JP2008024591A - Islet transplantation auxiliary - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the take rate of cells to be regarded as a problem and to decrease the number of the islet cells required in clinical islet transplantation which is expected as a radical therapy for diabetes. <P>SOLUTION: The islet transplantation auxiliary which largely contributes to islet transplantation contains activated protein C (APC) as the major component. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention belongs to the field of ethical drugs, and relates to a drug having an active ingredient derived from blood. Specifically, it relates to a new use of plasma proteins. More specifically, the present invention relates to a medicinal product for transplantation containing activated protein C (hereinafter sometimes referred to as APC) as a main active ingredient, and in particular, increases the survival rate of transplanted cells in islet transplant patients, In particular, the present invention relates to a technical field relating to improvement of life prognosis that can improve QOL, survival rate and the like for diabetic patients.

Diabetes is a typical lifestyle-related disease, and the number of patients in Japan alone is estimated to be over 6 million. Diabetes consists of type I diabetes (more often in children and younger people and almost no insulin is secreted from the pancreatic islets of Langerhans) and type II diabetes (more in adults, although insulin is secreted but its action and effectiveness are poor) are categorized. Insulin is secreted from the pancreas as needed to increase blood sugar, and has a function of lowering blood sugar level. Among insulin-dependent diabetes, type I diabetes has a frequency of 4-6%. The number of patients with type I diabetes is estimated to be 240-360,000. Despite the painful dietary restrictions and insulin injections since childhood, patients with type I diabetes may die from a variety of disastrous complications, which can lead to pancreatic (organ) and islet (Island of Langerhans) There is a long-awaited hope for radical treatment by transplantation. As complications of diabetes, in addition to neuropathy, there are an increasing number of patients with microvascular disorders peculiar to diabetes such as diabetic retinopathy and nephropathy, in addition to the risk of developing myocardial infarction and stroke As a factor, it is known that diabetes is a major cause. In Japan, more than 5,000 people diabetic retinopathy leads to blindness every year, and more than 7,000 people undergo dialysis after diabetic nephropathy. In addition, medical expenses related to diabetes have been increasing steadily, recently exceeding 1 trillion yen. 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.

In order to fundamentally treat type I diabetes, it is necessary to treat so that insulin is normally secreted from the islets. Transplantation is expected as a radical treatment for such diabetes. Pancreas transplantation is intended for patients with type I diabetes who have been depleted of endogenous insulin or patients with renal failure. Simultaneous Pancreas Kidney transplantation (SPK), aiming to normalize blood glucose, improve QOL, and prevent the development of secondary complications. Pancreatic (organ) transplantation performed to compensate for the endocrine mechanism of the pancreas in theory does not require the entire pancreas, and should be achieved only by transplantation of islet cells related to endocrine.

Islet transplantation, in which only the islets of Langerhans are removed from the pancreas and transplanted, is a theoretically superior endocrine replacement therapy for diabetics with impaired islet cells, and many experimental and clinical studies have been conducted since the late 1960s. Has been tried. This transplantation technique consists of 1) treating the pancreas removed from the donor with enzyme, 2) taking out islet cells with a cell separator, and 3) injecting and transplanting the islet cells into the patient's portal vein. When the islet cells enter the liver through the portal vein, they are engrafted in blood vessels stretched in the liver and secrete insulin. The results of clinical trials indicate that the organ suitable for islet transplantation is not the pancreas but the liver. Since islet transplantation is a transplantation method that involves simply instilling pancreatic islet cells, it does not require laparotomy or vascular anastomosis and does not require pancreatic juice treatment, compared to surgery that transplants the entire organ. Has the advantage that it is extremely safe and can be performed on high-risk patients. Moreover, even if rejection occurs, it has the advantage that it is a transplantation method that does not require re-extraction and has a very low burden on the patient.

In 1967, Lacy et al. Reported a method for isolating pancreatic islets by extracting the pancreatic islets by digesting the pancreas with collagenase (see Non-Patent Document 1). Although this method enabled islet isolation from small animals such as mice and rats, isolation of islets from large animal pancreas including humans was difficult. However, after that, Ricordi et al. Developed a unique automatic islet separation device (see Non-Patent Document 2), introduced a digestion process devised by the UCLA group into two stages of so-called warm digestion and cold digestion, Improvements etc. have made it possible to apply to humans.

Islet transplantation is a kind of “cell” transplantation, unlike other “organ” transplants, so that, like sperm banks, islets can be cryopreserved semipermanently, and the required amount of islets can be transplanted when needed A so-called islet bank can be created. The development of cryopreservation of pancreatic islets has been conducted since 1977, when Rajotte et al. Succeeded in treating diabetic rats using rat frozen thawed islets (see Non-Patent Document 3). At present, the method of slow freezing and rapid thawing using a program freezer is the standard method for cryopreservation of human islets.

Clinically, the University of Minnesota first performed islet transplantation in humans in 1974, but until recently the results were poor compared to other organ transplants. However, a protocol devised by the University of Alberta in 2000 (Edmonton Protocol: a protocol for clinical islet transplantation using an immunosuppressive method that does not use steroids) led to a very high insulin withdrawal rate (see Non-Patent Document 4). ) Pancreatic islet transplantation is becoming established as a breakthrough treatment for type I diabetes. This protocol consists of (1) selecting a well-brained brain death donor, (2) reducing the storage time from pancreas extraction to islet isolation, (3) transplanting a sufficient amount of isolated islets, and (4) using rapamycin as an immunosuppressant. It has the feature of adopting. When the islets engraft in the liver, they die over time, so mass transplantation of high-quality islets is the key to successful transplantation.
According to a report from the International Islet Transplant Registry (ITR), of 185 islet transplants performed between 2000 and August 2002, 120 arelets (Islet Transplant Alone: ITA), There were 23 cases of simultaneous transplantation (Simultaneous Islet-Kidney transplant: SIK), and 42 cases of islet after kidney transplantation (IAK). Examining cases that achieved insulin withdrawal for more than 1 week after transplantation, ITA was the best, with 62% for ITA, 45% for IAK, and 30% for SIK. This is because Edmonton Protocol has been used for ITA since 2000 (see Non-Patent Document 5).

In this way, the usefulness of islet transplantation as a treatment for diabetic roots is increasing, but in order to succeed in transplantation, the islet cell engraftment rate is low, and a large amount of islet cell infusion is required due to this. (Requires at least two donors), and further improvements are desired. That is, in normal organ transplantation, if there is one donor, transplantation is established for one recipient, but at present, in the case of islet transplantation, multiple recipients are required for one recipient. Yes, this is a big problem. If this is resolved and the donor: recipient ratio is improved to 1: 1, the transplant is expected to spread explosively and make a significant contribution to the treatment of diabetes.

APC, which is the main active ingredient as an adjuvant for islet transplantation in the present invention, circulates in blood vessels as its precursor protein C (hereinafter sometimes referred to as PC) in blood. Once the coagulation system is activated and thrombin is formed, thrombin binds to the membrane protein thrombomodulin (hereinafter sometimes referred to as TM) on vascular endothelial cells, and activates PC to produce APC having serine protease activity. Convert to APC selectively exerts a strong anticoagulant effect by selectively limiting and inactivating activated factor V and activated factor VIII of the blood coagulation system on cell membrane phospholipids (Non-Patent Document 6, Non-patent document 7). This anticoagulant effect by APC is enhanced when cofactor protein S (hereinafter sometimes referred to as PS) is present. A coagulation control mechanism involving PC, TM, PS, etc. is called a protein C anticoagulation system.

In addition, APC neutralizes vascular endothelial cell or platelet-derived tissue plasminogen activator inhibitor (hereinafter sometimes referred to as PAI) (see Non-Patent Document 8 and Non-Patent Document 9), It is also considered to be involved in enhancement of the fibrinolytic system by suppressing the activation of the anti-fibrinolytic factor Thrombin Activatable Fibrinolysis Inhibitor (hereinafter sometimes referred to as TAFI) (see Non-Patent Document 10). .

Furthermore, APC has been shown to be effective in a baboon sepsis model (see Non-Patent Document 11) and also has an action of suppressing cytokine production from leukocytes (see Non-Patent Document 12). Has been suggested. Clinically, in a large-scale clinical trial conducted by Eli Lilly for severe sepsis patients, the mortality rate was significantly reduced by administration of recombinant APC, and IL-6 levels of inflammatory cytokines were administered. It was shown that it decreased significantly from the first day (refer nonpatent literature 13).

Protein C anticoagulation system is known to be decreased in thrombotic diseases such as sepsis and disseminated intravascular coagulation syndrome (hereinafter sometimes referred to as DIC). It was shown to decline. That is, patients who received allogeneic kidney transplantation were monitored for 9 months and showed that the function of the protein C anticoagulation system (PC, PS, TM) was reduced when rejection occurred. It was reported to be useful for diagnosis (see Non-Patent Document 14).

Japanese Patent No. 3043558 JP-A-61-205487 JP-A-1-002338 Japanese Patent Laid-Open No. 1-085084 Lacy et al., Diabetes, vol.16, p.342-353, 1967 Ricordi et al., Diabetes, vol.37, p.413-420, 1988 Rajotte et al., Cryobiology, vol.14, p.116-120, 1977 N Engl J Med, vol. 343, p.230-238, 2000 Transplantation, vol.38, p.122-125, 2002 Biochemistry, vol.16, p.5824-5831, 1977 J. Biol. Chem., Vol. 258, p.1914-1920, 1982 Proc. Natl. Acad. Sci. USA, vol. 82, p.1121-1125, 1985 J. Biol. Chem., Vol. 276, p.15567-15570, 2001 Blood, vol. 88, p.2093-2100, 1996 J. Clin. Invest., Vol.79, p.918-925, 1987 Am. J. Physiol., P.L197-L202, 1997 N. Engl. J. Med., Vol. 344, p699-709, 2001 J. Exp. Med., Vol. 175, p81-90, 1992 Blood, vol. 63, p.115-121, 1984 Kisiel et al., J. Clin. Invest., Vol. 64, p.761-769, 1979 J. Clin. Invest., Vol. 79, p.918-925, 1987

In recent years, although the technique of islet transplantation has made great progress, the above-mentioned problems of requiring an improved cell engraftment rate and infusion of a large amount of islet cells are sufficient. It has not been overcome, and further improvement of the diabetic root treatment by islet transplantation is eagerly desired. Thus, it is an object of the present invention to improve the survival rate of cells during islet transplantation and to reduce the number of islet cells required.

In view of the above-mentioned backgrounds, the inventors of the present application focused on improving the engraftment rate of transplanted islet cells, and as a result of earnest research to find a drug that satisfies this, surprisingly, it has been attempted in the past. The APC administration of the anticoagulant that has not been found has the effect of significantly improving the survival rate of transplanted islet cells, and the present invention has been completed based on these findings.
That is, the present invention relates to a pancreas transplant survival rate, preferably an islet transplant survival rate improvement agent, characterized by containing APC as a main active ingredient. The agent for improving the survival rate of pancreatic transplantation of the present invention significantly increases the engraftment rate by administering at the time of pancreatic transplantation and islet transplantation, which is expected as a root treatment for diabetes, and normalizes insulin secretion. It is particularly effective in enabling control.

It has been found that the effect of APC can also be obtained by allogeneic islet transplantation. In clinical islet transplantation, if rejection is controlled, transplanted islet damage is reduced by administration of APC, and an improvement in engraftment is expected. In clinical islet transplantation, multiple (two to three) transplants are currently being performed to treat one diabetic recipient. This means that 2 to 3 donors are needed for one treatment. If APC is applied clinically and transplantation from one donor to one recipient is realized, it will be an epoch-making result.
The findings provided by the present invention significantly improve the success rate of islet transplantation. In other words, donor cells that were the biggest problem in the practical application of islet transplantation by enabling transplantation with a small number of cells that could not be transplanted in the past, transplantation with poorly conditioned cells or cells derived from different animals Can be easily secured. The invention of the present application exhibits such remarkable operational effects and is convinced that it is a very significant invention that contributes to this field.

The islet transplantation adjuvant that can be used in combination with the immunosuppressive agent provided in the present invention is characterized by having APC as its main active ingredient.
Examples of the immunosuppressive agent used as an islet transplantation adjuvant of the present invention include, but are not limited to, general-purpose drugs such as rapamycin, anti-CD4 antibody, FK506, and the like. The multi-drug combination is also included in the scope of the technical idea of the present invention. Moreover, there is no special restriction | limiting also about the administration aspect of the islet transplantation adjuvant of this invention, For example, it is thought that it is a suitable aspect that it administers for 1 week after a transplant from immediately before an islet cell transplant.

The method for producing APC used in this embodiment of the islet transplantation adjuvant of the present invention is not particularly limited. For example, PC isolated from human blood or obtained by gene recombination technology is activated. For example, a method for separating APC from human blood, or a method for directly preparing APC by a gene recombination technique. The activation method from PC to APC is not particularly limited, and for example, it can be carried out by a method of activation with thrombin separated from blood such as human or bovine or a method of activation with recombinant thrombin.

Examples of the method for producing APC derived from blood include the following methods. For example, a PC purified by affinity chromatography using an anti-PC antibody from human plasma using a method according to Japanese Patent No. 3043558 (see Patent Document 1) is activated with human thrombin and then used with cation chromatography. Purification method (see Non-Patent Document 15), Kisiel's steps of adsorption and elution of citrate Ba from human plasma, ammonium sulfate fractionation, DEAE-Sephadex column chromatography, dextran sulfate agarose chromatography and polyacrylamide gel electrophoresis A method of activating PC obtained by purification by APC (see Non-patent Document 16), affinity chromatography using an anti-PC antibody starting from a commercially available factor IX complex preparation containing PC Activated PC purified by APC And a method (see Non-Patent Document 17) to.

Examples of methods for preparing APC using gene recombination techniques include the methods described in JP-A-61-205487, JP-A-1-002338, or JP-A-1-085084 (patents). (Refer to Literature 2, Patent Literature 3, and Patent Literature 4).
In order to maintain the activity of the APC prepared by the above method to the maximum, the APC used in the present invention is lyophilized and stored together with a suitable stabilizer. In the present invention, APC as an active ingredient and a known appropriate excipient can be combined to produce the islet transplantation adjuvant of the present invention by a known method. The dosage of the drug having the APC of the present invention as the essential form can be appropriately changed depending on the age, disease state, etc. of the patient. For example, 100 μg to 10 mg / kg body weight / day of continuous administration for 1 to 6 days This is considered a preferred embodiment.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
The blood-derived APC used in this example is a single intravenous administration test in mice and dogs, a repeated intravenous administration test in mice, dogs and young dogs, a mouse reproduction test, and a local irritation test. Its safety has been confirmed by general pharmacological tests (effects on the respiratory circulatory system using beagle dogs) and virus inactivation tests.

(Allogeneic transplantation of islet cells for type I diabetic mice)
400 pancreatic islet cells isolated from C57BL / 6 mice (2 mice) were prepared by administering streptozocin to C57BL / 6 mice and specifically reducing the function of islets of Langerhans β cells. The number of cells that can be prepared from the pancreas was transplanted into the liver via the portal vein. After transplantation, blood glucose levels were normalized (Fig. 1), but 200 islet cells (cells that can be prepared in one mouse pancreas) Number) transplantation did not normalize blood glucose levels (FIG. 2). However, even in the transplantation of 200 islets, blood glucose levels of all mice were normalized when APC was intravenously injected through each 40 μg tail vein immediately before transplantation, 2 hours after transplantation, and 4 hours after transplantation (FIG. 3). It has been shown that APC prevents islet graft injury after transplantation, increases the number of engrafted islets, and controls blood glucose levels.

(Allogeneic transplantation of islet cells for type I diabetic mice)
When 400 islets of BALB / c mice were transplanted portally into the liver in diabetic C57BL / 6 mice used in Example 1, blood glucose levels normalized within 2 to 3 days, but allogeneic rejection As a result, the transplanted islets are destroyed, and the blood glucose level of the recipient rises again around 10 days after transplantation (FIG. 4). Thus, when the anti-CD4 antibody, an immunosuppressant, was administered once at 200 μg intraperitoneally at the time of transplantation, the rejection reaction was suppressed, and the recipient transitioned to normoglycemia after transplantation (FIG. 5). However, when 200 islet cells of BALB / c mice were transplanted into diabetic C57BL / 6 mice, blood glucose levels were not normalized after transplantation without anti-CD4 antibody administration, graft destruction, lymphocyte accumulation And rejection was confirmed (FIG. 6). However, even when anti-CD4 antibody was administered, islet grafts were present in the liver at the transplantation site, and rejection was controlled, but the number of engrafted islets was small and blood glucose levels were not normalized (FIG. 7).

(Effect of APC on allogeneic transplantation of islet cells in type I diabetic mice)
Immediately before transplanting 200 islet cells of BALB / c mice into the liver via portal vein in diabetic C57BL / 6 mice, 200 cells were injected with 40 μg of APC intravenously via the tail vein 2 hours and 4 hours after transplantation. The blood glucose level also normalized in allogeneic islets. Also. The rejection usually appearing within 1 week after transplantation was delayed and appeared 2 weeks later. As a result, the blood glucose level increased again 16 to 17 days after transplantation (FIG. 8). When transplanting 200 islet cells of BALB / c mice into diabetic C57BL / 6 mice, anti-CD4 antibody (200 μg administered once intraperitoneally at the time of transplantation) and APC (40 μg each immediately before transplantation, 2 hours after transplantation, 4 hours after transplantation) When APC was administered intravenously from the tail vein, blood glucose levels were normalized (FIG. 9).

Islet transplantation that is expected as a method for treating diabetic roots, but the islet transplantation auxiliary agent provided in the present invention makes it possible to improve the cell survival rate at the time of transplantation, and to reduce the number of transplanted cells associated therewith, Responds to social demands, especially medical economics. It also opens up the potential as a new immunosuppressant.

It is a figure which shows transition of the postoperative blood glucose level in the system of the allogeneic islet transplantation with respect to the streptozotocin induction type I diabetic mouse. 400 islets isolated from allogeneic syngeneic mice (C57BL / 6) in streptozotocin-induced type I diabetic mice (n = 10). Isografts, 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 the number of islets that can be isolated from one mouse islet is 200. Therefore, when two islets are used as donors, the blood glucose level is normalized after transplantation. It is a figure which shows transition of the postoperative blood glucose level in the system of the allogeneic islet transplantation with respect to the streptozotocin induction type I diabetic mouse. Data when the number of syngeneic islets transplanted is 200 under the same conditions as in FIG. The blood glucose level was not normalized at all after transplantation (n = 10). 200 indicates that the number of engrafted islets is not sufficient. It is a figure which shows the effect of APC at the time of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. Under the same conditions as in FIG. 2, APC 40 μg was intravenously administered 3 times in total, once before transplantation and 2, 4 hours after transplantation. All mice (n = 4) had normal blood glucose levels. It is thought that the damage after islet transplantation was prevented and the number of engrafted islets increased, which was reflected in the blood glucose level. It is a figure which shows transition of the postoperative blood glucose level in the system of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. Streptozotocin-induced type I diabetic mice (C57BL / 6) transplanted with 400 islets from allogeneic BALB / c mice via portal vein intrahepatic transplantation. The blood glucose level is normalized within a few days, but the transplanted islets are destroyed by allogeneic rejection, and the blood glucose levels of all recipients (n = 4) rise again around 10 days after transplantation. Even if there is enough islet volume in the same line, it does not work well in the other line. It is a figure which shows transition of the postoperative blood glucose level in the system of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. In the same conditions as in FIG. 4, 200 μg of anti-CD4 antibody as an immunosuppressant was intraperitoneally administered at the time of transplantation. Rejection was suppressed and blood glucose levels of all recipients (n = 6) remained normal after transplantation. It is a figure which shows transition of the postoperative blood glucose level in the system of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. Under the same conditions as in FIG. 4, the number of islets is 200. The blood glucose levels of all recipients (n = 5) were not normalized at all after transplantation. A histological search confirmed graft rejection and lymphocyte accumulation, and rejection. It is a figure which shows transition of the postoperative blood glucose level in the system of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. 6. Anti-CD4 antibody administered intraperitoneally (200 μg / injection) once at the time of transplantation under the same conditions as in FIG. 6 (200 islets). The blood glucose levels of all recipients (n = 4) were not normalized. Histological search revealed an islet graft in the liver of the transplant site. Although rejection was controlled, the number of engrafted islets was small and blood glucose levels were not normalized. It is a figure which shows the influence of APC administration at the time of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. Under the same conditions as in FIG. 6 (200 islets), APC 40 μg was intravenously administered three times, once before transplantation and 2, 4 hours after transplantation. The blood glucose level decreased in 200 allogeneic islets but increased again 16-17 days after transplantation due to rejection (n = 3). It is a figure which shows the effect of APC administration in the immunosuppressant combined use at the time of allogeneic islet transplantation with respect to streptozotocin-induced type I diabetic mice. The conditions were the same as in FIG. 6 (200 islets), but the anti-CD4 antibody was administered once at the time of transplantation, intraperitoneally (200 μg / injection), and APC 40 μg was administered once before transplantation, 2, 4 hours after transplantation. A total of 3 intravenous doses. The blood glucose levels of all recipients (n = 4) were normalized after transplantation.

Claims (3)

An islet transplantation adjuvant comprising activated protein C as a main active ingredient. The islet transplantation adjuvant according to claim 1, which has an effect of suppressing transplanted cell rejection at the time of islet transplantation. The islet transplantation adjuvant according to claim 1 or 2, which can be used in combination with an immunosuppressive agent.

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