JP6570053B2 - Non-injury site preparation containing sphingosine-1-phosphate receptor 2 activating compound - Google Patents

Description

  The present specification relates to a non-injury site administration preparation containing a sphingosine-1-phosphate receptor 2 activating compound.

  Multilineage-differentiating stress enduring cells that are present in the mesenchymal cell fraction and are expressed without surface manipulation as SSEA-3 (Stage-Specific Embryonic Antigen-3) as a surface antigen; Muse Cell) is known to be responsible for the pluripotency of the mesenchymal cell fraction (Patent Document 1, Non-Patent Documents 1 and 2). In addition, since Muse cells administered transvascularly are induced at the administration site of a compound that activates sphingosine-1-phosphate (S1P) receptor 2 (S1PR2) (for example, SIP itself), It has been reported that the compound is a migration factor for Muse cells (Patent Document 2).

  In addition, it has been found that Muse cells at the site of administration of SIP may be applicable to the treatment of various diseases based on tissue regeneration. In particular, it has been reported that when Muse cells are intravenously administered to a myocardial infarction, it accumulates in the heart, which is a disease site, and reduces the infarct region at the site (Patent Document 3).

International Publication WO2011 / 007900 Pamphlet International Publication WO2014 / 133170 Brochure International Publication WO2014 / 027684 Pamphlet

: Dezawa, M .; , Et al. , J .; Clin. Invest. , Vol. 113, p. 1701-1710 (2004): Dezawa, M .; , Et al. , Science, Vol. 309, p. 314-317 (2005)

  As shown in Patent Document 3, the use form of Muse cells for treatment of a disease is a form of cell transplantation in which a sample is collected from a patient suffering from a disease, or collected and cultured, and transplanted again to the individual. The usage form which takes is mentioned.

  However, when cell transplantation is involved, there are cases where it is difficult to proliferate to the required number of cells and to control the quality of cultured cells.

  Moreover, even if the S1PR2-activating compound is a migration factor, it is necessary to administer the compound to the site of injury. However, administration and delivery of the compound to the site of injury are not always easy.

  As described above, although cell therapy is highly useful, it is still difficult to prepare an appropriate Muse cell, and the hurdle to treatment is not low. There was also difficulty in administering S1PR2 activating compounds.

  This specification provides the formulation which can utilize a Muse cell in a more practical form in view of the said problem.

  Since the Muse cells are pluripotent stem cells that are endogenous in humans or the like, they are inherently contained in individuals or derived from bone marrow at the time of injury (hereinafter referred to as endogenous Muse cells). Has been found to be a suitable preparation for more effective delivery to the site of injury. The present specification provides the following means based on these findings.

(1) A non-injury site-administered preparation for the treatment of myocardial injury, comprising a compound that activates sphingosine-1-phosphate receptor 2 (S1PR2) as an active ingredient.
(2) The preparation according to (1), wherein the myocardial injury is treated without transplanting pluripotent stem cells.
(3) The preparation according to (1) or (2), wherein the myocardial injury is acute myocardial infarction.
(4) The preparation according to any one of (1) to (3), which is a preparation for transdermal administration.
(5) The preparation according to any one of (1) to (4), which is a subcutaneous injection or an intramuscular injection.
(6) The preparation according to any one of (1) to (5), wherein the daily dose is 1 mg / kg or more and 10 mg / kg or less.
(7) The preparation according to any one of (1) to (6), wherein the compound is an agonist for sphingosine-1-phosphate receptor 2.
(8) The endogenous pluripotent stem cell has the following characteristics:
The preparation according to any one of (1) to (7), having one or two of (a) positive for SSEA3 and / or CD105 and (b) negative for CD117 and / or CD146.
(9) A non-injury site-administered formulation for delivering endogenous pluripotent stem cells to an injury site, comprising a compound that activates S1PR2 as an active ingredient.
(10) The preparation according to (9), which is a transdermal administration preparation.
(11) The preparation according to (10), wherein the compound is an agonist for sphingosine-1-phosphate receptor 2.

It is a figure which shows the outline | summary of the protocol of the S1P administration test using a rabbit myocardial infarction model. It is a figure which shows the measurement result of the Muse cell in the blood of a rabbit myocardial infarction model. It is a figure which shows the effect of S1P administration with respect to a rabbit myocardial infarction model. It is a figure which shows the evaluation result of the amount of S1P in blood.

  The present disclosure relates to formulations for the treatment of myocardial injury and formulations for delivery of endogenous pluripotent stem cells to the site of injury.

  The preparation of the present disclosure (hereinafter simply referred to as the present preparation) can contain an S1PR2 activating compound (hereinafter simply referred to as the present compound). According to this preparation, the compound can be administered to a non-injury site and contained in the bloodstream, thereby mobilizing pluripotent stem cells such as endogenous Muse cells into the bloodstream and delivering them to the injury site. it can.

  The administration site of this preparation is a site different from the site of injury where tissue or cell injury occurs in an individual. This formulation uses this compound not to mobilize pluripotent stem cells such as endogenous Muse cells into the bloodstream, but as so-called migration factors. For this reason, endogenous Muse cells can be accumulated at an injured site by administering to a site where Muse cells are not desired to be accumulated.

  For this reason, this preparation mobilizes endogenous pluripotent stem cells such as endogenous Muse cells into the bloodstream and accumulates them at the injury site without cell transplantation and without administering this compound to the injury site. Thus, it is possible to contribute to the regeneration of tissues and cells at the site of injury.

  This preparation can be used for the treatment of myocardial injury. By administering this preparation to a site that is not an injury site of myocardial injury, endogenous pluripotent stem cells are mobilized in the bloodstream and delivered to the site of myocardial injury to suppress the expansion of the infarct region or reduce the infarct region be able to. Thereby, a myocardial disorder can be treated or improved.

  Hereinafter, embodiments of the present disclosure will be described in detail.

(Non-injury site preparation for treatment of myocardial injury containing S1PR2 activating compound)
The non-injury site administration preparation for treatment of myocardial injury of the present disclosure (hereinafter referred to as the present preparation for treatment) can contain an S1PR2 activating compound as an active ingredient. The therapeutic formulation can mobilize endogenous stem cells into the bloodstream and deliver them to the site of myocardial injury. Hereinafter, first, endogenous pluripotent stem cells will be described.

(Endogenous pluripotent stem cells)
The therapeutic preparation can act on endogenous pluripotent stem cells preexisting in the living body. In the present specification, the term “endogenous pluripotent stem cell” refers to a pluripotent stem cell that is inherently present in a living body or induced due to an injury caused in the living body without being transplanted. Means.

  Examples of endogenous pluripotent stem cells include cells that Dezawa, one of the inventors of the present invention, found in the human body and named them “Muse (Multilineage-differentiating Stress Ending) cells”. Muse cells can be obtained from skin tissues such as bone marrow fluid and dermal connective tissue, and are also scattered in connective tissues of each organ. Moreover, this cell is a cell which has the property of both a pluripotent stem cell and a mesenchymal stem cell.

  The endogenous pluripotent stem cells such as Muse cells are preferably at least one of SSEA3 positive and CD105 positive.

  For example, Muse cells are identified as double positives for the respective cell surface markers “SSEA-3 (Stage-specific embrionic antigen-3)” and “CD105”. Accordingly, Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators. Details such as a method for separating Muse cells, an identification method, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as the population of Muse cells, all SSEA-3 positive cells are CD105 It is known to be a positive cell. That is, when separating Muse cells from mesenchymal tissue or cultured mesenchymal stem cells in a living body, Muse cells can be purified and used simply by using SSEA-3 as an antigen marker.

  In the present specification, a cell population containing pluripotent stem cells (Muse cells) or Muse cells isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker is simply “ Sometimes referred to as “SSEA-3 positive cells”. In the present specification, “non-Muse cells” refer to cells other than “SSEA-3 positive cells”, which are cells contained in a mesenchymal tissue or cultured mesenchymal tissue in a living body.

  More specifically, Muse cells or cell populations containing Muse cells can be obtained by using an antibody against cell surface marker SSEA-3 alone, or using both antibodies against SSEA-3 and CD105. (Eg, mesenchymal tissue). Here, “living body” means a living body of a mammal. In the present disclosure, the living body does not include a fertilized egg or an embryo whose development stage is earlier than the blastula stage, but includes an embryo at a developmental stage after the blastula stage including a fetus or blastula. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. It is done.

  In the present disclosure, Muse cells are clearly distinguished from embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) in that they are derived from living tissue. The “mesenchymal tissue” refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, and connective tissues present in various organs. For example, Muse cells can be obtained from bone marrow or skin. For example, it is preferable to collect a mesenchymal tissue of a living body and separate and use Muse cells from this tissue. Moreover, you may isolate | separate a Muse cell from a cultured mesenchymal cell using the said isolation | separation means.

  Endogenous pluripotent stem cells such as Muse cells are CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC). ), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB Marker), and at least one of 11 markers selected from the group consisting of Dct (MB marker), for example, 2, 3, 4, 5, 6, 7, 8, Non-expression of 9, 10 or 11 markers can be separated as an index. For example, without limitation, non-expression of CD117 and CD146 can be separated as an index, and non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index, and The non-expression of 11 markers can be separated as an index.

  As described above, a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 positive or double positive of SSEA-3 positive and CD105 positive as an index. It is known that human adult skin and the like contain various types of stem cells and progenitor cells. However, Muse cells are distinct from these cells. That is, such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells. (ADSC). Therefore, Muse cells can be isolated using “non-expression” of a marker unique to these cells as an index.

In addition, the Muse cell having the above-described characteristics targeted in the present invention is as follows:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
It may have at least one property selected from the group consisting of (iii) showing no neoplastic growth; and (iv) having a self-renewal capability. Preferably, it has 2 or more, more preferably 3 or more, and still more preferably all characteristics.

  In the above (i), “the telomerase activity is low or absent” means that the telomerase activity is low or cannot be detected using, for example, TRAPEZE XL telomerase detection kit (Millipore). “Low” telomerase activity means, for example, telomerase having a telomerase activity comparable to that of somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less compared to Hela cells. It means having activity.

  Regarding (ii) above, the Muse cell has the ability to differentiate into three germ layers (endoderm, mesodermal, and ectoderm) in vitro and in vivo, for example, induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. In addition, when transplanted to the testis in vivo, it may show the ability to differentiate into three germ layers. Furthermore, it has the ability to migrate, engraft and differentiate into organs (heart, skin, spinal cord, liver, muscle, etc.) that have been damaged by implantation into a living body by intravenous injection.

  As for (iii) above, Muse cells have the property of proliferating at a growth rate of about 1.3 days in suspension culture, but stop growing in about 10 days. Further, when transplanted to the testis, Muse cells have cancer for at least half a year. It has the property of not becoming

  Regarding (iv) above, Muse cells have a self-renewal (self-renewal) ability. Here, “self-renewal” refers to culturing cells contained in an embryoid body-like cell mass obtained by suspension culture of one Muse cell, and forming an embryoid body-like cell mass again. . The self-renewal may be repeated once or multiple times.

(S1PR2 activating compound)
The therapeutic preparation can contain an S1PR2 activating compound as an active ingredient. S1PR2 is one of the receptors for sphingosine-1-phosphate (SIP). It has been reported that it is specifically expressed in Muse cells, which are endogenous pluripotent stem cells (Patent Document 1, etc.).

  S1PR2 receptor activating compounds include sphingosine-1-phosphate (S1P), sphingosine-1-phosphate derivatives, and sphingosine-1-phosphate receptor agonists. Here, “sphingosine-1-phosphate (S1P)” is represented by the following formula. SIP is a metabolite of sphingolipid that constitutes a cell membrane, and is excised from a cell membrane by a certain enzyme and released, and then bound to a G protein-coupled receptor expressed on the cell membrane, thereby causing cell migration and the like. It is known as a physiologically active substance that causes Moreover, as a receptor for S1P, an S1P receptor that is a G protein-coupled receptor is known, and it has been known that five types of S1PR1 to S1PR5 exist so far.

  As the S1PR2 activating compound, S1P derivatives can be used as long as they have the ability to recruit endogenous pluripotent stem cells to the bloodstream in addition to SIP. The S1P derivative is not particularly limited, but sphingosylcholine, galactosylsphingosine (psychosin), glucosylsphingosine (glucosicosine), sulfogalactosylsphingosine (lysosulfatide), N, N-dimethylsphingosine 1-phosphate, N, N, Mention may be made of N-trimethylsphingosine 1-phosphate, ceramide 1-phosphate, dihydrosphingosine 1-phosphate, phytosphingosine 1-phosphate, and dehydrophytosphingosine 1-phosphate, and salts thereof.

In addition, as the S1PR2 activating compound, an agonist for S1PR2 can be used. Examples of such agonists include the following compounds (1) to (5).
(1) 1- (2- (1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl) -2-oxoethyl) -5- (trifluoromethyl) pyridin-2 (1H) -one (for example Park, SW, et al., J. Am. Soc. Nephrol., 23, p.266-80 (2012)).
(2) 1- (2- (1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl) -2-oxoethyl) pyrrolidine-2,5-dione (3) 1- (2- (1- Benzyl-2,5-dimethyl-1H-pyrrol-3-yl) -2-oxoethyl) -3-methylimidazolidine-2,4,5-trione (4) 1- (1-benzyl-2,5-dimethyl -1H-pyrrol-3-yl) -2-((1-methyl-1H-tetrazol-5-yl) thio) ethanone (5) (S) -1- (2- (1-benzyl-2,5- Dimethyl-1H-pyrrol-3-yl) -2-oxoethyl) -2 ', 3'-dihydrospiro [imidazolidine-4,1'-indene] -2,5-dione

  Examples of the S1PR2 activating compound include substances that inhibit S1P lyase responsible for dephosphorylation reaction. In addition, S1P is dephosphorylated in the living body by sphingosine-1-phosphate lyase present in the endoplasmic reticulum and decomposed into trans-2-hexadecenal and ethanolamine phosphate. Usually, this dephosphorylation reaction is performed. It is known that the reaction of adding phosphoric acid to trans-2-hexadecenal is in an equilibrium state.

Examples of such inhibitors include the following compounds (6) to (8).
(6) (E) -1- (4-((1R, 2S, 3R) -1,2,3,4-tetrahydroxybutyl) -1H-imidazol-2-yl) ethanone oxime (eg, Bagdanoff, (See JT, et al., J. Med. Chem., Vol. 53, p. 8650-8862 (2010)).
(7) (1R, 2S, 3R) -1- (2- (isoxazol-3-yl) -1H-imidazol-4-yl) butane-1,2,3,4-tetraol (Bagdanoff et al., Supra)
(8) 1- (5-((1R, 2S, 3R) -1,2,3,4-tetrahydroxybutyl) -1H-imidazol-2-yl) ethanone (eg, Cayman Chemical Item Number 13222 Cayman Chemical Com. , Michigan, USA)

  The therapeutic preparation can be used for the treatment of myocardial injury. In the present specification, the myocardial injury refers to an injury caused by a decrease or cessation of blood flow to the myocardium caused by stenosis or occlusion of the coronary artery. Examples include angina pectoris, unstable angina pectoris, and myocardial infarction. Myocardial infarction specifically includes severe large myocardial infarction and associated heart failure. Here, “myocardial infarction” refers to myocardial necrosis caused by occlusion of a coronary artery. “Heart failure” refers to a syndrome caused by a failure of cardiac function that pushes out a sufficient amount of blood flow, resulting in decreased cardiac output and associated increase in venous pressure, and the various clinical symptoms that result. included. Myocardial infarction causes acute heart death and chronic heart death.

  The myocardial injury to which the therapeutic preparation is applied is advantageously acute myocardial infarction. This is because the therapeutic preparation is a preparation suitable for rapid treatment after onset. In the case of acute myocardial infarction, the mortality rate is as high as 35 to 50%, and 60 to 70% of deaths are deaths within 1 to 2 hours after onset. Also, if the first attack myocardial necrosis is large, the risk of recurrence of myocardial infarction is high. Therefore, in the treatment of myocardial infarction, prompt treatment is required when a seizure occurs, and it is important to keep the size of the necrotic myocardium, that is, the infarct size as small as possible.

  Moreover, it is advantageous that the myocardial injury to which the therapeutic preparation is applied is severe large myocardial infarction. In particular, it is advantageous to have a myocardial infarction that allows left ventricular remodeling of the myocardium to progress. There are various classifications for determining the severity of myocardial infarction. Such classification includes, for example, classification by time course, morphological classification (intramyocardial range, site, size of necrosis, etc.), myocardial necrosis, ventricular reconstruction after infarction, hemodynamic classification (Related to treatment, prognosis, etc.), classification by clinical severity, etc. Here, a myocardial infarction having a high degree of severity and having suffered from myocardial necrosis in a wider area is particularly referred to as “severe large myocardial infarction”. For example, complete occlusion of the proximal part of the left coronary artery. In severe large myocardial infarction, myocardial left ventricular remodeling progresses and heart failure occurs, and it is known that the prognosis is poor. “Left ventricular remodeling” after myocardial infarction means hypertrophy of myocardial cells at the non-infarcted site, an increase in stroma (extracellular matrix), which is compensated for the decrease in cardiac function due to non-thinning of the infarcted site occurring after myocardial infarction Refers to a series of changes such as enlargement of the heart lumen. Since long-term prognosis after myocardial infarction correlates with the degree of left ventricular dysfunction, suppressing left ventricular remodeling is essential to maintain and preserve left ventricular function.

  The therapeutic preparation is a preparation in which the S1PR2 activating compound is administered to a non-injured site. Here, the non-injured site refers to a site different from the damaged site of myocardial injury. In the present specification, the injury site of myocardial injury refers to degeneration or loss of various cells and tissues caused by trauma, inflammation, disease, ischemia, necrosis, tumor formation, aging, etc. in myocardial tissue or cells. Means the specific part lost by. For example, the damaged site in the myocardium includes a site in which cells or tissues are damaged due to blood flow being reduced or stopped due to stenosis or occlusion of blood vessels in the myocardium.

  This preparation is administered to a site different from the site of injury, that is, a site of non-injury. A non-injury site is a site other than the above-mentioned injury site. That is, this therapeutic preparation can accumulate endogenous pluripotent stem cells at the injured site without transplanting the pluripotent stem cells by administering to a site different from the injured site. .

  The non-injured site as the administration site is not particularly limited, but is preferably a site other than the heart. Therefore, for example, any site other than the local heart may be used as the administration site applied to the administration route of general preparations.

  In addition to oral administration, this therapeutic preparation is subcutaneous, intramuscular, intravenous, intrathecal, sublingual, rectal, vaginal, nasal, inhalation, transdermal, oral mucosa, implant, etc. Formulation forms intended for parenteral administration can be employed. Although it does not specifically limit as a formulation form, About subcutaneous administration, intramuscular administration, intravenous administration, intrathecal administration, injections, such as an injection, etc. are mentioned. In the case of transcutaneous, oral mucosa, implants, etc., patches and films are also possible.

  The therapeutic preparation is preferably in a form other than intravenous administration, and for example, subcutaneous preparation, intramuscular preparation, and transdermal preparation are suitable. Of these, transdermal administration preparations such as subcutaneous injections and intramuscular injections are suitable. This is because these preparations are suitable for mobilizing Muse cells in the bloodstream. For example, in the case of a subcutaneous injection or an intramuscular injection, although not particularly limited, the upper arm part or the buttocks can be used as the administration site.

  This therapeutic preparation can be produced using a known preparation method depending on the administration form and the like. When an injection is prepared, a pH adjusting agent, a buffer, a stabilizer, an isotonic agent, a local anesthetic and the like can be added, and a local injection can be produced using a conventional method. Examples of the pH adjusting agent and buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA (sodium edetate), thioglycolic acid, and thiolactic acid. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the isotonic agent include sodium chloride and glucose.

  The concentration of the S1PR2 activating compound included in the therapeutic preparation can be appropriately changed depending on the degree of damage at the injury site. For example, the concentration of the S1PR2 activating compound effective for mobilizing Muse cells into the bloodstream and accumulating them at the site of injury is not particularly limited, and is, for example, 1 nM to 100 μM.

  The dosage of the present therapeutic preparation varies depending on the severity of myocardial injury, the number of days of onset, the age of the individual, etc., but is 1 mg / kg body weight, 10 mg / kg body weight or more, and less than 1 day. It can be carried out over a suitable number of times (for example, about 10 times or less, 2 times or more and about 8 times or less). The dose may be 3 mg / kg body weight, daily or more 8 mg / kg body weight, or less than day, and preferably 4 mg / kg body weight, daily or more 6 mg / kg body weight, or less than day. For example, after the onset of acute myocardial infarction, it is preferable to administer at least once within 24 hours after the onset.

  This therapeutic preparation can treat myocardial injury by using an S1PR2 activating compound as an active ingredient and administering it to a non-injured site of myocardial injury. That is, this therapeutic preparation can accumulate Muse cells existing in the living body and / or Muse cells induced in the living body at the time of injury at the injured site while being administered to the non-injured site. By utilizing the regenerative capacity of the cells, the damaged site can be repaired, and the progress of the injury can be suppressed or the injury can be reduced.

  Although inferring and not limiting to the present disclosure, the therapeutic formulation increases the blood concentration of the S1PR2 activating compound, thereby allowing endogenous pluripotent stem cells such as Muse cells in the bone marrow to Can be mobilized during the flow. Muse cells once mobilized in the bloodstream are thought to accumulate at the site of injury based on their migration ability. Some of the inventors have already found that Muse cells are accumulated at the injury site using the S1PR2 activating compound as a migration factor. However, the migration factor induces a migration element (here, a Muse cell) on the high concentration side of the migration factor according to the concentration gradient of the factor.

  In contrast, the therapeutic preparation does not use an S1PR2 activating compound as a migration factor. As a result, this preparation for treatment is one in which the S1PR2 activating compound is administered to a non-injury site that is not an injury site, and endogenous pluripotent stem cells are accumulated at the injury site. This therapeutic preparation is described in Patent Document 2 in that the S1PR2 activating compound is not administered to the injured site, and that endogenous pluripotent stem cells can be accumulated at the injured site when administered to a non-injured site. It can be said that it is more advantageous than the pharmaceutical composition. Further, even those skilled in the art who have contacted Patent Document 2 cannot predict the degree of delivery of the Muse cells to the injured site by the S1PR2 activating compound to the non-injured site. It is also unthinkable that myocardial injury can be treated by such usage and dosage.

(Non-injury site preparation for delivering endogenous pluripotent stem cells to the injury site)
The non-injury site administration preparation of the present disclosure (hereinafter simply referred to as the present delivery preparation) can contain a compound that activates sphingosine-1-phosphate receptor 2 as an active ingredient. This delivery preparation can deliver endogenous pluripotent stem cells to the injured site by administration to the non-injured site. With respect to the S1PR2 activating compound and the non-injured site in this delivery preparation, the various embodiments already described for the preparation for treatment can be applied as they are.

  This delivery preparation is not particularly intended for applications such as diseases. The present invention is applied to a disease in which an injured site is generated in a living body, a state in which an injured has occurred, or a state in which such a disorder is foreseen. The site of injury referred to here is not limited to the above-mentioned myocardial injury, but causes various trauma, inflammation, disease, ischemia, necrosis, tumor formation, aging, etc. in various organs, organs and tissues in the living body. It means a specific site lost due to degeneration or loss of various cells and tissues.

(Method for assisting diagnosis of injury in living body or inspection method for injury in living body).
According to the present specification, it is possible to provide a method for assisting diagnosis of injury in a living body or a method for inspecting injury in a living body, which includes a step of measuring the amount of S1P in blood. According to this method, by measuring the amount of S1P in the blood, it is possible to make a positive determination or to assist in making a positive determination regarding the occurrence of damage in any part of the living body. An increase in the amount of S1P in the blood means mobilization of Muse cells. In other words, it means the occurrence of a repair point in the living body.

  The amount of S1P in the blood is not particularly limited, and can be measured by a known method such as MS, HPLC, coloration (fluorescence) method and the like.

  The following examples further illustrate the present invention, but the present invention is not limited to these examples.

  In this Example, S1P was administered subcutaneously to a rabbit myocardial infarction model based on the protocol shown in FIG. 1, and blood Muse cells and infarct areas were evaluated. Note that the experimental protocol using rabbits in this example was approved by the Ethics Committee on Animal Experiments at Gifu University and published by the National Institutes of Health (NIH) “Management and Use of Experimental Animals”. Was implemented in accordance with the “Guidelines on Revision” (revised 1996).

(Preparation of rabbit myocardial infarction model)
Japanese white rabbits (about 2-3 kg / animal) were anesthetized with 30 mg / kg sodium pentobarbital. In rabbits, arterial gas analysis was performed continuously and ventilation conditions were adjusted as appropriate to maintain arterial gas in the physiological range. The left carotid artery and jugular vein were cannulated and arterial pressure was monitored. After left thoracotomy in the third intercostal space, the heart was exposed and 4-0 silk ligation was performed under the branch of the coronary artery descending the center of the outer front of the left ventricle. A thin vinyl tube was passed through both ends of the suture, and the coronary artery branch was occluded by pulling the suture. The tube was then clamped using mosquito hemostatic forceps. Myocardial ischemia was confirmed by local cyanosis and electrocardiographic changes. The occlusion (ischemia) time is appropriately adjusted. After the suture was released, it was confirmed by a change of the myocardium to red throughout the risk area (see Yasuda et al., Am. J. Physiol. Heart. Circ. Physiol., 296: H1558-1565, 2009).

(Reduction effect of blood Muse cells and myocardial infarction size by subcutaneous administration of S1PR2 agonist)
Based on the protocol shown in FIG. 1, the infarction (ischemia) time due to ligation was 30 minutes (corresponding to 3 hours of ischemia in humans) in rabbits, and then the suture was released and reperfusion was started. After 24 hours of reperfusion, the control group is physiological saline, and the S1P administration group is 5 mg / kg of SID46371153 (the compound (1) described above) which is an agonist for sphingosine-1-phosphate receptor 2. The injection was performed intramuscularly on the back of the rabbit.

(Measurement of blood Muse cells)
48 hours after reperfusion, blood was collected from each group and SSEA3 and CD44 positive cells were counted by FACS. The results are shown in FIG.

  As shown in FIG. 2, in the S1P administration group, it was found that about 1.5 times as many Muse cells as the control group were mobilized in the blood.

(Evaluation of infarct area)
14 days after reperfusion, the effect of reducing the infarct size in each group was compared. More specifically, rabbits 14 days after reperfusion were treated with heparin (500 U / kg) and euthanized by intravenous injection of excess amounts of pentobarbital. The heart was removed and the effect of reducing the infarct size in the myocardial tissue was also examined histochemically by Masson trichrome staining.

  The excised heart was fixed in 10% formalin solution, and after embedding in paraffin, a section was prepared from each specimen in the cross-sectional direction so that an atrioventricular ring was obtained. Thereafter, Masson trichrome (MT) staining was performed according to a conventional method to visualize the myocardial infarction region. In this MT staining, a tissue composed of living cells is stained red, while a collagen fibrosis tissue is pale and observed as if the color has been lost. The results are shown in FIG.

  As shown in the upper part of FIG. 3, it can be seen that in the control group to which physiological saline was administered, the collagen fiberized tissue (infarct site) that was not stained with MT spread. On the other hand, in the left ventricular tissue of the S1P administration group, the pale part is small compared to the control, indicating that the infarct size has decreased. Characteristically, papillary muscles were also observed to recover from infarction.

  In addition, as shown in the lower part of FIG. 3, the infarct area of the control group was 33.46%, while that of the S1P administration group was 18.26%. That is, it was found that the infarct area was reduced to about half.

  From the above, it was found that the S1PR2 agonist administered subcutaneously can mobilize Muse cells into the blood, reach the injury site of the heart, and reduce the infarct area.

  In this example, blood S1P after the onset of acute myocardial infarction was evaluated. Control group (healthy person group: 63 people, average age 62.4 ± 12.9, male 38 people, female 22 people), coronary artery disease (CAD) (group (healthy human group; 63 people, average age 62.4 ± 12. 9, control group 7 out of acute myocardial infarction (AMI) onset group (healthy person group: 63, average age 62.4 ± 12.9, 38 males, 22 females) The blood concentration was measured for 5 subjects, 5 CAD groups and 7 AMI groups, and blood samples were collected regularly until 28 days after onset, and the maximum value was averaged. The measurement was performed by a general protocol using LC / MS / MS, and the results are shown in FIG.

  As shown in FIG. 4, it was found that the amount of S1P in the blood was high in the AMI group. Therefore, it was found that blood S1P increases in response to the occurrence of an injury site due to myocardial infarction or the like in a living body.

Claims (5)

  A preparation for the treatment of myocardial injury, comprising sphingosine-1-phosphate as an active ingredient and administered by subcutaneous injection or intramuscular injection at a non-injured site for the treatment of myocardial injury. Treating the myocardial injury is without transplantation pluripotent stem cells, formulations according to claim 1.   The preparation according to claim 1 or 2, wherein the myocardial injury is acute myocardial infarction.   The formulation in any one of Claims 1-3 whose daily dosage is 1 mg / kg or more and 10 mg / kg or less. The preparation is a preparation for mobilizing endogenous pluripotent stem cells into the bloodstream,
The endogenous pluripotent stem cell has one or two of the following characteristics: (a) positive for SSEA3 and / or CD105, and (b) negative for CD117 and / or CD146. Formulation in any one of 1-4.