WO2002005840A1

WO2002005840A1 - Medicinal compositions for promoting fixation of transplanted cells

Info

Publication number: WO2002005840A1
Application number: PCT/JP2001/006166
Authority: WO
Inventors: Yoshiki Sawa; Satoshi Taketani; Shigeru Miyagawa
Original assignee: Cardio, Inc.
Priority date: 2000-07-18
Filing date: 2001-07-17
Publication date: 2002-01-24
Also published as: JP2005097118A; AU2001271064A1

Abstract

In the treatment of severe myocardial infarction or myocardiopathy by transplanting myocardial cells into the affected site of severe myocardial infarction or myocardiopathy, the fixation of the transplanted myocardial cells can be promoted and apoptosis of the transplanted myocardial cells and fibrosis of the damaged myocardial site can be inhibited by administering HGF gene or HGF to the damaged myocardial site. Thus severe myocardial infarction or myocardiopathy can be effectively treated.

Description

Description Pharmaceutical composition for promoting cell transplantation

The present invention relates to a pharmaceutical composition for promoting cell colonization of transplanted cells to a disease-damaged site, comprising a hepatocyte growth factor (HGF) or an HGF gene as an active ingredient, and apoptosis of transplanted cells to a disease-damaged site. The present invention relates to a pharmaceutical composition for suppressing fibrosis at a diseased site. More specifically, a pharmaceutical composition containing HGF or an HGF gene as an active ingredient for promoting the transplantation of transplanted cardiomyocytes to a damaged myocardial site and suppressing apoptosis of transplanted cardiomyocytes or fibrosis at a damaged myocardial site The present invention relates to a pharmaceutical composition for medical use. The present invention also relates to a method for treating ischemic or diabetic organ diseases, which comprises injecting transplantation cells and HGF or the HGF gene into a diseased site. More specifically, the present invention relates to a method for treating ischemic or diabetic heart disease, which comprises injecting cardiomyocytes and HGF or HGF gene into a damaged myocardial site. In particular, the present invention relates to a method for treating myocardial infarction or cardiomyopathy by administering cardiomyocytes for transplantation and HGF or the HGF gene to a site of cardiac ischemia due to myocardial infarction or an injured myocardial site due to cardiomyopathy.

Background art

In the circulatory field, despite the remarkable improvement in medical technology in recent years, there remains a disease that cannot be treated except for heart transplantation. For example, severe heart failure, severe myocardial infarction or cardiomyopathy are such diseases.

Myocardial infarction is a disease in which ischemic ischemia occurs in the perfused area due to severe stenosis or occlusion due to various lesions of the coronary artery. There are several classifications for determining the severity of myocardial infarction, and by using these in combination, individual pathological conditions can be more clearly understood. For example, classification based on time course, morphological classification based on the extent and location of the myocardium, the size of the necrosis, ventricular reconstruction after myocardial death and infarction, and hemodynamics related to treatment and prognosis Classification, classification by clinical severity, and the like. What is judged to be high in severity according to these classifications is severe myocardial infarction according to the present invention. There is no accurate treatment method for severe myocardial infarction to date. It is a situation where only the plant is left.

Cardiomyopathy is a general term for diseases caused by structural and functional abnormalities of the myocardium.Secondary cardiomyopathy secondary to underlying diseases such as hypertension, metabolic disorders, and ischemia, and obvious underlying diseases It is classified as idiopathic cardiomyopathy (ICM) that develops without any. Pathological changes include myocardial hypertrophy, fibrosis, and degeneration.

As described above, cardiomyopathy has a fairly widespread spectrum, from relatively obvious causes to those with completely unknown causes. At present, research is being carried out to identify the cause and elucidate the pathogenic mechanism, and no reliable treatment method has yet been found.

Heart failure is a condition in which the heart itself is impaired, such as cardiac insufficiency, circulatory insufficiency, and reduced contractile force, and the inability to circulate the required quantity and quality of blood to the organs throughout the body is called heart failure. However, cardiomyopathy is a terminal symptom of heart disease. Severe heart failure is defined as severe heart failure, also known as end-stage heart failure.

There is no other way to treat patients with severe heart failure than heart transplantation, and even if transplantation is performed, there are various problems such as rejection, and it has not been perfect in many respects. Therefore, transplantation of own atrial cells and right ventricular cells into affected areas of the heart disorder is now being considered as a new trial for the treatment of severe heart failure, severe myocardial infarction and cardiomyopathy (Cell Engineering, 19, 860- 863 (2000)).

However, in such cardiomyocyte transplantation, the quality of transplantation of the transplanted cardiomyocytes determines the success or failure of the treatment. At present, the transplantation rate of transplanted cardiomyocytes is only about 20 to 30% at most.

Disclosure of the invention

An object of the present invention is to provide a pharmaceutical composition for promoting cell colonization of transplanted cells for treating ischemic or diabetic organ diseases. More specifically, it is an object of the present invention to provide a therapeutic method for transplanting cardiomyocytes suitable for treating severe myocardial infarction or cardiomyopathy, for which no effective therapeutic method has been found based on these drugs and compositions.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that colonization of transplanted cells is promoted by performing cell transplantation using HGF or an HGF gene in combination. In other words, HGF or the HGF gene was used in cardiomyocyte cell transplantation. It was found that when used, the colonization of the cardiomyocytes was promoted, and the apoptosis of the transplanted cardiomyocytes and the fibrosis of the cardiomyocytes at the damaged site of the transplantation destination were suppressed. The cardiac function was significantly improved, and a comparison of the cell findings showed that the cardiomyocytes were clearly mature.

As described above, the present inventor has found that by injecting HGF or HGF gene and cardiomyocytes directly into the affected myocardium, colonization of the transplanted cardiomyocytes and regeneration of the cardiomyocytes become possible, and without performing heart transplantation. It has been found that cardiomyocyte transplantation can be used to treat severe heart failure, severe myocardial infarction and cardiomyopathy.

Furthermore, the present inventors have found that the use of the HGF or the HGF gene of the present invention is also effective for cell transplantation therapy for ischemic organ disease. For example, not only cardiomyocyte transplantation to the above-mentioned myocardial infarction site, but also cerebral ischemia, transplantation of neural progenitor cells to the site of cerebral infarction or cells that can be differentiated into nerve cells, myocardial infarction site, skeletal muscle ischemia Transplantation of vascular endothelial cells or cells that can be divided into vascular endothelial cells into the site has become possible. In addition, it has been found that the use of the HGF or the HGF gene of the present invention is also effective for a cell transplantation method for diabetic organ disease. That is, it is known that in diabetic patients, various organ disorders such as impaired blood circulation of the kidney, knee, peripheral nerves, eyes, and limbs occur at a high rate. At present, various treatments for these diseases by cell transplantation are being studied. For example, attempts are being made to transplant insulin-secreting cells into the spleen with reduced insulin secretion, and transplantation of bone marrow-derived cells for impaired limb blood circulation.The low engraftment rate of 1S cells has become a problem. Was. However, by using the HGF or the HGF gene of the present invention, in cell transplantation to a diabetic patient, the engraftment can be promoted and the effect can be enhanced.

That is, the gist of the present invention is:

(1) a pharmaceutical composition comprising hepatocyte growth factor (HGF) or an HGF gene as an active ingredient, for promoting cell colonization of transplanted cells at a disease-damaged site;

(2) the pharmaceutical composition according to (1), wherein the diseased site is an ischemic or diabetic site;

(3) The pharmaceutical composition according to (1) or (2), wherein the diseased site is a damaged myocardial site or a cardiac ischemic site; (4) The pharmaceutical composition according to any of (1) to (3) above, wherein the diseased site is a cardiac ischemic site due to myocardial infarction or a damaged myocardial site due to cardiomyopathy;

(5) The pharmaceutical composition according to the above (2), wherein the diabetic disorder site is a knee with impaired insulin secretion ability or a blood vessel disorder 40.

(6) The pharmaceutical composition according to any one of (1) Riki et al. (5) above, which is administered together with a cell for transplantation to a site of disease.

(7) The pharmaceutical composition according to (6), which is administered together with a cardiomyocyte for transplantation to a damaged myocardial site or a cardiac ischemic site.

(8) The pharmaceutical composition according to (6) or (7) above, which is administered together with a cardiac muscle cell for transplantation to a site of cardiac ischemia due to myocardial infarction or a site of injured myocardium due to cardiomyopathy.

(9) The pharmaceutical composition according to (6), which is administered together with a hepatic insulin secreting cell having a reduced insulin secretion ability, or administered together with a cell capable of differentiating into a vascular endothelial cell to a limb having impaired circulation.

(10) The pharmaceutical composition of (1) above, which is administered together with cells for transplantation to a site of ischemic or diabetic disease to treat ischemic or diabetic organ disease.

(11) The pharmaceutical composition of the above (10), wherein the transplant cell is a cardiomyocyte, and for treating a heart disease,

(1 2) The medicament according to the above (10) or (11) for treating myocardial infarction or cardiomyopathy by administering together with myocardial cells for transplantation to a site of cardiac ischemia due to myocardial infarction or to an injured myocardium due to cardiomyopathy Composition,

(13) Treatment of diabetic organ disease by administration together with spleen hesin-secreting cells with reduced insulin secretion ability, or administration to limbs with impaired blood circulation together with cells that can be distributed to vascular endothelial cells. 10) a pharmaceutical composition,

(14) a pharmaceutical composition comprising HGF or an HGF gene as an active ingredient, for suppressing apoptosis of transplanted cells into a diseased lesion or fibrosis at the diseased lesion,

(15) The pharmaceutical composition according to the above (14), wherein the diseased site is an ischemic or diabetic site.

(16) The above-mentioned (14), wherein the diseased site is a damaged myocardial site or a cardiac ischemic site. Or a pharmaceutical composition of (15),

(17) The pharmaceutical composition according to any of (14) to (16), wherein the transplanted cells are cardiomyocytes,

(18) The pharmaceutical composition according to the above (15), wherein the diabetic disorder site is a stomach with impaired insulin secretion ability or a limb with impaired blood circulation,

(19) the pharmaceutical composition according to any of (1) to (is), wherein the HGF gene is in the form of a viral vector or a non-viral vector capable of expressing HGF;

(20) the pharmaceutical composition according to (19), wherein the HGF gene is in the form of HV J ribosome;

(2 1) The pharmaceutical composition according to any of (1) to (20) above, wherein the pharmaceutical composition is in the form of a sustained-release preparation comprising a biocompatible material,

(22) The pharmaceutical composition according to the above (21), wherein the biocompatible material is silicone, collagen, gelatin or a copolymer of glycolic acid and lactic acid.

(23) a method of promoting colonization of transplanted cells, which comprises administering HGF or an HGF gene to the diseased lesion when administering the cells for transplantation to the diseased lesion,

(24) The method according to (23), wherein the diseased site is an ischemic or diabetic site.

(25) The method according to (23) or (24) above, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

(26) The method according to any of (23) to (25) above, wherein the transplanted cells are cardiomyocytes,

(27) The method according to (24) above, wherein the diabetic disorder site is a limb with impaired insulin secretion or a limb with impaired blood circulation,

(28) The method according to (27) above, wherein the transplanted cells are cells capable of differentiating into insulin-secreting cells or vascular endothelial cells,

(29) a method for treating ischemic or diabetic organ disease, comprising administering an HGF or HGF gene together with transplanted cells to an ischemic or diabetic disease disorder site;

(30) the method of the above (29), wherein the HGF or the HGF gene is administered together with a cardiomyocyte for transplantation to a damaged myocardial site or a cardiac ischemic site to treat a heart disease; (31) Treat diabetic organ disease by administering HGF or HGF gene together with hepatic insulin secreting cells with reduced insulin secretion ability or cells capable of differentiating into vascular endothelial cells to limbs with impaired blood circulation The treatment method of the above (29),

(32) The therapeutic method according to any of (29) to (31) above, wherein the HGF gene is in the form of a viral vector or a non-viral vector capable of expressing HGF.

(33) The treatment method according to (32), wherein the HGF gene is in the form of HV J ribosome,

(34) The therapeutic method according to any of (27) to (33), wherein the HGF or the HGF gene is administered in the form of a sustained-release preparation comprising a biocompatible material,

(35) The method according to the above (34), wherein the biocompatible material is a copolymer of silicone, collagen, gelatin or glycolic acid / lactic acid.

(36) use of an HGF or HGF gene for producing a pharmaceutical composition for promoting transplantation of transplanted cells to a disease / lesion site,

(37) Use according to the above (36), wherein the disease lesion is an ischemic or diabetic lesion.

(38) The use of (36) or (37) above, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site;

(39) Use of any of (36) to (38) above, wherein the transplanted cells are cardiomyocytes,

(40) The use of the above (37), wherein the diabetic disorder site is a wisteria with reduced insulin secretion ability or a limb with impaired blood circulation,

(41) Use of the above (40), wherein the transplanted cells are cells capable of differentiating into insulin-secreting cells or vascular endothelial cells,

(42) use of HGF or an HGF gene for producing a pharmaceutical composition for suppressing apoptosis of transplanted cells into a diseased lesion or fibrosis at the diseased lesion,

(43) The use according to (42) above, wherein the diseased site is an ischemic or diabetic site.

(44) The above (42) or the above, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site. Or use of (43),

(45) Use of any of (42) to (44) above, wherein the transplanted cells are cardiomyocytes,

(46) The use of the above (43), wherein the diabetic disorder site is a wisteria with reduced insulin secretion ability or a limb with impaired blood circulation,

(47) Use of the above (46), wherein the transplanted cell is a cell capable of transferring to insulin-secreting cells or vascular endothelial cells,

(48) Use of any of the above ( ₃₆ ) to (47), wherein the HGF gene is in the form of a viral vector or a non-viral vector capable of expressing HGF,

(49) The use of the above (48), wherein the HGF gene is in the form of HV J ribosome, (50) the use of the above (36) to (49), wherein the pharmaceutical composition is in the form of a sustained-release preparation comprising a biocompatible material. Any use,

(51) Use of the above (50), wherein the biocompatible material is a copolymer of silicone, collagen, gelatin or glycolic acid / lactic acid,

It is.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing comparative data of improvement in cardiac function.

FIG. 2 is a graph showing the degree of improvement in myocardial blood flow at the injured myocardial site (infarct lesion). FIG. 3 is a tissue fragment micrograph showing the fixation of transplanted cardiomyocytes at the administration site stained with HF.

FIG. 4 is a micrograph of a tissue slice showing that cell adhesion factors (connexin43, Desmin) were confirmed between cardiomyocytes and that a Gap junction was formed.

FIG. 5 is a tissue fragment micrograph showing the viability of the transplanted cardiomyocytes.

FIG. 6 is a graph showing the inhibitory effect of cardiomyocyte fibrosis by the combined use of the HGF gene.

BEST MODE FOR CARRYING OUT THE INVENTION

The HGF used in the present invention is a known substance. As long as it is purified to the extent that it can be used as a medicine, HGF prepared by various methods can be used. For example, Toyobo Code No. HGF-101 etc.) May be used. As a method for producing HGF, for example, the HGF can be obtained by culturing primary cultured cells or cell lines that produce HGF, and separating and purifying it from a culture supernatant or the like. Alternatively, an HGF-encoding gene is incorporated into an appropriate vector by genetic engineering techniques, inserted into an appropriate host, and transformed to obtain the desired recombinant HGF from the culture supernatant of this transformant. (For example,

Nature, 342, 440 (1989), Japanese Patent Application Laid-Open No. Hei 5-111383, Biochem. Biophys. Res. Commun. 163, 967-989)). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, yeast, or animal cells can be used. The HGF thus obtained may have one or more amino acids in its amino acid sequence replaced, deleted and Z or added, as long as it has substantially the same action as natural HGF. Similarly, the sugar chain may be substituted, deleted and Z- or added.

The HGF gene used in the present invention refers to a gene capable of expressing HGF, and includes a part of the gene sequence as long as the expressed polypeptide is substantially the same as HGF. Also include a gene in which is deleted or replaced by another base, another base sequence is inserted into the base, or a base is bonded to the 5 'end and the Z or 3' end. Further, a gene that hybridizes with a gene encoding HGF under stringent conditions and encodes a protein having the same function as HGF may be used. Examples of such a gene include the HGF gene described in Nature, 342, 440 (1989), JP-A-5-111383, Biochem. Biophys. Res. Thus, these genes can be used in the present invention. These genes can be obtained by the well-known PCR method or can be chemically synthesized. In addition, these methods can be combined with, for example, site-directed mutagenesis, ordinary hybridization, and the like.

Next, the administration method and administration form of the HGF gene used in the present invention will be described.

When administering the HGF gene to a patient, the dosage form may be a non-viral vector dosage form, a viral vector dosage form, or a naked DNA It is roughly divided into three types of administration by the indirect injection method, and its preparation method and administration method are described in detail in experimental manuals and other documents. (Separate volume experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate volume experiment Medicine, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997). The details are described below.

A. When using non-viral vectors

A method of incorporating DNA encoding the HGF of the present invention into a gene expression vector and introducing a DNA molecule using a liposome (ribosome method, HVJ-ribosome method, catonic ribosome method, ribofectin method, ribofector method) Mining method), microinjection method, transfer of DNA molecule into cells together with carrier (metal particles) by gene gun (Gene Gun), etc. Is possible. Examples of expression vectors used here include pCAGGS (Gene 108, 193-200 (1991)), pBK-CMV, pcDN A3.1, pZeo SV (Invitrogen, Stratagene) and the like. Vector.

Among these, HVJ-ribosome encapsulates DNA in ribosomes made of lipid bilayer, and then fuses this liposome with inactivated Sendai virus (Hemagglutinating virus of Japan: HVJ). Things. The HV J-liposome method is characterized by having a very high fusion activity with the cell membrane as compared with the conventional liposome method, and is a preferable introduction form. HVJ-Liposome preparation method is described in the literature (Experimental Medicine Separate Volume, Basic Techniques for Gene Therapy, Yodosha, 1996, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997, J. Clin. Invest. 93, 1458- 1464 (1994), Am. J. Physiol. 271, R1212-1220 (1996)) and the like, and are described in detail in the examples described later. The HVJ is preferably the Z strain (available from ATCC), but basically other HVJ strains (eg, ATCC VR-907 and ATCC VR-105) can also be used.

B. When using viral vectors

A typical example of a virus vector is a method using a virus vector such as a recombinant adenovirus or retrovirus. More specifically, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus The DNA of the present invention is introduced into a DNA virus such as Nores, vaccinia-inoles, box-inoles, poliovirus, Cymbis virus, Sendai virus, SV40, immunodeficiency virus (HIV) or RNA virus, and the DNA is introduced into cells. By infecting the recombinant virus, it is possible to introduce the gene into cells. It is known that the adenovirus infection efficiency among the above virus vectors is much higher than when other virus vectors are used. From this viewpoint, it is preferable to use an adenovirus vector system.

C. DNA direct injection method (naked DNA method)

The direct DNA injection method is, for example, a method in which the above-mentioned expression plasmid, which is a non-viral vector, is dissolved in physiological saline and administered as it is.For example, Circulation, 96 (Suppl.Π), 382-388 (1997) Can be directly injected into tissues such as skeletal muscle, myocardium, subcutaneous, liver, and thyroid.

Next, the preparation form and dosage of the above-mentioned HGF gene used in the treatment of the present invention, and the administration route, preparation form and dosage when HGF is used, will be described.

As the above-mentioned preparation form of the HGF gene, various preparation forms (for example, liquid preparations) suitable for each of the above-mentioned administration forms can be taken. As a route of administration of HGF, it can be directly administered to tissues such as skeletal muscle, myocardium, spleen, liver, and thyroid.

The preparation may be, for example, an injection containing HGF or an HGF gene as an active ingredient. The injection can be prepared by a conventional method. For example, after dissolving in an appropriate solvent (buffer such as PBS, physiological saline, sterilized water, etc.), sterilizing by filtration with a filter or the like, and then aseptically It can be prepared by filling in a suitable container. A conventional carrier or the like may be added to the injection as needed. In addition, ribosomes such as HV J-ribosome used when administering the HGF gene can be in the form of a ribosome preparation such as a suspending agent, a freezing agent, and a centrifugal concentrated cryogenic agent. In particular, the HVJ ribosome preparation is a gene preparation prepared for administering the HGF gene, and is preferably administered parenterally. For example, administration methods using a non-invasive catheter or non-invasive syringe Can be. As an administration method using a non-invasive catheter, for example, injection of the HGF gene directly into the myocardium from the intraventricular cavity can be mentioned.

In order to facilitate the presence of HGF or HGF gene around the diseased site, it is possible to prepare a sustained-release preparation (mini-pellet preparation, etc.) and implant it near the affected part, or use an osmotic pump or the like. It can also be used for continuous and gradual administration to the affected area.

The biocompatible material that can be used in such a sustained-release preparation is not particularly limited as long as it is biocompatible. Specific examples of the non-degradable synthetic polymer include, for example, silicone and ethylene-butyl acetate. Examples include copolymers, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyatalylate, and polymethacrylate. Silicone is preferred from the viewpoint of easy molding. Specific examples of biodegradable synthetic polymers include, for example, collagen, gelatin, monohydroxycarponic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), hydroxy Polymers, copolymers, or mixtures thereof synthesized from one or more of tricarboxylic acids (eg, cunic acid, etc.) by non-catalytic dehydration polycondensation, poly (1-cyanoacrylate), polyamino acids (eg, polyamino acids) Poly-anhydrides such as mono-γ-benzyl-L-glutamic acid) and maleic anhydride copolymers (eg, styrene-maleic acid copolymer). The type of polymerization may be random, block, or graft. When monohydroxycarboxylic acids, hydroxydicarboxylic acids, or hydroxytricarboxylic acids have an optically active center in the molecule, D-, L-, or DL- Any of the bodies can be used. Preferably, a copolymer of glycolic acid and lactic acid can be used.

Among these, silicone, collagen, gelatin, or a glycolic acid / lactic acid copolymer is particularly preferred as a biocompatible material for a sustained-release preparation.

The dosage form of the sustained-release preparation is not particularly limited as long as it is a dosage form suitable for achieving the object of the present invention, and examples thereof include a rod shape (pellet shape, cylinder shape, needle shape, etc.), a tablet shape, and a disc shape. Shaped, spherical, and sheet-like preparations can also be made.

The HGF gene content of the preparation of the present invention can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, etc., and usually, a viral vector containing the DNA of the present invention. Alternatively, it is 0.0001 to 100 mg, preferably 0.001 to 10 mg as a non-viral vector, and it is preferable to administer it once every several days to several months. The dose of the HGF gene is selected from the range of about 1 to about 4000 μg, preferably about 10 to about 400 g per adult patient, as the amount of the HGF gene encoding HGF.

When HGF is used as an active ingredient, the HGF content of the preparation of the present invention can be appropriately adjusted in the same manner according to the disease to be treated, the age and weight of the patient, etc., but usually 6 // g to 600 mg, preferably Is 60 g to 6 Omg, which is preferably administered once every few days to several months.

As the transplant cell used in the present invention, any cell can be appropriately used as long as the cell can be transplanted to a site of a damaged tissue associated with an ischemic or diabetic organ disease. For example, in heart diseases such as severe heart failure and severe myocardial infarction, cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle-derived cells (particularly satellite cells), bone marrow cells (particularly myocardium) Bone marrow cells differentiated into like cells) and the like. Furthermore, transplantation cells can be appropriately selected for other organs. For example, transplantation of neural progenitor cells into cerebral ischemia / cerebral infarct site or cells capable of differentiating into neural cells, differentiation into vascular endothelial cells or vascular endothelial cells into myocardial infarction site / skeletal muscle ischemic site Transplantation of natural cells is an example of the transplanted cells. Furthermore, there are cells used for cell transplantation for diabetic organ damage. For example, cells for cell transplantation therapy, which are variously examined for diseases such as impaired blood circulation of the kidney, knee, peripheral nerves, eyes, and limbs, can be mentioned. In other words, attempts are being made to transplant insulin-secreting cells into the spleen with reduced insulin secretion ability, and to transplant cells that can differentiate into vascular endothelial cells or bone marrow-derived cells for impaired limb blood circulation. Such cells can be used.

The dose of the transplanted cells can be appropriately adjusted depending on the disease to be treated, the age of the patient, the transplanted cells to be treated, and the like. Usually, the dose of the transplanted cells of the present invention is 1 × 10 ⁴ to 10 ^11. It is preferable to administer HGF or the HGF gene once a few days to several months at the same time. For example, when cardiomyocytes are used, the range is 1 × 10 ⁴ to 10 ¹¹ , preferably ¹ × 10 ⁶ to 10 ^9. The dose is selected from the box.

In addition to the HGF or HGF gene introduced in the present invention, an endogenous cardioprotective factor or a cardiomyocyte regeneration factor can be used in combination. For example, it has been reported that factors such as TGF- and heat shock protein (HSP), which are highly expressed during cardiomyocyte injury, reduce myocardial injury and are involved in myocardial repair. I can do it. In addition, it has been reported that growth factors such as EGF repair various cell damages in tissues, and these genes can also be used. Furthermore, in addition to these myocardial protective factors and regeneration factors, factors involved in myocardial protection and regeneration can be considered.

In the present invention, the HGF or HGF gene can be introduced into cardiac myocardial cells alone or in combination as described above, and highly expressed, thereby synthesizing a target protein necessary for damaged cardiomyocytes and the like. Thus, by activating the damaged myocardial cells and the like, the myocardial cells and the like can be restored and regenerated, and the recovery and normalization of the cardiac function that has fallen into cardiomyopathy can be performed. Therefore, it can be used not only for patients with severe cardiomyopathy, but also for those with mild ongoing disease.

By applying the preparation and treatment method of the present invention, it is possible to not only perform aggressive myocardial transplantation treatment for heart disease patients with severe heart failure, severe myocardial infarction, cardiomyopathy, etc. Alternatively, it has been found that effective treatment for organ diseases caused by diabetes can be achieved by means of cell transplantation. For example, for skeletal muscle damage due to ischemia or diabetes, or knee damage or liver damage due to ischemia or diabetic, use the cell transplantation instead of the organ transplantation to indicate the damaged area of the damaged organ. It became possible to treat.

The present invention is particularly effective in heart disease. According to the present invention, it becomes possible to repair the injured myocardium that has fallen into heart failure and to improve the heart function. Thus, a new rescue path was possible for patients with severe heart failure, severe myocardial infarction, cardiomyopathy, etc., for whom there was no cure other than heart transplantation.

Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Effect of HGF gene administration on transplantation of cultured cardiomyocytes to rat ischemic heart

The left anterior descending branch of a Lewis rat (3 weeks old, male) was ligated to produce a rat myocardial infarction model. Two weeks later, only the medium was injected into the ischemic site around the infarct area, and a control group (n = 5) was obtained. In the cardiomyocyte transplantation group (n = 7), IX 10 cultured cardiomyocytes were injected together with the medium, and in the cardiomyocyte transplantation + HGF gene administration group (n = 5), the same amount of cultured cardiomyocytes was injected and the same. 4 g of the HGF gene was administered by the HVJ-liposome method to the injection site.

The HGF gene used was prepared as follows. First, 2 ml of an HVJ-liposome preparation was prepared from 200 _§ of 11 GF cDNA plasmid. The HGF cDNA plasmid content in the liposome preparation was 20% of the plasmid used (40 μg of plasmid was contained in the preparation). 0.2 ml of this 2 ml HV J-liposome preparation was administered.

Administration was performed by injecting and administering cardiomyocytes and the HGF gene to three sites of the injured myocardium, respectively.

Improvement of cardiac function and improvement of myocardial blood flow at 4 and 8 weeks after cardiomyocyte transplantation were evaluated by the following methods.

① Improvement of heart function:

For ultrasonic dimensional echocardiography! The ejection fraction (EF) was measured. In the evaluation of cardiac function improvement, the degree of improvement was represented by setting the value of one solid before transplantation to 1. Cardiac function did not improve at all in the control group, but mild cardiac function improvement was observed in the cardiomyocyte transplant group (cellTx) as shown in FIG. In addition, marked improvement was seen in the cardiomyocyte transplantation + HGF gene administration group (cellTx + HGF).

②Improved myocardial blood flow at the affected myocardial site (infarct lesion):

It was evaluated by contrast echocardiography. Figure 2 shows an example of the results. A comparison between the control (Ligation model) and the case of cardiomyocyte transplantation + HGF gene administration (cellTx + HGF) showed an improvement in myocardial blood flow at the injured myocardial site (infarct lesion).

③ Adherence of transplanted cardiomyocytes:

Sections of the administration site were prepared and confirmed by microscopic histological evaluation. HF dyeing As shown in Fig. 3 after the color, in the cardiomyocyte transplantation + HGF gene administration group (cellTx + HGF), the transplanted cardiomyocytes settled in the injured myocardium (infarct lesion), and the transplanted cardiomyocytes developed well. However, the increase in cell diameter was histologically observed.

In addition, as shown in Fig. 4, cell adhesion factors such as connexin43 and Desmin were confirmed between cardiomyocytes, and formation of Gap junction was observed.

生存 Viability of transplanted cardiomyocytes:

The apoptosis cells were searched by TUNEL staining. Fig. 5 shows the stained image. According to the results, apoptosis of the transplanted cardiomyocytes was observed in the cardiomyocyte transplantation group (cellTx), but not in the cardiomyocyte transplantation + HGF gene administration group (cellTx + HGF).

抑制 Inhibitory effect of cardiomyocyte fibrosis by combined use of HGF gene:

Cell sections from the injured myocardium (infarct lesion) were stained with Masson's trichrome to evaluate fibrosis. Cardiomyocytes were stained red, and fibroblasts and fibrotic areas were stained blue. This was processed by computer and calculated and evaluated based on the ratio of the blue area to the total area. As shown in FIG. 6, compared to the cardiomyocyte transplantation group (cellTx), fibrosis was significantly suppressed in the cardiomyocyte transplantation + HGF gene administration group (cellTx + HGF).

Example 2

Effect of HGF on transplantation of cultured cardiomyocytes to rat ischemic heart

The left anterior descending branch of a Lewis rat (3 weeks old, male) is ligated to prepare a rat myocardial infarction model. Two weeks later, only the medium is injected into the ischemic area around the infarct area, and a control group (n = 5 to 10) is used. In the cardiomyocyte transplantation group (n = 5 to 10), 1 × 10 ⁷ cultured cardiomyocytes were injected together with the medium, and in the cardiomyocyte transplantation + HGF administration group (n = 5 to 10), the same amount of cultured cardiomyocytes And 1 to 100 ^ / 1¾ HGF is administered to the same injection site. Administration is performed by injecting and administering cardiomyocytes and HGF to the three sites of the injured myocardium, respectively.

The following methods are used to evaluate the improvement of cardiac function and the improvement of myocardial blood flow at 4 and 8 weeks after cardiomyocyte transplantation.

① Improvement of heart function:

Ultrasonic three-dimensional echocardiography) ejection fraction (EF) Is measured. The evaluation of cardiac function improvement can also be evaluated by expressing the degree of improvement assuming that the value of one solid before transplantation is 1. Compared with the control group and the cardiomyocyte transplantation group (cellTx), marked improvement is seen in the cardiomyocyte transplantation + HGF administration group (cellTx + HGF).

Evaluate by contrast echocardiography. Compared with the control group, cardiac muscle blood flow was improved in the injured myocardial site (infarct lesion) in the case of cardiomyocyte transplantation + HGF administration (cellTx + HGF).

③ Adherence of transplanted cardiomyocytes:

Make HE-stained sections at the site of administration and confirm by microscopic histological evaluation. In the cardiomyocyte transplantation + HGF administration group (cellTx + HGF), the transplanted cardiomyocytes are established at the injured myocardium (infarct lesion), the sarcomere of the transplanted cardiomyocytes is well developed, and the cell diameter is increasing.

生存 Viability of transplanted cardiomyocytes:

Apoptosis cells can be evaluated by TUNEL staining. Apoptosis of the transplanted cardiomyocytes is observed in the cardiomyocyte transplantation group (cellTx), but not in the cardiomyocyte transplantation + HGF administration group (cellTx + HGF).

効果 Inhibitory effect of cardiomyocyte fibrosis by combined use of HGF:

Cell sections of the injured myocardium (infarct lesion) are stained with Masson's trichrome to evaluate fibrosis. Cardiomyocytes are stained red, and fibroblasts and fibrotic areas are stained blue. This is processed by computer, and the ratio of the blue area to the total area is calculated and evaluated. Compared with the cardiomyocyte transplantation group (cellTx), fibrosis is suppressed in the cardiomyocyte transplantation + HGF administration group (cellTx + HGF).

Example 3

Administration effect of sustained release preparation of HGF on transplantation of cultured cardiomyocytes to rat ischemic heart

(1) Preparation of sustained-release HGF preparation

Was dissolved HGF 2% collagen-containing aqueous solution, 0.6 to 6 0 _§ / 111 1 of 110 F solutions were prepared to prepare a solution form of HGF sustained release formulation.

(2) Administration effect of sustained-release HGF preparation

A rat myocardial infarction model was prepared as in Example 2, two weeks later, divided into three groups, For the control group, 0.2 ml of the medium alone is administered into the ischemic area around the infarct area. In the cardiomyocyte transplantation group, 1 × 10 ⁷ cultured cardiomyocytes are injected together with the medium into the ischemic site around the infarct area. In the cardiomyocyte transplantation + HGF sustained release preparation administration group, 0.2 ml of a liquid HGF sustained release preparation is administered. Administration is performed by injecting and administering cardiomyocytes and HGF to three sites of the affected myocardium, respectively.

After administration of the HGF preparation, the improvement of cardiac function and the improvement of myocardial blood flow at 4 weeks and 8 weeks after the administration are evaluated in the same manner as in the previous section.

It has excellent effects such as improvement of cardiac function, improvement of blood flow at the injured myocardium, fixation of transplanted cardiomyocytes, and survival of transplanted cardiomyocytes.

Industrial applicability

The HGF gene or HGF can promote the colonization of cardiomyocytes transplanted into the affected area of the injured myocardium, and can also suppress apoptosis of the transplanted cardiomyocytes and fibrosis at the damaged site. Therefore, the HGF gene or HGF can be effectively used for treatment of severe myocardial infarction or cardiomyopathy by cardiomyocyte transplantation.

Claims

The scope of the claims

I. A pharmaceutical composition comprising liver hepatocyte growth factor (HGF) or the HGF gene as an active ingredient, for promoting cell colonization of transplanted cells at a disease / lesion site.

2. The pharmaceutical composition according to claim 1, wherein the diseased site is an ischemic or diabetic site.

3. The pharmaceutical composition according to claim 1 or 2, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the diseased site is a site of cardiac ischemia due to myocardial infarction or a site of damaged myocardium due to cardiomyopathy.

5. The pharmaceutical composition of claim 2, wherein the diabetic disorder site is a knee with reduced insulin secretion ability or a limb with impaired blood circulation.

6. The pharmaceutical composition according to any one of claims 1 to 5, for administration together with cells for transplantation to a site of a disease.

7. The pharmaceutical composition according to claim 6, which is administered to a site of a damaged myocardium or a site of cardiac ischemia together with a cardiomyocyte for transplantation.

8. The pharmaceutical composition according to claim 6 or 7, which is administered to a site of cardiac ischemia due to myocardial infarction or a site of injured myocardium due to cardiomyopathy together with cardiac muscle cells for transplantation.

9. The pharmaceutical composition according to claim 6, which is to be administered together with insulin-secreting cells of a visceral mouse whose insulin secretion ability has been reduced, or to a limb having impaired blood circulation together with cells capable of dividing vascular endothelial cells.

10. The pharmaceutical composition according to claim 1 for treating an ischemic or diabetic organ disease by administering it to an ischemic or diabetic disease disorder site together with cells for transplantation.

11. The pharmaceutical composition according to claim 10, wherein the cells for transplantation are cardiomyocytes and are used for treating heart disease.

12. The pharmaceutical composition of claim 10 or 11 for treating myocardial infarction or cardiomyopathy by administering it to a site of cardiac ischemia due to myocardial infarction or to a site of impaired myocardium due to cardiomyopathy together with cardiomyocytes for transplantation.

1 3. Administered together with hepatic insulin secreting cells with reduced insulin secretion 10. The pharmaceutical composition according to claim 10, wherein the composition is administered to a limb having blood circulation disorders together with cells capable of differentiating vascular endothelial cells to treat diabetic organ diseases.

14. A pharmaceutical composition for inhibiting apoptosis of transplanted cells into a diseased lesion or fibrosis at a diseased lesion, comprising a HGF or HGF gene as an active ingredient.

15. The pharmaceutical composition according to claim 14, wherein the diseased site is an ischemic or diabetic site.

16. The pharmaceutical composition according to claim 14 or 15, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the transplanted cells are cardiomyocytes.

18. The pharmaceutical composition according to claim 15, wherein the diabetic disorder site is a spleen with reduced insulin secretion ability or a limb with impaired blood circulation.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the HGF gene is in the form of a viral vector or a non-viral vector capable of expressing HGF.

20. The pharmaceutical composition of claim 19, wherein the HGF gene is in the form of HVJ ribosome.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the pharmaceutical composition is in the form of a sustained-release preparation comprising a biocompatible material.

22. The pharmaceutical composition according to claim 21, wherein the biocompatible material is silicone, collagen, gelatin or a copolymer of glycolic acid and lactic acid.

23. A method for promoting colonization of transplanted cells, which comprises administering HGF or an HGF gene to the diseased lesion when administering the cells for transplantation to the diseased lesion.

24. The method according to claim 23, wherein the diseased lesion is an ischemic or diabetic lesion.

25. The method according to claim 23 or claim 24, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

26. The method according to any one of claims 23 to 25, wherein the transplanted cells are cardiomyocytes.

2 7. Spleen or blood circulation disorder in which diabetic lesion has decreased insulin secretion ability 25. The method of claim 24, which is a harmful limb.

28. The method according to claim 27, wherein the transplanted cell is a cell that can be separated into insulin-secreting cells or vascular endothelial cells.

29. A method for treating ischemic or diabetic organ disease, comprising administering HGF or an HGF gene together with transplanted cells to an ischemic or diabetic disease disorder site.

30. The method according to claim 29, wherein the HGF or HGF gene is administered to a site of a damaged myocardium or a site of cardiac ischemia together with a cardiomyocyte for transplantation to treat a heart disease.

31. Treat diabetic organ disease by administering HGF or HGF gene together with hepatic insulin secreting cells with reduced insulin secretion ability or cells capable of dividing vascular endothelial cells to limbs with impaired blood circulation 30. The method of claim 29, wherein the method comprises:

32. The method according to any one of claims 29 to 31, wherein the HGF gene is in the form of a viral vector or a non-viral vector capable of expressing HGF.

33. The method of claim 32, wherein the HGF gene is in the form of an HV J ribosome.

34. The method according to any one of claims 27 to 33, wherein HGF or the HGF gene is administered in the form of a sustained-release preparation comprising a biocompatible material.

35. The method according to claim 34, wherein the biocompatible material is silicone, collagen, gelatin, or a copolymer of glycolic acid and lactic acid.

36. Use of HGF or an HGF gene for producing a pharmaceutical composition for promoting transplantation of transplanted cells to a site of disease.

37. Use according to claim 36, wherein the disease site is an ischemic or diabetic site.

38. The use according to claim 36 or 37, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

39. Use according to any of claims 36 to 38, wherein the transplanted cells are cardiomyocytes.

40. Use according to claim 37, wherein the diabetic disorder site is a knee with reduced insulin secretion capacity or a limb with impaired blood circulation.

41. Use according to claim 40, wherein the transplanted cells are cells capable of differentiating into insulin secreting cells or vascular endothelial cells.

42. Apoptosis of transplanted cells into diseased lesion or fiber at diseased lesion Use of HGF or an HGF gene for producing a pharmaceutical composition for suppressing the activation.

43. Use according to claim 42, wherein the disease site is an ischemic or diabetic site.

44. Use according to claim 42 or 43, wherein the diseased site is a damaged myocardial site or a cardiac ischemic site.

45. The use according to any one of claims 42 to 44, wherein the transplanted cell is a cardiomyocyte.

46. Use according to claim 43, wherein the diabetic disorder site is the excretory limb with impaired insulin secretion or blood circulation disorders.

47. Use according to claim 46, wherein the transplanted cells are cells capable of differentiating into insulin-secreting cells or vascular endothelial cells.

48. Use according to any of claims 36 to 47, wherein the HGF gene is in the form of a viral or non-viral vector capable of expressing HGF.

49. Use according to claim 48, wherein the HGF gene is in the form of the HV J ribosome.

50. Use according to any of claims 36 to 49, wherein the pharmaceutical composition is in the form of a sustained release formulation comprising a biocompatible material.

51. Use according to claim 50, wherein the biocompatible material is a copolymer of silicone, collagen, gelatin or glycolic acid / lactic acid.