JP2011026220A - Pharmaceutical composition for treating myocardial infarction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for a drug which repairs myocardial tissues by allowing an effective amount of G-CSF or EPO to act on the ischemic myocardial part in order to bring about the myocardial tissue repairing and regenerating capability possessed by G-CSF and EPO without side effects to a maximum. <P>SOLUTION: The intravenously administering pharmaceutical composition for treating myocardial infarction includes a liposome having an average particle diameter of ≥70 and ≤150 nm whose surface has a sialyl Lewis X tetrasaccharide chain (Neu5Ac. alpha. 2-3 Gal. beta. 1-4 (Fuc. alpha. 1-3) GlcNAc) and erythropoietin and/or granulocyte colony-stimulating factor which is included in the liposome and is intravenously administered. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition for treating myocardial infarction using a liposome capable of realizing non-invasive and effective drug delivery to a local site of infarcted myocardium.

  For example, in myocardial infarction such as severe large myocardial infarction, it is known that left ventricular remodeling proceeds and heart failure occurs, resulting in poor life prognosis. Therefore, if the necrotic myocardial tissue can be regenerated and left ventricular remodeling can be improved, the prognosis will improve. One method for regenerating infarcted myocardial tissue and improving left ventricular remodeling is cytokine therapy. Specific regenerative repair cytokines include erythropoietin (EPO) and / or granulocyte colony stimulating factor (G-CSF). G-CSF and EPO have been shown to repair myocardial tissue and revascularize when administered systemically after myocardial infarction. EPO has already been applied to renal anemia, and G-CSF has been clinically applied to granulocytopenia. The ethical hurdle for use as a repair and regeneration cytokine is low, and it is extremely minimally invasive with intravenous injection. Because the patient is burdensome and less burdensome, clinical application for myocardial tissue revascularization treatment is possible.

  To date, as a drug delivery system (DDS) of such drugs to infarcted myocardial tissue, gelatin gel patches are applied to the infarcted myocardial local site by a surgical technique, and the drug is applied to the local site from the patch. Release is performed (Non-patent Document 1). On the other hand, sialic acid X (sialyl Lewis X-type tetrasaccharide chain; Neu5Ac.alpha.2-3 Gal.beta.1-4 (Fuc.alpha.1-3) GlcNAc which binds to E-selectin etc. on the surface as a kind of DDS ) Target-directed liposomes to which (SLX) is bound are known (Patent Document 1).

International Publication WO2005 / 011633 Pamphlet

Kobayashi H & Minatoguchi S et al. Cardiovasc Res 2008

Systemic administration of G-CSF and EPO has a problem in that complications such as myocardial infarction and stroke may occur due to increased white blood cell count, increased red blood cell count, and increased hematocrit. In addition, an effective drug DDS for myocardial infarction has not yet been established.
Therefore, the present invention is for myocardial tissue repair capable of causing an effective amount of G-CSF or EPO to act on the ischemic myocardial region in order to maximize the ability of G-CSF and EPO to repair and regenerate myocardial tissue without side effects. It is an object of the present invention to provide a composition for other drugs.

  The present inventors have studied various DDS of G-CSF and EPO in the infarcted myocardial region, and SLX that binds to E-selectin etc. is provided on the surface, and these drugs are encapsulated in liposomes that can pass through capillaries. Thus, the present inventors have found for the first time that the drug can be selectively delivered to the infarcted myocardial region and that the encapsulated drug acts effectively, thereby completing the present invention. In the infarcted myocardial region, although it is known that the expression of selectin and the like is enhanced, it is not known to reach the infarcted myocardial region when liposomes with SLX on the surface are administered intravenously, Even those skilled in the art could not have predicted. In addition, it has not been known that the drug is effectively taken up after reaching the infarcted myocardium. According to the disclosure of the present specification, the following means are provided.

(1) Liposomes having an average particle size of 70 nm or more and 150 nm or less comprising sialyl Lewis X-type tetrasaccharide chain (Neu5Ac.alpha.2-3 Gal.beta.1-4 (Fuc.alpha.1-3) GlcNAc) on the surface; A pharmaceutical composition for treating myocardial infarction administered intravenously, comprising erythropoietin and / or granulocyte colony-stimulating factor encapsulated in the liposome.
(2) A method for treating myocardial infarction, comprising administering the pharmaceutical composition for treating myocardial infarction according to (1) via a vein.

It is a figure which shows the selective accumulation | storage to the infarct site of SLX-liposome by fluorescence. The fluorescent part (red) is indicated by an arrow. The stained tissue for determining the infarct area is shown as a cross-sectional view of the heart at the papillary muscle level. The black purple part is the non-risk area, the red part and the white part are the dangerous areas, the red part is the area that is ischemic but then recovered, and the white part is the infarct (necrosis) after ischemia The region that was

  The disclosure herein relates to a pharmaceutical composition for the treatment of myocardial infarction. According to the pharmaceutical composition disclosed in the present specification, by using a liposome known to bind to E-selectin or P-selectin, the liposome can reach the infarcted myocardial region more than expected, Moreover, it was found that the tissue in the infarcted myocardium can be regenerated and repaired by releasing the drug.

(Pharmaceutical composition for treatment of myocardial infarction)
The pharmaceutical compositions disclosed herein can contain EPO. In this specification, EPO includes physiologically active substances having different amino acid compositions having physiological activity like EPO or a derivative of EPO. In addition, the pharmaceutical composition disclosed herein can contain G-CSF. In this specification, G-CSF includes physiologically active substances having different amino acid compositions having physiological activity similar to that of G-CSF or a derivative of G-CSF.

  The pharmaceutical composition disclosed in the present specification contains liposomes having an average particle size of 70 nm to 100 nm with sialic acid X on the surface. Liposomes usually mean closed vesicles composed of a lipid layer assembled in a membrane and an aqueous layer inside. In the present specification, examples of the liposome include those in which a sugar chain is covalently bonded to the surface, that is, the lipid layer via a linker protein such as human serum albumin. Examples of the sugar chain include sialyl Lewis X-type tetrasaccharide chain (Neu5Ac.alpha.2-3 Gal.beta.1-4 (Fuc.alpha.1-3) GlcNAc). Examples of the linker protein include animal serum albumin such as human serum albumin (HSA) and bovine serum albumin (BSA).

  Examples of the lipid constituting the liposome of the present invention include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, gangliosides or glycolipids or phosphatidylglycerols, cholesterol, and the like. As phosphatidylcholines, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine and the like, and as phosphatidylethanolamines, dimyristoyl phosphatidylethanolamine, Dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, etc. include phosphatidic acids such as dimyristoyl phosphatidic acid, dipalmitoyl phosphine. Thydic acid, distearoylphosphatidic acid, dicetyl phosphoric acid, etc. are gangliosides, ganglioside GM1, ganglioside GD1a, ganglioside GT1b, etc., and glycolipids are galactosylceramide, glucosylceramide, lactosylceramide, phosphatide, globoside. As the phosphatidylglycerols, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol and the like are preferable.

  The liposome used in the present invention may be a normal one, but its surface is preferably made hydrophilic. The liposome itself can be produced according to a known method, and examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-rehydration method. Moreover, it is also possible to adjust the particle diameter of the liposome using an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like. The production method of the liposome of the present invention will be specifically described. For example, first, phosphatidylcholines, cholesterol, phosphatidylethanolamines, phosphatidic acids, gangliosides or glycolipids, or phosphatidylglycerol. A mixed micelle of a lipid and a surfactant sodium cholate is prepared.

  Among them, the formulation of phosphatidylethanolamines is essential as a hydrophilization reaction site, and the formulation of gangliosides or glycolipids or phosphatidylglycerols is essential as a linker protein binding site. And a liposome is produced by performing ultrafiltration of the mixed micelle obtained by this. Subsequently, the liposome surface is hydrophilized using a divalent reagent for crosslinking and tris (hydroxymethyl) aminomethane on the lipid phosphatidylethanolamine of the liposome membrane.

  Hydrophilization of liposomes is achieved by a conventionally known method, for example, a method of preparing liposomes using a phospholipid obtained by covalently bonding polyethylene glycol, polyvinyl alcohol, maleic anhydride copolymer or the like (Japanese Patent Laid-Open No. 2001-302686). In the present invention, tris (hydroxymethyl) aminomethane can be used to make the liposome surface hydrophilic.

A sugar chain is further bound to the liposome used in the present invention via a linker protein. As this means, first, the liposome is bound with an oxidizing agent such as NaIO ₄ , Pb (O ₂ CCH ₃ ) ₄ , or NaBiO _3. The ganglioside present on the liposome membrane surface is oxidized by treatment, and then the linker protein and the ganglioside on the liposome membrane surface are combined by a reductive amination reaction using a reagent such as NaBH ₃ CN or NaBH ₄ . This linker protein is also preferably made hydrophilic, in which a compound having a hydroxy group is bound to the linker protein. For example, bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissk Cynimidyl propionate, disuccinimidyl suberate, 3,3'-dithiobissulfosuccinimidyl propionate, ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyls What is necessary is just to couple | bond tris (hydroxymethyl) aminomethane with the linker protein on a liposome using bivalent reagents, such as a succinate.

In the liposome used in the present invention, a sugar chain is bound to a linker protein on the liposome. For this purpose, the reducing end of the sugar constituting the sugar chain is an ammonium salt such as NH ₄ HCO ₃ or NH ₂ COONH _4. And then bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimidyl propionate, disuccinimidyl suberate, 3,3'-dithiobissulfate A linker protein bound on the liposome membrane surface using a bivalent reagent such as fosuccinimidyl propionate, ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate, Liposome having a sugar chain on its surface by binding to the glycosylated aminated saccharide That. These sugar chains are commercially available.

  In the present invention, sialyl Lewis X-type tetrasaccharide chain (SLX) is known to bind to E-selectin, and in particular, it is known to have strong directivity to lymph node, brain, liver and spleen. . However, the inventors have found that the infarcted myocardial region can be an effective target.

  The average particle diameter of the liposome is preferably 150 nm or less in consideration of allowing the capillary blood to pass through. More preferably, it is 140 nm or less, More preferably, it is 120 nm or less, More preferably, it is 100 nm or less. Moreover, it is preferable that an average particle diameter is 30 nm or more, More preferably, it is 50 nm or more, More preferably, it is 70 nm or more. The zeta potential of the liposome is -100 to 10 mV, preferably -70 to 0 mV, more preferably -60 to -10 mV. In the process of producing liposomes contained in the pharmaceutical composition disclosed in the present specification, liposomes that are not bound to the surface, liposomes that are not hydrophilicized and have sugar chains bound thereto, and sugar chains that are not bound. There are four types of liposomes, namely hydrophilicized liposomes and hydrophilicized sugar chain-bound liposomes. These four types of liposomes can be used in an isotonic solution such as physiological saline. And the zeta potential range.

  The liposome contains EPO and / or G-CSF. In order to encapsulate these drugs in liposomes, a well-known method may be used. For example, by forming liposomes using a solution containing a drug or the like and lipids of phosphatidylcholines and phosphatidylethanolamines. Drugs are encapsulated in liposomes.

  The pharmaceutical composition of the present invention contains pharmacologically acceptable carriers, diluents or excipients in addition to liposomes bound to the sialyl Lewis X-type sugar chain surface, as well as liposomes to which no sugar chain is bound. You may go out. The pharmaceutical composition of the present invention is preferably administered intravenously. That is, it can be used as an intravenous injection or a drip. Such a composition is produced by a known method and contains carriers, diluents and excipients usually used in the pharmaceutical field. For example, an injection is prepared by dissolving, suspending or emulsifying the sugar chain-bound liposome of the present invention in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, isotonic solutions containing physiological saline, glucose and other adjuvants are used. Suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants and the like You may use together. As the oily liquid, sesame oil, soybean oil or the like is used, and as a solubilizing agent, benzyl benzoate, benzyl alcohol or the like may be used in combination.

  Although the dosage of the pharmaceutical composition disclosed in the present specification can be appropriately determined depending on the severity of myocardial infarction and the like, a pharmaceutically effective amount of the composition of the present invention is administered to a patient. Here, “administering a pharmaceutically effective amount” refers to administering an appropriate level of a drug to a patient to regenerate and repair at least part of the infarcted myocardial region. The frequency of administration of the pharmaceutical composition of the present invention is appropriately selected according to the patient's symptoms.

  Hereinafter, the present invention will be specifically described by way of examples. The following examples do not restrict the present invention.

(Selective accumulation of SLX-liposomes at the infarct site)
In order to investigate whether SLX-liposomes accumulate selectively in the infarct region, a myocardial infarction model of coronary artery 30 minutes ischemia, 1 day, 2 days, 7 days reperfusion was prepared using Japanese white rabbits Then, human serum albumin labeled with the fluorescent substance Cy5.5 is encapsulated in liposomes, and SLX-Cy5.5-liposome with Sialyl Lewis X (SLX) bound to the surface is injected intravenously from the ear vein immediately after the onset of myocardial infarction. Whether or not the fluorescent substance was accumulated in the myocardial infarction part after 1 day, 2 days and 7 days after the infarction was observed with a fluorescence microscope. In addition, 1 ml of liposome having a lipid concentration of 2.6 mg / ml was administered. The results are shown in FIG. In FIG. 1, the green part indicates cardiomyocytes, the blue part indicates cell nuclei, and the red part indicates SLX-Cy5.5-liposomes.

  As shown in FIG. 1, Cy5.5 shown in red is not accumulated at all in the non-infarcted myocardial region, whereas Cy5 shown in red at any time point after infarction 1, 2, and 7 in the infarcted region. .5 was observed, indicating that SLX-Cy5.5-liposomes selectively accumulate at the infarcted myocardial site.

(Evaluation of myocardial infarction size)
Japanese white rabbits (approx. 2 kg) were used to create a myocardial infarction model with 30 minutes of coronary artery ischemia, 1 day, 2 days, and 7 days of reperfusion, no control group, EPO systemic administration group, SLX- (EPO) -liposome group, G-CSF systemic administration group, and SLX- (G-CSF) -liposome group were prepared. For each of these groups, the heart was removed 14 days after the infarction, the ischemic region and the non-ischemic region were determined by Evans blue dye, and the infarct region was determined by TTC staining. FIG. 2 shows the stained tissue. Infarct size was determined as a percentage of the infarct area relative to the risk area of the entire heart. The dose of the active ingredient in each group was as follows.
EPO systemic administration group: EPO 1500 IU / kg / day × 5 day = 7500 IU / kg (about 15000 IU / fowl)
SLX- (EPO) -liposome group: EPO 2000 IU / ml × 1 ml = 2000 IU / wing
G-CSF systemic administration group: 10 μg / kg / day × 5 day = 50 μg / kg (about 100 μg / feather)
SLX- (G-CSF) -liposome group: G-CSF 20 μg / ml × 1 ml = 20 μg / wing

The infarct size in each group was as follows.
Control group (n = 10): 26.8 ± 1.4%
EPO-systemic administration (n = 10): 24.4 ± 1.5%
SLX-EPO-liposome group (n = 10): 17.0 ± 0.8%
G-CSF systemic administration group (n = 10): 10.0 ± 2.0%
SLX-G-CSF-liposome group (n = 10): 18.0 ± 0.8%

  As shown in FIG. 2, it was confirmed that SLX- (EPO) -liposome and SLX- (G-CSF) -liposome administration resulted in a reduction in infarct size as compared with the control. G-CSF systemic large-scale administration (10 μg / kg / day x 5 days) reduces infarct size, but EPO small-scale systemic administration (1500 IU / kg x 5 days) shows no change in infarct size. I have confirmed.

  As described above, according to the present invention, it was possible to obtain a therapeutic effect on the infarcted myocardial region of myocardial infarction beyond expectations by encapsulating the drug in a specific liposome.

Claims (2)

Liposomes having an average particle size of 70 nm or more and 150 nm or less comprising a sialyl Lewis X-type tetrasaccharide chain (Neu5Ac.alpha.2-3 Gal.beta.1-4 (Fuc.alpha.1-3) GlcNAc) on the surface;
Erythropoietin and / or granulocyte colony-stimulating factor encapsulated in the liposome,
A pharmaceutical composition for the treatment of myocardial infarction, which is administered intravenously.   A method for treating myocardial infarction, wherein the pharmaceutical composition for treating myocardial infarction according to claim 1 is administered via a vein.