JP2008189574A - Auxiliary agent for transplanting pancreatic islet - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic and/or an ameliorating agent having high therapeutic effects or to provide a method for therapy and/or amelioration thereof. <P>SOLUTION: The agent comprises thrombomodulin as an active ingredient and is used for administering to a patient suffering from diabetes undergoing pancreatic islet transplantation and normalizing blood glucose level after the pancreatic islet transplantation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug used for islet transplantation in a diabetic patient, comprising thrombomodulin as a main active ingredient, a drug for treating and / or improving diabetes, or the treatment and / or improvement of diabetes by islet transplantation. It relates to a drug for assisting the effect.

  Diabetes is a chronic complex metabolic disease that prevents the body from properly maintaining and using carbohydrates, fats, and proteins. Various genetic and environmental factors are characterized by high levels of blood glucose caused by a deficiency in insulin production or reduced action. According to the actual survey of diabetes conducted by the former Ministry of Health and Welfare in 1997, there were 6.9 million people who were strongly suspected of having diabetes, and when 6.8 million people who could not deny the possibility of diabetes were added, about 13.7 million people were classified as risk groups. Has been estimated. The global diabetic population estimated by WHO is about 150 million in 2000 and is expected to reach about 300 million in 2025.

  Diabetes causes complications such as retinopathy, nephropathy, and neuropathy when it progresses, promotes the onset and progression of stroke and ischemic heart disease, and significantly lowers the patient's QOL. According to a survey conducted by the Japanese Society for Dialysis Medicine in 1998, 35.7% of patients newly introduced with dialysis in 1998 were caused by diabetic nephropathy. It has increased more than twice. In addition, according to a disease survey study of visual impairment of the former Ministry of Health and Welfare conducted in 1988, 3,000 people are visually impaired annually due to diabetic retinopathy. Thus, diabetes has become a major social problem, and eradication of diabetes has become an important issue in Japan as well as in the West.

  Many cases of diabetes are classified into type I that develops frequently in young people and type II that develops frequently in adults, and about 5 to 10% of diabetic patients are type I. Type I diabetes occurs when pancreatic β cells that produce insulin are specifically destroyed due to abnormal autoimmunity (Non-patent Document 1). Thus, diabetic patients rely on external insulin injections to prevent hyperglycemia (hyperglycemia) complications related to life and death. Chronic and long-lasting organ damage complications due to hyperglycemia cannot be prevented unless the very strict treatment of blood glucose measurement and insulin administration (insulin intensive treatment) is adhered to. Insulin intensive therapy increases the risk of acute hypoglycemia with acute and severe consciousness-changing states that can be fatal by overdosing insulin. Insulin therapy solves the urgent problem of rapid death in type I diabetes and plays a huge role in that it can now be offered to any patient. However, on the other hand, it cannot be a fundamental treatment for type I diabetes, and very difficult problems such as the risk of hypoglycemia and long-term complications remain.

  As a fundamental therapy for type I diabetes, regenerative replacement therapy for islet β cells is performed by transplanting the pancreas or pancreas and kidney simultaneously. Transplantation allows for very strict blood glucose control and avoids the development of hypoglycemia and even long-term complications. It is a treatment method that is expected to cure type I diabetes, as well as to free patients from daily annoyance by insulin intensive therapy. However, pancreatic transplantation has problems such as large surgical invasion and complications due to the accompanying exocrine glands that can be transplanted.

  The pancreas existing in the retroperitoneum is composed of exocrine glands and endocrine glands. Exocrine glands occupy 98% or more of the total pancreas volume, and endocrine glands are 2% or less. The endocrine tissue found by Langerhans in 1869 is called an islet. The islets are a group of endocrine cells, and are composed of α cells, β cells, PP cells, δ cells, and the like. Insulin, the only hormone in the body that acts to lower blood sugar, is secreted from the beta cells of the islets. Islet transplantation aims to replace and regenerate a hypoglycemic system once it has been isolated by isolating the islet from the pancreas and transplanting it to a patient with type I diabetes.

  Islet transplantation involves injecting pancreatic islet cells into the patient's portal vein after isolating pancreatic islets by enzymatic treatment of the pancreas removed from the donor. Surgical laparotomy and vascular anastomosis are unnecessary and are extremely invasive, so it is safer for diabetic patients and can also be performed in patients who have been exempted from pancreatic transplantation due to the progression of vascular lesions. In addition, since insulin is released into the portal circulation, it is physiologically advantageous. Furthermore, there are many advantages over pancreas transplantation, such as so-called banking by cryopreservation of isolated islets.

  In type I diabetes, it is very difficult to control blood glucose by insulin therapy, and islet transplantation is considered the most effective treatment. In addition, insulin therapy is actively used in the treatment of type II diabetes. This is based on the concept of administering insulin to rest the patient's own islet β-cells and waiting for the β-cell function to recover. Has been. This is because even if type II diabetes progresses, insulin deficiency will occur due to glucose toxicity and β-cell exhaustion, so type II diabetes is not currently indicated for islet transplantation. In the case where a large amount of transplanted islets can be supplied, insulin-dependent diabetes mellitus with insulin resistance as a trigger is likely to be indicated.

  As described above, the reason for the development up to the clinical research of islet transplantation is that a method for separating a large amount of islets from the pancreas mainly using large animals has been developed. In 1984, Ricordi et al. Devised a unique automatic islet separation device, which made it possible to separate a large amount of islets from porcine or human pancreas (Non-patent Document 2). Patent Document 1 describes a method for isolating and preparing islets used for islet transplantation.

  Islet transplantation, which started from the experimental medical stage, did not perform well at the beginning, but since March 1999, the University of Alberta in Edmonton, Canada has been using an innovative immunosuppressant called the Edmonton Protocol (Edmonton Protocol). Islet transplantation was performed, and it was reported that all 7 cases of pancreatic islet transplantation were withdrawn from daily insulin administration in patients with type I diabetes (Non-patent Document 3). Came to be done.

  The usefulness of islet transplantation as a treatment for type I diabetes is increasing, but it is necessary to infuse a large amount of islet cells for successful transplantation, and in order to obtain high quality islet cells, Preparation is an issue. In 2006, Shapiro AM et al. Reported the outcome of patients who received islet transplantation using the Edmonton protocol (Non-Patent Document 4). Among patients whose insulin treatment became unnecessary due to islet transplantation, 2 years after transplantation And 76% of patients resumed insulin treatment. For these reasons, further improvement is desired in islet transplantation as a radical treatment method for patients with type I diabetes.

  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 inactivates activated factor V and activated factor VIII of blood coagulation factor in vivo, and is involved in the production of plasminogen activator having a thrombolytic action. It is known (Non-Patent Document 5). Therefore, thrombomodulin is said to be useful as an anticoagulant or thrombolytic agent by promoting 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 on (Non-patent Document 6).

  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). An amino acid sequence of 575 residues including (exemplified) has been clarified (Patent Document 2). 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 7).

  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 2). There are some other reports as examples of soluble thrombomodulin (Patent Documents 3 to 10). Or as a natural type, soluble thrombomodulin derived from human urine is exemplified (Patent Documents 11 and 12).

  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 8). However, there is no difference in activity and physical properties, and both can be considered substantially the same.

  Conventional uses of thrombomodulin include, for example, myocardial infarction, thrombosis (eg, acute or chronic cerebral thrombosis, arterial or venous acute or chronic peripheral thrombosis, etc.), 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 Complications, intravascular blood coagulation syndrome (DIC), angina pectoris, transient ischemic attack, pregnancy toxemia, liver VOD (Liver veno-occlusive disease); fulminant hepatitis and hepatic vein occlusion after bone marrow transplantation ), And is expected to be used for the treatment and prevention of diseases such as deep vein thrombosis (DVT). Moreover, liver disorders (Patent Document 13), absorptive bone disease (Patent Document 14), wound healing (Patent Document 15), and the like can be cited as applications for diseases other than diseases associated with hypercoagulation such as thrombosis and DIC. Furthermore, wound healing (Patent Document 16), protection of brain tissue (Patent Document 17), and the like are disclosed as applications in which thrombomodulin is used in combination with other active ingredients.

JP-T-2-504222 JP-A 64-6219 JP-A-2-255699 Japanese Patent Laid-Open No. 3-133380 JP-A-3-259084 JP-A-4-210700 JP-A-5-213998 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 Atkinson MA et al, N Engl J Med 1994; 331: 1428-1436 Ricordi C et al, Diabetes 1988; 37: 413-420 Shapiro AM et al, N Engl J Med 2000; 343: 230-238 Shapiro AM et al, N Engl J Med 2006; 355: 1372-4 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

  An object of the present invention is to provide a drug having a higher therapeutic effect for normalizing blood glucose level after islet transplantation in a diabetic patient or a method for normalizing blood glucose level after islet transplantation in a diabetic patient. Another subject of the present invention is a drug that more effectively improves the survival rate of transplanted islet cells in islet transplantation of diabetic patients, a drug that extends the survival time of transplanted islet cells in pancreatic islet transplantation of diabetic patients, or It is to provide a method. Furthermore, another subject of the present invention is to provide a drug having a higher therapeutic effect for treating and / or improving the condition of diabetes of a diabetic patient who has undergone pancreatic islet transplantation, or a method for treating and / or improving the same. It is in.

  In recent years, pancreatic islet transplantation as a fundamental treatment for type I diabetes has improved its therapeutic results due to technological progress, but it requires a large amount of islet cells, and withdrawal from post-transplant insulin treatment has been prolonged. Since it is not maintained, multiple islet transplantation is required, and further improvement in treatment results is required by improving the islet transplantation method. As a result of diligent research to find a drug that satisfies the engraftment rate of transplanted islet cells, the present inventors have surprisingly administered thrombomodulin that has not been attempted before. Thus, it has been found that the engraftment rate of transplanted islet cells is significantly improved. Moreover, it discovered that the survival period of the islet transplanted cell after islet transplantation can be extended by administering thrombomodulin. Furthermore, it has been found that the blood glucose level after islet transplantation can be normalized by administering thrombomodulin, and the present invention has been completed based on these individual findings. That is, the present invention relates to a drug containing thrombomodulin as a main active ingredient, administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation, and normalizing blood glucose level after pancreatic islet transplantation. The agent of the present invention can remarkably increase the engraftment rate of transplanted islet cells by being administered at the time of islet transplantation, which is expected as a root treatment for diabetes, and normalizes insulin secretion, and islet transplantation after islet transplantation It becomes possible to prolong the survival period of cells. Moreover, it becomes possible to normalize the blood glucose level of a diabetic patient after islet transplantation and to treat and / or improve diabetes more effectively.

Specifically, the present invention includes the following.
[1] A drug comprising thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation to normalize blood glucose level after pancreatic islet transplantation The drug;
[1-2] A drug containing thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient who receives pancreatic islet transplantation and / or a diabetic patient who receives pancreatic islet transplantation to treat and / or improve diabetes Of the drug;
[1-3] A drug containing thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation, and treatment and / or improvement effect of diabetes by pancreatic islet transplantation The drug for assisting;
[1-4] A drug containing thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient who receives pancreatic islet transplantation and / or a diabetic patient who receives pancreatic islet transplantation to improve the survival rate of transplanted islet cells Of the drug;
[1-5] A drug containing thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient who receives pancreatic islet transplantation and / or a diabetic patient who receives pancreatic islet transplantation, and extends the survival period of transplanted islet cells The drug;
[1-6] A drug containing thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation to normalize plasma insulin concentration after pancreatic islet transplantation The drug to do;

[2] The drug according to any one of [1] to [1-6], wherein the thrombomodulin is a soluble thrombomodulin;

[3] Peptide obtained from a transformed cell prepared by transfecting a host cell with DNA encoding the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or SEQ ID NO: 11 The drug according to any one of [1] to [2], which is
In addition, when the item number to be cited is shown as a range as in [1] to [2] and a term having a branch number such as [1-2] is arranged in the range, [1] -2] means that a term having a branch number such as is also cited. The same applies to the following.
[3-2] 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 shown in SEQ ID NO: 1 or SEQ ID NO: 3. ] The drug according to any one of [2];
[3-3] 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, [1] ] The drug according to any one of [2];
[3-4] The thrombomodulin is a peptide obtained by a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, ] The drug according to any one of [2];

[4] The thrombomodulin is any one of a peptide having positions 19 to 132 in SEQ ID NO: 1 or SEQ ID NO: 3, a peptide having a homologous mutant sequence of the above sequence and a thrombomodulin activity, or a mixture thereof The drug according to any one of [1] to [2] or [3-2], which is
[4-2] The thrombomodulin is a peptide having a sequence at positions 19 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3, or a peptide having a homologous mutation sequence of the above sequence and having thrombomodulin activity [1 ] To [2] or the drug according to any one of [3-2];
[4-3] The thrombomodulin is a peptide having a sequence of positions 19 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3, or any one of the above [1] to [2], or [3-2] Listed drugs;
[4-4] A peptide in which the thrombomodulin has a sequence at positions 19 to 132 or 17 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3, a peptide having a homologous mutation sequence of the above sequence and having thrombomodulin activity Or the drug according to any one of [1] to [2], or [3-2], which is any one of a mixture thereof;
[4-5] [1] to [2] above, wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO: 1 or SEQ ID NO: 3, or a mixture thereof. Or the drug according to any one of [3-2];
[4-6] The above [1] to [2] or [3-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO: 1 or a mixture thereof. A drug according to any one of
[4-7] The above [1] to [2] or [3-2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO: 3, or a mixture thereof. A drug according to any one of
[4-8] The above [1] to [2], or [3-2], wherein the thrombomodulin is a peptide comprising a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO: 1, or a mixture thereof. A drug according to any one of
[4-9] [1] to [2], or [3-2] above, wherein the thrombomodulin is a peptide consisting of a sequence at positions 19 to 132 or 17 to 132 in SEQ ID NO: 3, or a mixture thereof. A drug according to any one of

[5] The thrombomodulin is any one of a peptide having a sequence at positions 19 to 480 in each of SEQ ID NO: 5 or SEQ ID NO: 7, a peptide having a homologous mutant sequence of the above sequence and having a thrombomodulin activity, or a mixture thereof The drug according to any one of [1] to [2] or [3-3],
[5-2] The thrombomodulin is a peptide having a sequence of positions 19 to 480 in each of SEQ ID NO: 5 or 7 or a peptide having a homologous mutation sequence of the above sequence and having thrombomodulin activity [1 ] To [2] or the drug according to any one of [3-3];
[5-3] The thrombomodulin is a peptide having a sequence at positions 19 to 480 in each of SEQ ID NO: 5 or 7; [1] to [2], or any one of [3-3] Listed drugs;
[5-4] a peptide having the 19th to 480th positions or the 17th to 480th positions in SEQ ID NO: 5 or 7 respectively in SEQ ID NO: 5 or 7; a peptide having a homologous mutation sequence of the above sequence and having thrombomodulin activity Or the drug according to any one of [1] to [2], or [3-3], which is any one of a mixture thereof;
[5-5] [1] to [2], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 480 or 17 to 480 in SEQ ID NO: 5 or SEQ ID NO: 7, respectively, or a mixture thereof. Or the drug according to any one of [3-3];
[5-6] The above [1] to [2] or [3-3], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 480 or 17 to 480 in SEQ ID NO: 5, or a mixture thereof. A drug according to any one of
[5-7] [1] to [2], or [3-3], wherein the thrombomodulin is a peptide having a sequence at positions 19 to 480 or positions 17 to 480 in SEQ ID NO: 7, or a mixture thereof. A drug according to any one of
[5-8] The above [1] to [2], or [3-3], wherein the thrombomodulin is a peptide consisting of a sequence at positions 19 to 480 or 17 to 480 in SEQ ID NO: 5, or a mixture thereof. A drug according to any one of
[5-9] The above [1] to [2], or [3-3], wherein the thrombomodulin is a peptide comprising a sequence at positions 19 to 480 or 17 to 480 in SEQ ID NO: 7, or a mixture thereof. A drug according to any one of

[6] A peptide having the 19th to 516th positions in each of SEQ ID NO: 9 or SEQ ID NO: 11 in the thrombomodulin, one or more amino acids of the amino acid sequence of the peptide being substituted, deleted, or added The drug according to any one of [1] to [2] or [3-4], which is any one of a peptide having an amino acid sequence and having thrombomodulin activity, or a mixture thereof;
[6-2] The thrombomodulin is a peptide having the sequence of positions 19 to 516 in each of SEQ ID NO: 9 or SEQ ID NO: 11, or one or more amino acids in the amino acid sequence of the peptide are substituted, deleted, or The drug according to any one of [1] to [2] or [3-4], which is a peptide having an added amino acid sequence and having thrombomodulin activity;
[6-3] The thrombomodulin according to any one of [1] to [2] or [3-4] above, wherein the thrombomodulin is a peptide having a sequence at positions 19 to 516 in SEQ ID NO: 9 or SEQ ID NO: 11, respectively. Drugs;
[6-4] a peptide wherein the thrombomodulin has a sequence of positions 19 to 516, positions 19 to 515, positions 17 to 516, or positions 17 to 515 in each of SEQ ID NO: 9 or SEQ ID NO: 11, The drug according to any one of [1] to [2] or [3-4] above, which is a peptide having a homologous sequence mutation sequence and having thrombomodulin activity, or a mixture thereof;
[6-5] a peptide in which the thrombomodulin has a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9 or SEQ ID NO: 11, respectively The drug according to any one of [1] to [2] or [3-4], which is a mixture thereof;
[6-6] The aforementioned thrombomodulin is a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9, or a mixture thereof [1] to [2] or the drug according to any one of [3-4];
[6-7] The thrombomodulin is a peptide having a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 11, or a mixture thereof [1] to [2] or the drug according to any one of [3-4];
[6-8] The thrombomodulin is a peptide comprising a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9, or a mixture thereof [1] to [2] or the drug according to any one of [3-4];
[6-9] The above [1] to [1], wherein the thrombomodulin is a peptide consisting of a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9. 2] or the drug according to any one of [3-4];
[6-10] The aforementioned thrombomodulin is 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, or a mixture thereof [1] to [2] or the drug according to any one of [3-4];
[6-11] The above [1] to [1], wherein the thrombomodulin is a peptide consisting of a sequence at positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 11. 2] or the drug according to any one of [3-4];

[7] A drug comprising a combination of the drug according to [1] or any one of [2] to [6-11] and an immunosuppressive agent, wherein the drug receives pancreatic islet transplantation and / or Or the agent for normalizing blood glucose level after islet transplantation administered to a diabetic patient who has undergone islet transplantation;
[7-2] A drug comprising a combination of the drug according to [1] or any one of [2] to [6-11], an immunosuppressant, and a therapeutic drug for diabetes, wherein the drug is an islet The drug for normalizing blood glucose level after islet transplantation administered to a diabetic patient receiving transplant and / or a diabetic patient receiving islet transplant;
[7-3] A drug comprising a combination of the drug according to [1-2] or any one of [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for treating and / or improving diabetes by being administered to a diabetic patient and / or a diabetic patient who has undergone pancreatic islet transplantation;
[7-4] A drug comprising a combination of the drug according to any one of [1-2] or [2] to [6-11], an immunosuppressant, and a therapeutic drug for diabetes, the drug For treating and / or ameliorating diabetes by being administered to diabetic patients undergoing islet transplantation and / or diabetic patients receiving islet transplantation;
[7-5] A drug comprising a combination of the drug according to [1-3] or any one of [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for assisting the treatment and / or amelioration effect of diabetes by islet transplantation, which is administered to a diabetic patient and / or a diabetic patient who has undergone pancreatic islet transplantation;
[7-6] A drug comprising a combination of the drug according to [1-3] or any one of [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for assisting the treatment and / or amelioration effect of diabetes by islet transplantation, which is administered to a diabetic patient and / or a diabetic patient who has undergone pancreatic islet transplantation;
[7-7] A drug comprising a combination of the drug according to [1-4] or any one of [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for improving the survival rate of transplanted islet cells administered to a diabetic patient and / or a diabetic patient who has undergone islet transplantation;
[7-8] A drug comprising a combination of the drug according to any one of [1-4] or [2] to [6-11], an immunosuppressant, and a therapeutic drug for diabetes, the drug For administering a pancreatic islet cell to a diabetic patient and / or a diabetic patient receiving a pancreatic islet transplant;
[7-9] A drug comprising a combination of the drug according to [1-5] or any one of [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for prolonging the survival time of transplanted islet cells administered to a diabetic patient and / or a diabetic patient who has undergone islet transplantation;
[7-10] A drug obtained by combining the drug according to any one of [1-5] or [2] to [6-11], an immunosuppressant, and a therapeutic drug for diabetes, For administering to a diabetic patient undergoing islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation to prolong the survival period of transplanted islet cells;
[7-11] A drug comprising a combination of the drug according to any one of [1-6] or [2] to [6-11] and an immunosuppressant, wherein the drug undergoes islet transplantation The drug for normalizing plasma insulin concentration after islet transplantation administered to a diabetic patient and / or a diabetic patient who has undergone islet transplantation;
[7-12] A drug comprising a combination of the drug according to any one of [1-6] or [2] to [6-11], an immunosuppressant, and a therapeutic drug for diabetes, the drug For administering to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation to normalize plasma insulin concentration after pancreatic islet transplantation;

[8] The drug according to any one of [1] to [7-12] above, wherein the diabetic patient receiving pancreatic islet transplantation is a type I diabetic patient;

[1 ′] a method for normalizing blood glucose level after islet transplantation, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[2 ′] the method according to [1 ′] above, wherein the dragonfly modulin is a soluble thrombomodulin;
[3 ′] The [1 ′] or the above, wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO: 9 or 11. The method according to [2 ′];
[4 ′] The thrombomodulin is any one of a peptide having a sequence at positions 19 to 516 in each of SEQ ID NO: 9 and SEQ ID NO: 11, or a peptide having a homologous mutation sequence of the above sequence and having a thrombomodulin activity. [1 ′] or the method according to [2 ′];

[5 ′] a method for normalizing blood glucose level after islet transplantation, comprising administering thrombomodulin and an immunosuppressive agent to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[5'-2] Normalizing blood glucose level after islet transplantation, including administering thrombomodulin, immunosuppressive agent, and diabetes therapeutic agent to diabetic patients receiving islet transplantation and / or diabetic patients receiving islet transplantation Method;

[6 ′] The method according to any one of [1 ′] to [5′-2] above, wherein the diabetic patient receiving pancreatic islet transplantation is a type I diabetic patient;
[7 ′] The method according to [1 ′], which has the characteristics according to any one of [1] to [8];
[8 ′] a method for treating and / or improving diabetes comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[8′-2] The method according to [8 ′], wherein the method has the characteristics according to any one of [1] to [8];

[9 ′] a method for assisting in the treatment and / or amelioration of diabetes by islet transplantation, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[9′-2] The method according to [9 ′], which has the characteristics according to any one of [1] to [8];
[10 ′] A method for improving the survival rate of transplanted islet cells, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[10′-2] The method according to [10 ′], wherein the method has the characteristics according to any one of [1] to [8];

[11 ′] A method for prolonging the survival period of transplanted islet cells, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[11′-2] The method according to [11 ′], wherein the method has the characteristics according to any one of [1] to [8];
[12 ′] a method for normalizing plasma insulin concentration after islet transplantation, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[12′-2] The method according to [12 ′], wherein the method has the characteristics according to any one of [1] to [8];

[13 ′] a method for assisting in the treatment and / or amelioration of diabetes by islet transplantation, comprising administering thrombomodulin to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation;
[13′-2] The method according to [13 ′], which has the characteristics according to any one of [1] to [8].

  The use of the thrombomodulin-containing drug of the present invention for a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation can normalize the blood glucose level after pancreatic islet transplantation in diabetic patients. According to the method, the blood glucose level after islet transplantation of a diabetic patient can be effectively normalized. In addition, the engraftment rate of transplanted islet cells after islet transplantation can be improved and the survival period of the transplanted islet cells can be extended by the agent containing thrombomodulin of the present invention. Furthermore, the insulin concentration in plasma can be normalized, and diabetes can be treated and / or improved more effectively.

The present invention will be specifically described below.
In the present invention, specific examples of islet transplantation include pancreas single transplantation or pancreatic kidney simultaneous transplantation performed for patients who have been depleted of endogenous insulin or patients with renal failure as described above. The agent of the present invention can also be used for the aforementioned islet transplantation.

  The islet transplantation method in the present invention includes a method in which 1) the pancreas removed from the donor is treated with an enzyme, 2) only the islet cells are taken out with a cell separator, and 3) the islet cells are injected into the portal vein of the patient and transplanted. A preferred example is given.

  Examples of the blood glucose level in the present invention include blood glucose levels in plasma. Further, normalization of blood glucose level is not particularly limited as long as blood glucose level is lowered to a state where it can be determined that the pathology of diabetes is improved. For example, insulin administration can be performed by lowering blood glucose level. A specific example is to maintain a state in which the amount is reduced, or to maintain a state where insulin administration is unnecessary by lowering the blood glucose level, and lowering the blood glucose level and not requiring insulin administration It is preferable to maintain the state. In general, insulin is preferably administered at 0.25 unit or less per kg body weight, and preferably at 0.1 unit or less. However, when the blood glucose level is normalized, the dose of insulin is In some cases, the dose may be less than the above preferred dose, or the administration of insulin may be unnecessary.

  Normalized blood glucose level is usually the blood glucose level (fasting blood glucose level) obtained by blood sampling after fasting for 10 to 14 hours, or the blood glucose level after taking 75 g of glucose (hereinafter referred to as glucose load). It may be called test blood glucose level). As a glucose tolerance test blood glucose level, a blood glucose level in plasma obtained by taking blood 1 hour or 2 hours after taking glucose is preferably used, and a blood glucose level 2 hours later is more preferably used. Specifically, the upper limit of fasting blood glucose level is preferably 160 mg / dL or less, more preferably 140 mg / dL or less, further preferably 130 mg / dL or less, particularly preferably 110 mg / dL or less, and the lower limit of 60 mg / dL. dL or more is preferable, 70 mg / dL or more is more preferable, and 80 mg / dL or more is most preferable. The glucose tolerance test blood glucose level is preferably, for example, 220 mg / dL or less, more preferably 200 mg / dL or less, further preferably 180 mg / dL or less, and 140 mg / dL or less as the upper limit of the blood glucose level in 2 hours after glucose loading. Is particularly preferable, and the lower limit is preferably 60 mg / dL or more, more preferably 70 mg / dL or more, and still more preferably 80 mg / dL or more. Setting the blood glucose level within the above range is also a preferable aspect of normalizing the blood glucose level.

  In order to assist the treatment and / or improvement effect of diabetes by islet transplantation in the present invention, the treatment and / or improvement effect of diabetes is enhanced by using the agent of the present invention in the treatment of diabetes by islet transplantation. Or increasing the probability of treatment and / or improvement of diabetes. It may also mean treating and / or ameliorating diabetes itself.

  The engraftment of transplanted islet cells in the present invention includes that the cells introduced in the islet transplantation survive in the introduced organ, and the engraftment rate indicates how much the introduced cells are alive at a certain point in time. One index.

  As an index showing the improvement of the engraftment rate of transplanted islet cells, a homeostatic model assessment of β-cell function (hereinafter referred to as HOMA) is evaluated by combining the insulin production ability of the transplanted islet cells and the decrease in blood glucose level based on the insulin production. -May be referred to as -β value), and can be calculated by the following equation. That is, HOMA- [beta] value (%) = 20 * FPI / (FPG-3.5). Here, FPI indicates fasting plasma insulin concentration (μU / mL), and FPG indicates fasting blood glucose level (mmol / L).

  Specifically, the value of the improved engraftment rate of transplanted islet cells is preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, and the upper limit as the HOMA-β value. Is not limited, but is preferably 100% or less. Setting the survival rate of the transplanted islet cells within the above range is also a preferable embodiment for improving the survival rate of the transplanted islet cells.

  In addition, as an index indicating improvement in the survival rate of transplanted islet cells, the plasma concentration of C peptide produced when insulin is produced from proinsulin, which is a precursor of insulin, in the production of insulin by islet cells may be used. preferable. Specifically, the value of the improved engraftment rate of the transplanted islet cells is preferably 0.5 ng / mL or more, more preferably 0.7 ng / mL or more as the lower limit of the plasma concentration of C peptide, 0 The upper limit is preferably 3.5 ng / mL or less, more preferably 2.8 ng / mL or less, and further preferably 2.2 ng / mL or less. Setting the engraftment rate of transplanted islet cells within the above range is also a preferable embodiment for improving the engraftment rate of transplanted islet cells.

  Specifically, the survival period of the transplanted islet cells in the present invention is preferably 1 month or more as a lower limit, 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 limited and is preferably 5 years or less. Setting the survival period of the transplanted islet cells within the above range is also a preferable embodiment of extending the survival period of the transplanted islet 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.

  The survival period of the transplanted islet cells is exemplified as a period until the transplanted islet cells do not produce insulin. For example, the plasma C peptide concentration or fasting blood glucose level after the islet transplantation is abnormal. It can be evaluated as a period until it shows. Examples of the state in which the plasma C-peptide concentration or fasting blood glucose level is abnormal are exemplified by the plasma C-peptide concentration being 0.3 ng / mL or less, or the fasting blood glucose level being 270 mg / dl or more. .

  The diabetic patient in the present invention may be a diabetic patient who has undergone pancreatic islet transplantation or a diabetic patient who has undergone pancreatic islet transplantation. In the former case, the agent of the present invention may be administered to a diabetic patient before receiving islet transplantation, and in the latter case, the agent of the present invention may be administered to a diabetic patient after receiving islet transplantation. In addition, “a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation” also includes “a diabetic patient undergoing pancreatic islet transplantation”. .

  In addition, the diabetic patient in the present invention is not particularly limited as long as insulin production is highly impaired, but specific examples include type I diabetic patients or type II diabetic patients, and type I diabetic patients. Is a preferred example. In addition, type II diabetic patients may be preferred. As an indicator of insulin production, an abnormality in plasma insulin concentration or C peptide concentration is exemplified. Examples of the state in which insulin production is highly impaired include a state in which the plasma insulin concentration is 2 μU / mL or less, or a state in which the plasma C peptide concentration is 0.3 ng / mL or less.

  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 actions (1) to (4) above.

  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. Such amino acid sequences are called homologous mutation sequences. As described later, these mutations can be easily obtained by using ordinary gene manipulation techniques.

  The sequence of SEQ ID NO: 3 is 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.

  Thus, 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. Preferable examples include, but not limited to, a peptide comprising a sequence at positions 19 to 132 or 17 to 132 in each of SEQ ID NO: 1 or SEQ ID NO: 3, or a peptide consisting of a homologous variant of the above sequence and having at least thrombomodulin activity. 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.

  As described above, 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 at least a peptide sequence having thrombomodulin activity. Preferable examples include, but not limited to, a peptide composed of the 19th to 480th positions or the 17th to 480th positions in SEQ ID NO: 5 or 7 respectively, or a peptide composed of homologous mutant sequences 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 11 or a homologous mutant sequence thereof, and includes at least a peptide sequence having 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. However, one or more of the amino acid sequence of the peptide of interest, that is, one or more amino acids, more preferably several (for example, 1 to 20, preferably 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 sequences of SEQ ID NO: 9 or 11 include the 19th to 516th positions, the 19th to 515th positions, and the 17th position in SEQ ID NO: 9 or 11 respectively. A more preferred example is a peptide consisting of the sequence at positions ˜516 or 17th to 515. Further, a peptide comprising the sequence of positions 19 to 516, 19 to 515, 17 to 516, or 17 to 515 in SEQ ID NO: 9 or 11 is represented by SEQ ID NO: 9 or 11 A mixture for each is also given as a more preferred example.

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.
Moreover, as a mixing ratio of the peptide ending at position 515 and the peptide ending at position 516 in each of SEQ ID NO: 9 or SEQ ID NO: 11, (70:30) to (90:10) are exemplified, and (75:25 ) To (85:15) are preferable examples.
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 need to have the amino acid sequence described above, and may or may not have a sugar chain. 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. The facts described in JP-A No. 11-341990 are known about the binding position and type of sugar chain, 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 base sequence encoding the sequence, a base 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 a human tissue type plasminogen activator can be used (International Publication 88/9811).

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. In addition, a strain (Escherichia coli DH5 / pSV2TM J2) (FERM BP-5570) re-deposited with this strain at the Institute for Life Science (currently National Institute of Advanced Industrial Science and Technology (AIST)) can also be used. 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, see 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. For example, it can be extracted and purified from urine, blood, other body fluids, etc. 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 thrombomodulin is produced using DNA having the base sequence of SEQ ID NO: 10, diversity of the C-terminal amino acid may be observed, and a peptide having a short amino acid residue may be produced. That is, the C-terminal amino acid may be modified such that the amino acid at position 515 becomes the C-terminal and the position 515 is amidated. Therefore, 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 is preferably C-terminal. In another embodiment, the amino acid at position 516 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 mixture of the peptide which consists of the 19th-516th position in sequence number 9, the 19th-515th position, the 17th-516th position, or the 17th-515th position is mentioned.

  Subsequently, the method for isolating and purifying thrombomodulin from the culture supernatant or the culture obtained as described above can be carried out in accordance with a known method [edited by Takeo Horio, basic experiment method for proteins and enzymes, 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 are obtained as a single band, or isolated purified product gel filtration HPLC or reverse phase HPLC results are simple. 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 by Q-Sepharose Fast Flow of an ion exchange column and has 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.

  The drug of the present invention is not particularly limited as long as it contains thrombomodulin as an active ingredient, and if necessary, the drug used for islet transplant patients is manufactured and used as a single or multiple preparations. You can also. That is, according to the present invention, a drug comprising a combination of thrombomodulin and an immunosuppressive agent, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation, and blood glucose level after pancreatic islet transplantation The agent for normalizing the disease, the agent for treating and / or improving diabetes, the agent for assisting the treatment and / or amelioration of diabetes by islet transplantation, and improving the survival rate of islet transplanted cells Or an agent for prolonging the survival of islet transplanted cells.

  Examples of immunosuppressants include corticosteroids, alkylating agents, antimetabolites, alkaloids, calcineurin inhibitors, cyclin-dependent kinase inhibitors, antithymocyte globulin, monoclonal antibodies to CD antigen, prednisolone, methylprednisolone, Hydrocortisone, triamcinolone, betamethasone, dexamethasone, cyclophosphamide, methotrexate, azathioprine, mycophenolate, mizoribine, vincristine, vinblastine, etoposide, mitomycin C, cyclosporine, tacrolimus, sirolimus, rapamycin, atour CD, m Examples include rituximab, basiliximab, and daclitumab, mycophenolate, azathioprine, tacroli Scan, sirolimus, rapamycin, Bashikikishimabu, or daclizumab is preferred but not limited to. In addition, as other immunosuppressive agents, infliximab, gusperimus, or anti-lymphocyte globulin may be mentioned as a preferred example.

  The drug comprising a combination of thrombomodulin and an immunosuppressive agent according to the present invention may be in the form of a compounded mixture in which the respective components are combined into one, or the respective components are not mixed. Alternatively, it may be in a non-mixed combination that can be separately administered from a plurality of containers.

  The drug comprising a combination of the thrombomodulin of the present invention and an immunosuppressive agent can be simultaneously administered to a patient as a complete mixture of the active ingredients of thrombomodulin and the immunosuppressive agent. In addition, the thrombomodulin and the immunosuppressive agent in the drug of the present invention can be administered to the patient simultaneously or sequentially as separate components. When administered sequentially, the administration order of thrombomodulin and the immunosuppressive agent is not limited, 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. Although thrombomodulin can be administered before, after, or during islet transplantation, it is preferably administered before or after islet transplantation, and more preferably after islet transplantation. There is also another embodiment in which it is more preferable to administer before islet transplantation. Furthermore, it may be more preferable to administer during islet transplantation. The immunosuppressive agent is preferably administered during or after islet transplantation. The administration sequence or administration site can be appropriately changed according to the condition of the subject patient.

  Further, according to the present invention, there is provided a drug comprising a combination of thrombomodulin, an immunosuppressive agent, and a therapeutic drug for diabetes, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation. The drug for normalizing blood glucose level after transplantation, the drug for treating and / or improving diabetes, the drug for assisting the treatment and / or amelioration effect of diabetes by islet transplantation, The agent for improving engraftment or the agent for prolonging the survival time of islet transplanted cells is provided.

  As a therapeutic agent for diabetes used as one component of the present invention, an insulin sensitivity increasing agent (PPARγ agonist), an insulin secretagogue (SU agent, glinide), a hepatic glucose release inhibitor, a sugar absorption delaying agent (α-glucosidase inhibition) Agents), DPP-IV inhibitors, glucagon-related peptides pioglitazone, rosiglitazone, troglitazone, glibenclamide, gliclazide, glipizide, glimepiride, repaglinide, nateglinide, mitiglinide, metformin, voglibose, acarbose, vildagliptin, exerigatid Vildagliptin, exenatide, or liraglutide is exemplified as a preferred antidiabetic agent, but is not limited thereto.

  As another aspect of the present invention, it is also preferable to use insulin or an insulin derivative as a therapeutic agent for diabetes. Insulin and insulin derivatives include super fast-acting, fast-acting, mixed (biphasic), intermediate, and long-acting types such as insulin lispro, insulin aspart, insulin gllysin, human insulin, human neutral insulin, human isoform Examples include, but are not limited to, pheninsulin, insulin glargine, or insulin detemir. These types of drugs can be used alone or in appropriate combination.

  The drug comprising a combination of the thrombomodulin, the immunosuppressive agent, and the antidiabetic agent of the present invention may be in the form of a combined preparation in which the respective components are combined together, and 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.

  A drug comprising a combination of the thrombomodulin, immunosuppressive agent and antidiabetic agent of the present invention can be simultaneously administered to a patient as a complete mixture of each active ingredient of thrombomodulin, immunosuppressant and antidiabetic agent. In addition, the thrombomodulin, the immunosuppressive agent, and the antidiabetic agent in the drug of the present invention can be administered to the patient simultaneously or sequentially as separate components. When administered sequentially, the administration sequence of the antidiabetic agent is not particularly limited as long as the antidiabetic agent is administered before or after islet transplantation, but the antidiabetic agent may be administered after administration of thrombomodulin and an immunosuppressant. preferable. There is also another embodiment in which it is preferable to administer thrombomodulin and an immunosuppressive agent after administration of a therapeutic agent for diabetes. The therapeutic agent for diabetes can be administered before or after islet transplantation, or during islet transplantation, but is preferably administered before or after islet transplantation. The administration sequence or administration site can be appropriately changed according to the condition of the subject patient.

  The agent 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, for example, a solution containing at least one selected from an immunosuppressive agent and a therapeutic agent for diabetes and a thrombomodulin-containing solution are mixed as usual. Thereafter, the additive is added and mixed, or the additive is mixed with at least one selected from water, distilled water for injection or an immunosuppressant dissolved in an appropriate buffer, or a therapeutic agent for diabetes, and then contains thrombomodulin. A method in which a solution is prepared by a method of adding and mixing the solution and freeze-dried can be mentioned. When the chemical | medical agent of this invention is a chemical | medical agent which combines each chemical | medical agent component, it is preferable to manufacture each chemical | medical agent by adding a support | carrier with an appropriate manufacturing method. The drug 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 in an ampoule 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, and then frozen and dried under reduced pressure. Or it can prepare as an aqueous solution injection formulation as it is.

The agent of the present invention is desirably administered by a parenteral administration method, for example, intravenous administration, intramuscular administration, subcutaneous administration or the like. Oral administration, rectal administration, intranasal administration, sublingual administration and the like are also possible. When the drug of the present invention is a drug formed by combining each drug component, each drug component is preferably administered by an appropriate administration method.

In the case of intravenous administration, a method in which a desired amount is administered at once or intravenous infusion is exemplified.
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 drug in the present invention varies depending on the age, weight, degree of disease, 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 agent of the present invention containing thrombomodulin thus obtained was administered during allogeneic transplantation of pancreatic islet cells to type I diabetic mice, it was excellent against an increase in blood glucose level caused by dysfunction of Langerhans islet β cells. Prophylactic and therapeutic effects were observed. That is, from the effects on these model animals, the agent of the present invention is a transplantation adjuvant that can improve the cell survival rate at the time of transplantation and reduce the number of transplanted cells in the islet transplantation for type I diabetic patients. It was confirmed to be useful as. As described above, when the drug 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 drug of the present invention is very useful in that a diabetic patient who is likely to bleed can exhibit a safe and high effect without increasing the risk of bleeding even when receiving islet transplantation. It has been reported that thrombomodulin, which is an active ingredient of the drug 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 the coagulation time (for example, Mohri M et al., Thromb Haemost 1999; 82: 1687-93) using a sample obtained by adding the agent of the present invention to plasma. The measurement of partial thromboplastin time (APTT) is exemplified. Moreover, as an evaluation method of the bleeding state in islet transplantation, using the amount of blood (transfusion) or plasma preparation as an index is exemplified.

  The drug 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 with little surgical invasion, which is a feature of islet transplantation. Moreover, since the chemical | medical agent of this invention does not raise the risk of bleeding as mentioned above, the test | inspection for evaluating that a dosage 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 by these at all.
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 rats 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 (hereinafter sometimes abbreviated as TMD123) 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 (hereinafter sometimes abbreviated as TMD123M) 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 (hereinafter sometimes abbreviated as TME456) 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 (hereinafter sometimes abbreviated as TMD12) 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.

[Test Example 1]
<Allogeneic syngeneic transplantation of islet cells in type I diabetic mice>
Male C57BL / 6 mice (Charles River, 7-10 weeks old) were administered 180 mg / kg of streptozocin (Sigma) to create a type I diabetic mouse model in which the function of the islets of Langerhans was specifically reduced. Three days after the administration of streptozotocin, collagenase treatment was performed with reference to the method of Sutton R et al. Jpn 1979; 26: 495-499), 200 islet cells isolated from C57BL / 6 mice were allogeneically transplanted intraportally into the liver. The transplanted mice were divided into groups, and physiological saline was rapidly administered to one test group. In the other test group, thrombomodulin (TMD123, 10 mg / kg) was rapidly administered intravenously once before islet transplantation, once every 12 hours and 24 hours after transplantation. Before and after islet transplantation (two to three times a week), heparinized blood was collected from the tail vein, and the blood glucose level in the obtained plasma was measured using an autoanalyzer (Beckman). As a result, in the physiological saline administration group (FIG. 1), the blood glucose level was not normalized in 200 islet cells, but the blood glucose level was normalized by administration of thrombomodulin (FIG. 2). Thrombomodulin has been shown to prevent islet graft injury after transplantation and control blood glucose levels.

It is a figure which shows transition of the postoperative blood glucose level of the physiological saline solution administration group (N = 5) in the allogeneic islet transplantation with respect to the streptozotocin induction type I diabetic mouse. Streptozotocin-induced type I diabetic mice transplanted with 200 islets isolated from allogeneic syngeneic mice (C57BL / 6) via portal vein intrahepatic transplantation. Syngeneic Transplants, in which no allograft rejection occurs. The vertical axis represents non-fasting blood glucose level, and the horizontal axis represents the number of days after transplantation. It shows that the fasting blood glucose level in the physiological saline administration group remains elevated. It is a figure which shows transition of the postoperative blood glucose level of the thrombomodulin (TMD123) administration group (N = 5) in the allogeneic islet transplantation with respect to a streptozotocin-induced type I diabetic mouse. Streptozotocin-induced type I diabetic mice transplanted with 200 islets isolated from allogeneic syngeneic mice (C57BL / 6) via portal vein intrahepatic transplantation. Syngeneic Transplants, in which no allograft rejection occurs. The vertical axis represents non-fasting blood glucose level, and the horizontal axis represents the number of days after transplantation. It is shown that blood glucose level after transplantation is normalized by three doses of TMD123 (10 mg / kg), immediately before transplantation, 12 hours, and 24 hours later.

Claims (8)

A drug comprising thrombomodulin as an active ingredient, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation, and normalizing blood glucose level after pancreatic islet transplantation. The agent according to claim 1, wherein the thrombomodulin is a soluble thrombomodulin. The thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 11, Drugs. The thrombomodulin is a peptide having a sequence of positions 19 to 516 in each of SEQ ID NO: 9 or SEQ ID NO: 11, or an amino acid sequence in which one or more amino acids of the amino acid sequence of the peptide are substituted, deleted, or added The drug according to any one of claims 1 to 3, which is a peptide having thrombomodulin activity. A drug comprising a combination of the drug according to any one of claims 1 to 4 and an immunosuppressant, wherein the drug is administered to a diabetic patient receiving pancreatic islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation, The drug for normalizing blood glucose level after transplantation. A drug comprising a combination of the drug according to any one of claims 1 to 4, an immunosuppressive agent, and a therapeutic drug for diabetes, wherein the drug is a diabetic patient who receives pancreatic islet transplantation and / or a diabetic patient who receives pancreatic islet transplantation The drug for normalizing blood glucose level after islet transplantation. The drug according to any one of claims 1 to 6, wherein the diabetic patient undergoing islet transplantation is a type I diabetic patient. A drug comprising thrombomodulin as an active ingredient for treating and / or improving diabetes by being administered to a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation.

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