a method for evaluating that the dosage is appropriate include a bleeding risk evaluation method using the above-described coagulation time method as an index.

[Explanation of Sequence Listing]
SEQ ID NO: 1: amino acid sequence encoded by the gene used for the production of TME456 SEQ ID NO: 2: nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 SEQ ID NO: 3: amino acid sequence encoded by the gene used for the production of TME456M : 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: production of TMD12M Amino acid sequence encoded by the gene used in SEQ ID NO: 8: nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 SEQ ID NO: 9: amino acid sequence encoded by the gene used for production of TMD123 SEQ ID NO: 10: amino acid sequence of SEQ ID NO: 9 SEQ ID NO: 11 encoding the gene used for the production of TMD123M Amino acid sequence SEQ ID NO: 12 the child encoding: nucleotide sequence SEQ ID NO: 13 that encodes the amino acid sequence of SEQ ID NO: 11: mutant synthetic DNA for use in performing site-directed mutagenesis

  EXAMPLES Hereinafter, although an Example and a test example demonstrate this invention concretely, this invention is not limited at all by these.

  The thrombomodulin in the present invention used in the test examples was produced according to the method of Yamamoto et al. (Method described in JP-A No. 64-6219). The production example is shown below. The thrombomodulin obtained in this production example was tested for safety through single and repeated intravenous administration tests using mice and monkeys, mouse reproduction tests, local irritation tests, safety pharmacology tests, virus inactivation tests, etc. Sex has been confirmed.

[Production Example 1]
<Acquisition of thrombomodulin>
The above-described method, that is, a DNA encoding the amino acid sequence of SEQ ID NO: 9 (specifically, consisting of the base sequence of SEQ ID NO: 10) is transfected into Chinese hamster ovary (CHO) cells, and the transformed cells From the culture solution, a high-purity purified product obtained by collecting the active fraction with a 20 mmol / L phosphate buffer solution (pH 7.3) containing 50 mmol / L NaCl was obtained by the above-described conventional purification method. Further, ultrafiltration was performed to obtain a thrombomodulin (sometimes abbreviated as TMD123 in this specification) solution having a concentration of 11.2 mg / mL.

<Preparation of additive solution>
In a 10 L stainless steel container, 480 g of arginine hydrochloride (manufactured by Ajinomoto Co., Inc.) was weighed, and 5 L of water for injection was added and dissolved. A 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.3.

<Chemical solution preparation and filling>
The total amount of the above additive solution was put in a 20 L stainless steel container, and 2398 mL of the TMD123 solution obtained above (corresponding to 26.88 g as the protein mass of soluble thrombomodulin, however, 12% excess was charged) was added and stirred. Further, water for injection was added to make the total volume 12 L, and the mixture was uniformly mixed and stirred. This chemical solution was sterilized by filtration through a filter (MCGL10S manufactured by Millipore) having a pore size of 0.22 μm. Each 1 mL of the filtrate was filled into a vial, and a rubber stopper was half stoppered.

<Freeze drying>
The freeze-drying process was carried out under the following conditions in the order of freeze-drying → filling with nitrogen → all stoppers with rubber stopper → clamping the cap, and a TMD123-containing preparation containing 2 mg of soluble thrombomodulin and 40 mg of arginine hydrochloride in one container was obtained.

<Freeze drying conditions>
Precooling (from room temperature to 15 ° C over 15 minutes) → Main cooling (15 ° C to -45 ° C over 2 hours) → Hold (2 hours -45 ° C) → Start of vacuum (18 hours -45 ° C) → Temperature rise ( Over 20 hours -45 ° C to 25 ° C) → Holding (15 hours 25 ° C) → Temperature rise (25 ° C to 45 ° C over 1 hour) → Holding (5 hours 45 ° C) → Room temperature (45 ° C over 2 hours) To 25 ° C) → Refilled nitrogen filling (up to -100mmHg) → All stoppers → Cap tightening

[Production Example 2]
DNA encoding the amino acid sequence of SEQ ID NO: 11 (specifically, consisting of the base sequence of SEQ ID NO: 12) was transfected into Chinese hamster ovary (CHO) cells, and the above-mentioned culture medium of the transformed cells was used. A thrombomodulin (may be abbreviated as TMD123M in this specification) solution purified by a conventional purification method is obtained, and a lyophilized preparation of TMD123M is obtained by the same method as described above.

[Production Example 3]
A DNA encoding the amino acid sequence of SEQ ID NO: 1 (specifically, consisting of the base sequence of SEQ ID NO: 2) was transfected into Chinese hamster ovary (CHO) cells, and the above-mentioned culture medium of the transformed cells was used. Thrombomodulin (which may be abbreviated as TME456 in the present specification) purified by a conventional purification method is obtained, and a freeze-dried preparation of TME456 is obtained by the same method as described above.

[Production Example 4]
DNA encoding the amino acid sequence of SEQ ID NO: 3 (specifically, consisting of the base sequence of SEQ ID NO: 4) was transfected into Chinese hamster ovary (CHO) cells, and the above-mentioned culture medium of the transformed cells was used. Thrombomodulin (hereinafter sometimes abbreviated as TME456M) purified by a conventional purification method is obtained, and a freeze-dried preparation of TME456M is obtained by the same method as described above.

[Production Example 5]
DNA encoding the amino acid sequence of SEQ ID NO: 5 (specifically, consisting of the base sequence of SEQ ID NO: 6) was transfected into Chinese hamster ovary (CHO) cells, and the above-mentioned culture solution of the transformed cells was used. Thrombomodulin (which may be abbreviated as TMD12 in the present specification) purified by a conventional purification method is obtained, and a freeze-dried preparation of TMD12 is obtained by the same method as described above.

[Production Example 6]
DNA encoding the amino acid sequence of SEQ ID NO: 7 (specifically, consisting of the base sequence of SEQ ID NO: 8) was transfected into Chinese hamster ovary (CHO) cells, and the above-mentioned culture medium of the transformed cells was used. Thrombomodulin (hereinafter sometimes abbreviated as TMD12M) purified by a conventional purification method is obtained, and a freeze-dried preparation of TMD12M is obtained by the same method as described above.

[Example 1]
<Effects on hematopoietic cell transplantation in MDS patients>
A 68-year-old man who developed MDS was administered whole body radiation, fludarabine and busulfan, and then transplanted hematopoietic cells from an unrelated person with a mismatched HLA locus. Tacrolimus and mycophenolic acid were used to prevent graft-versus host disease (GVHD). The body weight increased from the 13th day after the transplantation, and the increase in the body weight reached 6% on the 18th day after the transplantation. With the appearance of dyspnea and wet rales, chest X-ray and computed tomography revealed infiltrative shadows in the entire middle and lower lung fields, and the patient was diagnosed with ES combined with noncardiogenic pulmonary edema. Administration of the ATIII preparation (1,500 U / day) was started due to the complication of coagulation abnormalities. However, since it continued to deteriorate and was in a hypercoagulated state, administration of TMD123 was started from the 18th day of transplantation. On the 4th day from the start of TMD123 administration, the arterial oxygen partial pressure increased, and on the 6th day from the start of TMD administration, the moist rales disappeared and pulmonary edema was improved. On the 8th day from the start of TMD123 administration, the infiltration shadow in the lung field decreased, and on the 14th day from the start of TMD123 administration (end of TMD123 administration), the infiltration shadow in the lung field normalized. In addition, the body weight and SFMC started to decrease after the fifth day after the start of administration, and the body weight the day after the end of TMD123 administration was reduced to the same level as before the hematopoietic cell transplantation.
In addition, TMD123 manufactured according to manufacture example 1 was used.

Claims (8)

A medicament comprising thrombomodulin as an active ingredient for preventing and / or treating an engraftment syndrome associated with hematopoietic cell transplantation. The medicament according to claim 1, wherein the thrombomodulin is a soluble thrombomodulin. The thrombomodulin is a peptide obtained from a transformed cell (the transformed cell is a transformed cell transfected with DNA encoding the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11 ). The medicine according to 1 or 2. The thrombomodulin is a peptide comprising any one of the following amino acid sequences (i-1) or (i-2), and the peptide is a peptide having thrombomodulin activity: Medicine;
(I-1) the amino acid sequence of positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11; or (i-2) one or more of the amino acid sequences of (i-1) above An amino acid sequence in which the amino acids are substituted, deleted, or added. The medicament according to any one of claims 1 to 4, wherein the engraftment syndrome is a pathological condition involving at least one selected from the group consisting of rash, pulmonary edema, and diarrhea. The medicament according to any one of claims 1 to 5, wherein the hematopoietic cell transplantation is hematopoietic cell transplantation in a hematopoietic malignant tumor. And medicament according to any one of claims 1 to 6, a medicament comprising a combination of at least one selected from the group consisting of immunosuppressive agents and hematopoietic malignancies therapeutic agents, engraftment associated with hematopoietic cell transplantation The medicament for preventing and / or treating a syndrome. The medicament according to any one of claims 1 to 6, for administration together with at least one selected from the group consisting of an immunosuppressant and a hematopoietic malignant tumor therapeutic agent.