JP5727167B2 - Novel control method for isolated islets and transplanted islet disorders targeting NCX - Google Patents

Description

  The present invention relates to an islet cell protective agent.

  The number of diabetic patients in Japan is said to be about 8.9 million (2007 Diabetes Survey Report), of which about 100,000 diabetic patients must continue to inject insulin throughout their lives. As an ultimate treatment for releasing such severely diabetic patients from insulin injection, attempts have been made to transplant islet cells that make insulin into the liver of diabetic patients, permanently establish them, and completely cure diabetes.

  There are more than 500 clinical reports on the above treatment methods from 2000 in the world, and there are 18 cases in Japan from the first case to the present in 2004. However, there are two major unsolved problems in the above treatment. The first is that the islet cell donors are limited, and the lack of islet cells is a serious problem, and therefore an efficient islet isolation method is required. The second is that even if an immunosuppressive agent is used, the transplanted islet cells are destroyed by early rejection that occurs within a few hours after transplanting another organ. That is, because of this destruction, transplantation of all islet cells collected from one person does not provide a therapeutic effect, and transplantation is performed 2-3 times, that is, islet cells collected from 2 to 3 persons are not transplanted into one person. There is a problem that the therapeutic effect cannot be obtained.

  According to Patent Document 1, in the “Edmonton Protocol” in which healthy islets are transplanted into diabetic patients, the islets have a fragile three-dimensional structure, and a large amount of oxygen is required to improve proliferation and survival rate. It is described to do. Patent Document 1 also states that during this process, islets are damaged or destroyed if oxygen delivery conditions are not optimal, affecting the yield of healthy islets removed from a given donor pancreas. . In Patent Document 1, prior to cell isolation and transplantation, one or more emulsified perfluorocarbons (ePFCs) are injected into the donor pancreas to increase the health and engraftment of the islets, which are severely isolated. It is supposed to be able to withstand the treatment.

  Patent Document 2 describes a transplant islet disorder inhibitor for islet transplantation containing an IL-6 inhibitor as an active ingredient. In Patent Document 2, it is said that administration of an anti-IL receptor antibody suppresses inflammatory cytokine production in wet cells after transplantation.

  In Patent Document 3, by administering an anticoagulant activated protein (APC) immediately before transplantation, 2 hours after transplantation, and 4 hours from the tail vein of a mouse in which the function of the islets of Langerhans is specifically reduced, It is said that the prevention of post-reproductive islet graft damage was prevented, the number of engrafted islets was increased, and the control of blood glucose level was shown.

  In Patent Document 4, it is said that a blood glucose level after islet transplantation of a diabetic patient can be normalized by using a drug containing thrombomodulin for a diabetic patient receiving islet transplantation and / or a diabetic patient receiving pancreatic islet transplantation. ing. In addition, drugs containing thrombomodulin can improve the survival rate of transplanted islet cells after islet transplantation, prolong the survival time of transplanted islet cells, and normalize insulin concentration in plasma It is said that diabetes can be treated and / or improved more effectively.

JP 2008-532547 A International Publication No. 2007/043641 Pamphlet JP 2008-24591 A JP 2008-189574 A WO99 / 20598 pamphlet International Publication No. 98/43943 Pamphlet Japanese Patent Laid-Open No. 10-218844 Japanese Patent Laid-Open No. 10-265460 JP 11-49752 A

Annual Review of Physiology, 52, 467-485, 1990 Nippon Yakurigaku Zashi, 111, 105-115, 1998 The Journal of Pharmacology and Experimental Therapeutics, 296, 412-419, 2001 The Journal of Pharmacology and Experimental Therapeutics, 298, 249-256, 2001 European Journal of Pharmacology 458, 155-162, 2003

  However, the technique of the above-mentioned document leaves room for improvement in the following points.

  The technique of Patent Document 1 can increase islet oxygenation prior to cell isolation and transplantation, but does not take into account rejection occurring on the recipient side after transplantation.

  The techniques of Patent Documents 2 to 4 are useful as a treatment for improving the establishment rate of islet cells by administration to the recipient side, but protection of islet cells before cell isolation and transplantation is not considered.

  As described above, there has been no known technique capable of improving the islet cell colonization rate by suppressing the damage caused to the islet cells before the transplantation and suppressing the rejection occurring after the transplantation.

The present invention
[1] A pancreatic islet cell protective agent in islet isolation and / or islet transplantation, comprising a compound showing NCX (sodium / calcium exchange system) inhibitory activity or a pharmaceutically acceptable salt thereof,
[2] A compound exhibiting NCX inhibitory activity is represented by the formula (1)

[In the formula (1), R ¹ , R ² and R ³ are the same or different and each represents a hydrogen atom or a halogen atom;

Indicates. R ⁴ represents a hydrogen atom, a substituted or unsubstituted C ₁ -C ₆ alkyl group, or a substituted or unsubstituted C ₁ -C ₆ alkoxy group, Z represents a nitro group, an amino group, or NHC (O) CH ₂ R ⁵ R ⁵ represents a hydrogen atom, a substituted or unsubstituted C _{1 to} C ₆ alkyl group, a substituted or unsubstituted C _{1 to} C ₆ alkoxy group, a halogen atom, a hydroxy group, a C _{2 to} C ₇ acyloxy group, NR ⁶ R ⁷ or

R ⁶ and R ⁷ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted C ₁ -C ₆ alkyl group, or an N-methyl-4-piperidinyl group, and R ⁸ represents a hydrogen atom, a hydroxy group, or C _{2 to} C _{7 represents an} alkoxycarbonyl group, Y represents a methylene group, an epoxy group, a thio group or an NR ⁹ group, and n represents an integer of 1 to 4. R ⁹ represents a hydrogen atom, a substituted or unsubstituted C ₁ -C ₆ alkyl group, or a substituted or unsubstituted phenyl group. An islet cell protective agent according to [1], which is a compound represented by
[3] Islet cell protection according to [1] or [2], wherein the compound exhibiting NCX inhibitory activity is 2- [4-[(2,5-difluorophenyl) methoxy] phenoxy] -5-ethoxyaniline Or [4] islet cell protective agent according to any one of [1] to [3] used for the treatment of diabetes.

  According to the present invention, islet cells can be protected from hypoxia by using a phenoxyaniline derivative and a phenoxypyridine derivative exhibiting NCX inhibitory activity of formula (1), and from rejection after islet transplantation. Islet cells can be protected. Therefore, it is possible to improve the islet cell colonization rate by suppressing rejection caused after transplantation while suppressing damage to the islet cells before transplantation.

  ADVANTAGE OF THE INVENTION According to this invention, while suppressing the damage to an islet cell before transplantation, the rejection reaction which arises after transplantation can be suppressed, and the establishment rate of an islet cell can be improved.

It is a result which shows the effect of SEA0400 with respect to the hypoxia induction cell death of an islet cell. It is a result which shows the effect of SEA0400 with respect to HMGB1 release | release from an islet cell. It is a result which shows the effect of SEA0400 with respect to the plasma glucose level after a pancreatic islet cell transplant in a STZ induction diabetic mouse. It is the result of having quantified the amount of mRNA of NCX1, NCX2 and NCX3 in mouse heart, brain and pancreatic islet by real-time PCR. It is the result of having confirmed the presence of the protein of NCX1, NCX2, and NCX3 in the pancreatic islet of a mouse | mouth by Western blotting. It is a result which shows the change of the plasma glucose level after transplanting the islet cell obtained by isolation operation in SEA0400 presence to a STZ induction diabetic mouse. It is a result which shows the grade of the cell damage at the time of culture | cultivating the islet cell obtained by isolation operation in SEA0400 presence under hypoxic conditions.

  Embodiments of the present invention will be described below.

  One aspect of the present invention is an islet cell protective agent in islet isolation and / or islet transplantation, comprising a compound exhibiting NCX inhibitory activity or a pharmaceutically acceptable salt thereof.

In the present invention, the “compound exhibiting NCX inhibitory activity” is preferably one that inhibits NCX activity derived from brain (particularly glial cells) by 50% or more at a concentration of 3 μM. In addition, the measuring method of NCX activity is described in Non-Patent Document 4 (especially, see page 250, right column, “Cell Culture”, “Na ⁺ -Ca ²⁺ Exchange Activity”) and the literature cited therein. .

The “compound exhibiting NCX inhibitory activity” is more preferably a compound that specifically inhibits NCX for the purpose of further preventing side effects. In the present invention, the “compound that specifically inhibits NCX” refers to a compound that hardly inhibits other ion channels, transporters, enzymes, and receptors at a concentration that inhibits NCX. Specifically, for example, 3 μM Ca ²⁺ channel, Na ⁺ channel, K ⁺ channel, Na ⁺ / H ⁺ transporter, norepinephrine transporter, Na ⁺ , K ⁺ -ATPase, Ca ²⁺ -ATPase, phosphosease, phospholipase A ₂ nitric-oxides synthesase, constitutive nitric-oxide synthase, adenosine r ceptor, adrenergic receptor, glutamate receptor, bradykinin receptor, LTB4 receptor, it is preferable not to PAF receptor inhibited more than 50%.

  NCX has a plurality of subtypes, for example, NCX1, NCX2, and NC3 subtypes. Compounds that inhibit any one or more of these subtypes are preferred. Since the expression level of NCX1 is very high in islets, compounds that inhibit NCX1 are particularly preferred.

In addition, the measuring method using each ion channel, a transporter, an enzyme, and a receptor is described in the nonpatent literature 4 and the literature referred to this.
Examples of compounds that specifically inhibit NCX include phenoxyaniline derivatives and phenoxypyridine derivatives. For example, the compound shown by Formula (1) is mentioned.

In the formula (1), the C ₁ -C ₆ alkoxy group means a linear or branched alkoxy group having 1 to 6 carbon atoms, for example, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy Group, isobutoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, neopentyloxy group, tert-pentyloxy group, 1-methylbutoxy group, 2-methylbutoxy group, 1,2-dimethyl Examples thereof include a propoxy group, a hexyloxy group, and an isohexyloxy group.

The substituent of the substituted C ₁ -C ₆ alkoxy group, chloro group, fluoro group, a nitro group, an amino group, dimethylamino group, a carboxyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a hydroxy group, a cyano group, And a carbamoyl group.
A halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

The C ₁ -C ₆ alkyl group means a linear or branched alkyl group having 1 to 6 carbon atoms, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec -Butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, hexyl group, isohexyl group, etc. .

The substituent of the substituted C ₁ -C ₆ alkyl group, chloro group, fluoro group, a nitro group, an amino group, dimethylamino group, a carboxyl group, a methoxycarbonyl group, an ethoxycarbonyl group, a phenyl group, a methoxy group, an ethoxy group, Examples thereof include a hydroxy group, a cyano group, and a carbamoyl group.

The C ₂ -C ₇ acyloxy group means a linear or branched acyloxy group having 2 to 7 carbon atoms, and the acyl moiety may be cyclic or contain an aromatic group. Examples thereof include an acetoxy group, a propionyloxy group, an isopropionyloxy group, a cyclohexylenyloxy group, and a benzoyloxy group.

The C ₂ -C ₇ alkoxycarbonyl group means a linear or branched alkoxycarbonyl group having 2 to 7 carbon atoms, and the alkoxyl moiety may be cyclic or contain an aromatic group. Good. Specifically, for example, methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, isopropoxycarbonyl group, butoxycarbonyl group, isobutoxycarbonyl group, sec-butoxycarbonyl group, tert-butoxycarbonyl group, pentyloxycarbonyl group, iso Pentyloxycarbonyl group, neopentyloxycarbonyl group, tert-pentyloxycarbonyl group, 1-methylbutoxycarbonyl group, 2-methylbutoxycarbonyl group, 1,2-dimethylpropoxycarbonyl group, hexyloxycarbonyl group, isohexyloxycarbonyl Groups and the like.

  As the substituent of the substituted phenyl group, chloro group, fluoro group, nitro group, amino group, dimethylamino group, carboxyl group, methoxycarbonyl group, ethoxycarbonyl group, methyl group, ethyl group, methoxy group, ethoxy group, hydroxy group , A cyano group, a carbamoyl group, and the like.

  “Pharmaceutically acceptable salts thereof” include salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, maleic acid, succinic acid, Mention may be made of salts with organic acids such as fluoroacetic acid, dichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, gluconic acid, benzenesulfonic acid and citric acid. In addition, the compound of this invention can exist also as various solvates. Moreover, it may be a hydrate from the viewpoint of applicability as a medicine.

  Specific examples of “compounds that specifically inhibit NCX” include:

And 2- [4-[(2,5-difluorophenyl) methoxy] phenoxy] -5-ethoxyaniline (SEA0400) represented by In addition, the compound represented by Formula (1) is compoundable by the manufacturing method of patent documents 5-9 (especially Examples 1-17 of patent document 5, Examples 1-24 of patent document 6). And Examples 1 to 8 of Patent Document 7, Examples 1 to 37 of Patent Document 8, and Examples 1 to 25 of Patent Document 9).

  In the present invention, “islet isolation” is a method of collecting pancreatic islets from the pancreas, specifically digesting the pancreas by injecting a digestive enzyme such as collagenase from the pancreatic duct of the removed donor pancreas, This refers to a treatment (purification step) in which islets are separated from exocrine cells, which are other main constituent cells of the pancreas (digestion step), and then the islets are purified. As a method for purifying the islets, for example, a specific gravity centrifugation method using a specific gravity difference between endocrine cells and exocrine cells is used. By administering the islet cell protective agent of the present invention to the donor pancreas, islet cells can be protected from damage in such islet isolation treatment.

  In the present invention, “islet transplantation” refers to transplanting isolated islet cells. As a method of transplantation, for example, there is a technique of injecting islet cells through a tube (catheter) placed in the portal vein percutaneously transhepatically. By administering the islet cell protective agent of the present invention to donor pancreas or isolated islet cells, normal islet cells with little damage can be transplanted.

  The islet cell protective agent of the present invention can be used in the treatment of diabetes. Diabetes includes type 1 diabetes, type 2 diabetes, pancreatic diabetes, gestational diabetes and the like. Diabetes mellitus caused by a specific cause such as when it occurs secondarily due to pancreatitis or the use of a steroid or when a specific gene is abnormal is also included in the above-mentioned diabetes. The “diabetic patient” in the present invention is preferably a patient who develops type 1 diabetes. The secretion of insulin, a hormone that lowers blood sugar, is extremely reduced or almost no longer secreted, so there is a high risk of abnormally increased sugar in the blood and diabetic ketoacidosis. Therefore, it is almost always necessary to have a strong treatment such as insulin injection. Moreover, diabetic complications such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, cerebrovascular disorder, ischemic heart disease, lower limb arterial occlusion become a problem if blood glucose levels are not sufficiently controlled for a long time . Therefore, the transplantation method of the present invention is also effective in preventing or treating these diabetic complications.

One aspect of the present invention is a method of adding a compound exhibiting NCX inhibitory activity or a pharmaceutically acceptable salt thereof to a donor pancreas or a pancreatic islet isolated from a donor in vitro, and transplanting the pancreatic islet to a diabetic patient after the addition. And a method for suppressing islet cell damage after islet transplantation in a diabetic patient.
Moreover, the aspect of this invention may include the process of administering the compound which shows NCX inhibitory activity, or its pharmaceutically acceptable salt to the diabetic patient who performs an islet transplant.

  The islet cell protective agent of the present invention can be used in islet transplantation. Islet transplantation requires an islet cell donor (donor), and includes brain death donor islet transplantation, cardiac arrest donor islet transplantation, and living islet transplantation. The islet cell donor is a mammal. The mammal is preferably a human. In addition, the diabetic patient (recipient) transplanted with the islet of the present invention is a mammal, preferably a human. The islet cell donor and the diabetic patient may be of the same type or different type, but are preferably the same type.

  Examples of the method for administering or adding an islet cell protective agent in islet isolation or islet transplantation according to the present invention include a method for administration into the body of a pancreatic islet cell donor, a method for direct administration to the pancreas before extraction from the islet cell donor, and a pancreatic islet A method of adding all or part of the cell donor to the pancreas in vitro, a method of adding it to the islet cells being isolated, and a method of adding to the islet cells after isolation and before being transplanted to a diabetic patient The method of doing is mentioned. When the safety of the islet cell protective agent of the present invention is ensured, a method of administration into the body of a diabetic patient before transplantation and a method of administration into the body of a diabetic patient after transplantation are also possible.

  Examples of the method of administering the islet cell protective agent of the present invention to the donor pancreas include a method of administering from the pancreatic duct. In this case, the islet cell protective agent of the present invention is preferably used in a form dissolved in a preservation solution or a buffer solution. Examples of the preservation solution or buffer used at this time include UW (University of Wisconsin) solution, ET-Kyoto solution, and Hank's solution. Further, in the digestion step of islet isolation treatment, it may be dissolved in a buffer solution containing a digestive enzyme and injected together from the pancreatic duct. The dosage can be 1 μM to 100 μM when administered to the human pancreas.

  In addition, when administering to the islet cells being isolated, after the pancreas is digested to separate the islets, the purification step is performed in the presence of the islet cell protective agent of the present invention when performing the purification step by specific gravity centrifugation. It can be performed.

  Moreover, as a method of administering the islet cell protective agent of the present invention to the isolated islet cells, a method of dipping in a liquid in which the islet cell protective agent of the present invention is dissolved may be mentioned. In this case, it is preferable to dissolve the islet cell protective agent and use a liquid that is isotonic with the islet cells. For example, it is preferable to use a culture solution (for example, D-MEM). BSA (bovine serum albumin) may be added as necessary. Moreover, you may use the liquid used at the time of islet transplantation to a diabetic patient. The liquid in which the islet cells are soaked may contain nutrients (eg, sugars, amino acids) for maintaining the life of the islet cells. The concentration of the drug for treating cells needs to be a concentration that sufficiently inhibits NCX, preferably 0.1 μM to 1000 μM, more preferably 1 μM to 100 μM, and more preferably 5 μM to 100 μM. preferable. The dipping time is preferably 30 minutes to 72 hours, more preferably 5 hours to 48 hours. The temperature at the time of soaking is preferably 30 ° C. to 40 ° C., but more preferably the body temperature of the islet cell donor. When transplanting pancreatic islets to diabetic patients, it is preferable to remove the cytoprotective agent as much as possible. However, when the safety of the cytoprotective agent is ensured, removal of the drug from the pancreatic islet is not essential.

When administering the islet cell protective agent of the present invention to a pancreatic islet cell provider or a diabetic patient, an appropriate pharmaceutical composition form (tablet, pill, capsule, granule, dry syrup) is appropriately used using a known carrier, diluent or the like. And can be used orally or parenterally. For example, examples of the injection include subcutaneous injection, intramuscular injection, and intraperitoneal injection. When the islet cell protective agent of the present invention is administered to a diabetic patient, it may be before islet transplantation, at the same time as islet transplantation, or after islet transplantation. Moreover, administration of an islet cell protective agent may be performed once, and can also be administered continuously. Further, when the islet cells are administered from the portal vein of a diabetic patient, the islet cell protective agent of the present invention can be administered simultaneously with the islet cells or at different times. It can be produced by an agitation granulation method, a fluidized bed granulation method, or a crushing granulation method using additives such as an excipient, a disintegrant, a binder, a lubricant, and a coating base. In addition, antioxidants, coating agents, colorants, flavoring agents, surfactants, plasticizers, and the like can be added as necessary. Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

  When the pancreatic islet cell protective agent of the present invention is directly administered to a diabetic patient, the dosage varies depending on age, body weight, dosage form, etc., but is usually 0.1 to 1000 mg / day for an adult, and this is once a day. Or it can be divided into several doses.

  The islet cell protective agents of the present invention may be administered as part of a pharmaceutical composition with at least one known chemotherapeutic agent.

  In addition, at least one known chemotherapeutic agent may be administered to a diabetic patient after transplanting the islet cell administered with the islet cell protective agent of the present invention. For example, at least one known immunosuppressive agent may be administered.

  When the islet cell protective agent of the present invention is directly administered to a diabetic patient, at least one known chemotherapeutic agent may be administered in combination or simultaneously. For example, at least one known immunosuppressive agent may be administered in combination or may be administered simultaneously.

  In the present invention, “islet cell protection” means that the survival rate of transplanted islet cells is improved. Whether the islet cell engraftment rate has improved can be confirmed by measuring the blood glucose level in the living body, as described in the Examples. For example, the blood glucose level can be measured with a Beckman glucose analyzer after plasma separation.

  In addition, “islet cell protection” includes the improvement of the islet cell survival rate by protecting the islet cells from hypoxia. That is, “islet cell protection” includes suppression of cell death of islet cells in a hypoxic state. As used herein, “hypoxia” refers to an oxygen concentration of 5% or less, preferably an oxygen concentration of 3% or less, more preferably an oxygen concentration of 1 in a liquid or gas to which islet cells are exposed. % Or less. The normal oxygen state usually has an oxygen concentration of 15% to 25%.

  In addition, “islet cell protection” includes the improvement of the islet cell survival rate by suppressing the innate immune response associated with transplantation. More specifically, it includes suppression of rejection and an improvement in the islet cell engraftment rate. Suppression of rejection preferably means early rejection, and early rejection refers to rejection that occurs immediately after transplantation to within 1 week, preferably within 24 hours immediately after transplantation. More specifically, “islet cell protection” of the present invention includes inhibiting the release of HMGB1 (high mobility group1) from islet cells. It is known that early transplant rejection occurs when HMGB1 is released out of the cell (The Journal of Clinical Investigation, 120 (3): 735-743, 2010).

  By administering the islet isolation and / or islet cell protective agent of the present invention, it can be considered that the engraftment rate of the islet cells is improved when the blood glucose level continuously decreases. Islet engraftment can be determined by whether the blood glucose level of the diabetic recipe becomes normal after transplantation. For example, when 200 islets that can be isolated from one donor are transplanted in the control group in the examples of the present invention, the blood glucose level remains high, and the engraftment rate in this case is regarded as 0%. I can do it. In addition, in the SEA0400 administration group in the examples of the present invention, the blood glucose levels of all recipients are normalized regardless of the same number of islet transplants as in the control group. In this case, the engraftment rate is 100%. Can be considered.

Next, the effect of the present invention will be described.
According to the phenoxyaniline derivative and phenoxypyridine derivative having an NCX inhibitory action of the present invention, it is possible to protect against ischemia during islet isolation and / or islet transplantation and to suppress rejection. Therefore, the fixing property of the islet cells after transplantation can be improved.

NCX, a transporter present in the cell membrane, plays a major role in the regulation of intracellular free Ca ²⁺ concentration, an important ion that regulates the contraction of the myocardium and various smooth muscles, the release of neurotransmitters, and gene expression. It is known to fulfill (see Non-Patent Document 1). This drug that inhibits NCX is known to suppress an abnormal increase in intracellular Ca ²⁺ concentration in cells such as myocardium, kidney, and brain, and to have a cytoprotective action on these cells (non-patented). References 2 and 3).

  It is known that phenoxyaniline derivatives (Patent Documents 5 to 7) and phenoxypyridine derivatives (Patent Documents 8 and 9) are compounds that inhibit NCX, and these phenoxyaniline derivatives and phenoxypyridine derivatives are high in NCX. It was also known to have selectivity (see Non-Patent Document 4). Furthermore, it was also known that SEA0400, a phenoxyaniline derivative, has been confirmed to be effective against brain and heart ischemia-reperfusion models (see Non-Patent Documents 4 and 5).

  However, the action of phenoxyaniline derivatives and phenoxypyridine derivatives having NCX inhibitory activity on islet cells has not been reported.

  Under these technical backgrounds, the present inventors have found that the phenoxyaniline derivative and phenoxypyridine derivative having the NCX inhibitory action of the present invention suppress the rejection that occurs when collecting or transplanting pancreatic islets, The present inventors have found that the fixability is improved and have completed the present invention.

  The compound of the formula (1) having NCX inhibitory activity of the present invention exhibits an excellent cytoprotective action against islet cells, and further suppresses rejection caused when isolating and transplanting islets, Can improve the establishment of islet cells.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable.

(Test Example 1) Effect of SEA0400 on hypoxia-induced cell death of pancreatic islet cells Pancreatic islet cells were prepared by a known method (Transplantation. 42: 689-691, 1986; Endocrinol. Jpn. 26: 495-49, 1979; Nature. 244: 447, 1973) and isolated from the pancreas of male C57BL / 6 mice (10-15 weeks old, Charles River Japan Co., Ltd.) by collagenase treatment. Specifically, the pancreas is digested by injecting collagenase (Nitta Gelatin Co., Ltd., 2 mg / ml in Hanks solution, 2-3 ml) from the pancreatic duct (37 ° C., standing for 30-40 minutes), and the pancreatic islets are exocrine. After separating from the tissue, the islets were isolated by specific gravity centrifugation using the difference in specific gravity between endocrine cells and exocrine cells. Isolated islet cells were seeded in a petri dish (200-500 cells / dish, culture medium (D-MEM + 0.2% BSA)) and placed in a chamber. Inside the chamber is usually gas (mixed gas of air (95%) and carbon dioxide (5%)) or low oxygen gas (mixed gas of oxygen (1%), carbon dioxide (5%) and nitrogen (94%)) ) In the presence, the temperature was 37 ° C., and the islet cells were cultured for 6 hours in the environment. The partial pressure of oxygen in the chamber in the presence of low oxygen gas was about 1/6 that of normal gas. The case where normal gas was used was defined as “normal oxygen state”, and the case where low oxygen gas was used was defined as “low oxygen state”. The NCA inhibitor SEA0400 (final concentration 10 μM) was added to the petri dish just before being placed in the chamber after the islet cells were seeded in the petri dish. As a control, a solution in which DMSO (dimethyl sulfoxide) was added to a petri dish instead of SEA0400 was used.
Cells cultured for 6 hours in the chamber were double-stained with Hoechst 33342 (HO342, Sigma-Aldrich Japan) and PI reagent (propidium iodide, Sigma-Aldrich Japan). Cell death was confirmed by observing with a fluorescence microscope (manufactured by Leica, DM IRB) (FIG. 1). HO342 is a UV-excitable nucleic acid staining dye that exhibits blue fluorescence (shown as dark gray in FIG. 1), while PI is only taken up by cells with damaged plasma membranes. A cell into which PI has been taken up has a red color (displayed as white or light gray in FIG. 1), meaning that cell death has occurred. In the presence of normal gas, islet cell death was hardly observed. On the other hand, cell death (PI positive) was observed in cells cultured for 6 hours in the presence of hypoxic gas (control of hypoxia). This hypoxia-induced cell death was significantly suppressed by the coexistence of 10 μM SEA0400 (hypoxic SEA0400-added sample). Thus, SEA0400 showed cytoprotective action in vitro against islet cells.

(Test Example 2) Effect of SEA0400 on HMGB1 release from pancreatic islet cells As in Test Example 1, pancreatic islet cells are usually gas (mixed gas of air (95%) and carbon dioxide (5%)) or low in the chamber. The cells were cultured for 6, 12 or 24 hours in the presence of oxygen gas (a mixed gas of oxygen (1%), carbon dioxide (5%) and nitrogen (94%)). The NCA inhibitor SEA0400 (final concentration 10 μM) was added to the petri dish just before being placed in the chamber after the islet cells were seeded in the petri dish. As a control, in the same manner as in Test Example 1, a material added to a DMSO (dimethyl sulfoxide) petri dish instead of SEA0400 was used.
The culture supernatant was collected from the cells cultured in the chamber, and the amount of HMGB1 released into the culture solution was measured. The amount of HMGB1 was quantified using an HMGB1 ELISA kit (Sinotest Co., Ltd.). As shown in FIG. 2, the amount of HMGB1 released from pancreatic islet cells increased under hypoxic conditions (Hypoxia in FIG. 2). This increase in the amount of HMGB1 was significantly suppressed by the coexistence of 10 μM SEA0400 (Hypoxia SEA0400 in FIG. 2). Although extracellular release of HMGB1 is considered to be a phenomenon associated with early rejection of transplanted islet cells, SEA0400 was shown to suppress this extracellular release of HMGB1.

(Test Example 3) Effect of SEA0400 on plasma glucose level after islet cell transplantation in STZ-induced diabetic mice Streptozotocin (STZ) -induced diabetic mice are male C57BL / 6 mice (10-15 weeks old, Charles River Japan, Inc.) Streptozotocin (180 mg / kg) was intravenously administered. In diabetic mice on day 3 after STZ administration, 200 islets / animal isolated according to the method of Test 1 were transplanted intraportally into the liver. Isolated islet cells were immersed in SEA0400 solution (1 μM or 10 μM) for 1 hour, 3 hours or 24 hours 1 hour, 3 hours or 24 hours before transplantation into mice (200 cells / dish, culture medium) (D-MEM + 0.2% BSA)). Measurement of plasma glucose level after transplantation was performed by collecting blood from orbital sinus of a mouse and using a Beckman glucose analyzer (Beckman Japan) after plasma separation.
The results are shown in FIG. When the islets were not transplanted, all mice (7 mice) remained hyperglycemic (not shown), but when only the islet cells were transplanted, all mice (3 mice) had this hyperglycemic state. It was not improved (control group). On the other hand, all mice transplanted with 10 μM SEA0400 for 1 hour, 3 hours or 24 hours of islet cells (4 for 1 hour treated islet cells, 4 for 3 hour treated islet cells, for 24 hours treated islet cells) In 4 mice), a significant hypoglycemic effect was observed.
From the above results, it was shown that SEA0400 reduces transplanted islet injury, improves islet engraftment, and corrects hyperglycemia. That is, it was shown that SEA0400 can be used as an islet cell protective agent.

(Test Example 4) Quantification of NCX1, NCX2 and NCX3 mRNA levels in mouse heart, brain and pancreatic islets From heart, brain and pancreatic islets of male C57BL / 6 mice (10-15 weeks old, Charles River Japan, Inc.) mRNA extraction and quantification of NCX1, NCX2 and NCX3 mRNA amounts by real-time PCR are described in Jing-Ping Li et al, Endocrinology. 2007, May; 148 (5): 2116-25.
As shown in FIG. 4, it was confirmed that NCX1 was highly expressed in islet cells. From this result, it was shown that a compound that strongly inhibits NCX1, such as SEA0400, can be used as an islet cell protective agent.

(Test Example 5) Confirmation of protein expression of NCX1, NCX2 and NCX3 in mouse islets According to a conventional method (see Biochemistry 37 (49): 17230-17238, 1998), an isolated pancreatic islet extract (FEBS Lett. 329, 227-231, 1993). As a result, it was revealed that NCX1, NCX2, and NCX3 exist in the islet cells.

(Test Example 6) Effect of SEA0400 on hypoxic injury of islet cells associated with islet isolation operation Islet cells digest pancreas by injecting collagenase from the pancreatic duct, isolate the islets from exocrine tissues, and then endocrine cells. Although it is isolated by specific gravity centrifugation using the specific gravity difference of exocrine cells, it is considered that the islet is placed in a hypoxic state and damaged during this isolation process. Therefore, according to the method described in Test Example 1, islet cells were isolated by injecting a mixed solution obtained by adding SEA0400 (final concentration 10 μM) to the collagenase solution from the pancreatic duct. Subsequently, 200 islet cells immediately after isolation were transplanted into the portal vein of streptozotocin-diabetic mice prepared by the method described in Test Example 3. A control group was prepared by adding DMSO to a collagenase solution instead of SEA0400. In the control group, hyperglycemia was maintained in all mice (5 mice), but in the group transplanted with islet cells isolated using collagenase solution containing SEA0400, all mice (6 mice) had normoglycemia. (Fig. 6). This finding indicates that the hypoxic injury of the islet cells accompanying the islet isolation operation was controlled by SEA0400, and the survival rate after transplantation was improved.

(Test Example 7)
In the same manner as in Test Example 6 above, the islet cells were isolated in a state where DMSO (control group) or SEA0400 10 μM (SEA0400 group) was added to the collagenase solution, and the isolated islet cells were seeded in a petri dish, Placed in the chamber (200 pieces / dish, culture medium (D-MEM + 0.2% BSA)). After culturing for 6 hours in normoxia or hypoxia as in Test Example 1, each cell was subjected to PI staining and double staining with HO342, and cell death was confirmed by observation with a fluorescence microscope (FIG. 7). ). As described in Test Example 1, HO342 is a UV-excitable nucleic acid staining dye that exhibits blue fluorescence (shown as dark gray in FIG. 7), while PI is taken up only by cells whose plasma membrane is damaged. It is. A cell into which PI has been taken up has a red color (displayed as white or light gray in FIG. 7), meaning that cell death has occurred. As shown in the figure, in the control group of islet cells cultured under hypoxia, a large number of PI positive cells were found in the center, but in the SEA0400 group of pancreatic islet cells, the number of positive cells was clearly apparent to the extent that PI positive cells were scattered. It was decreasing. This finding indicates that the islet cells obtained by the isolation operation in the presence of SEA0400 are resistant to the subsequent damage caused by hypoxia.

  From the above, according to the present invention, it is possible to provide a new islet cell protective agent that improves islet engraftment.

Claims (2)

An islet cell protective agent in islet isolation and / or islet transplantation, comprising a compound exhibiting NCX (sodium-calcium exchange system) inhibitory activity or a pharmaceutically acceptable salt thereof ,
The islet cell protective agent, wherein the compound exhibiting NCX inhibitory activity is 2- [4-[(2,5-difluorophenyl) methoxy] phenoxy] -5-ethoxyaniline . The islet cell protective agent according to claim 1, which is used for the treatment of diabetes.